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Genotype-phenotype correlations in hereditary multiple exostoses in British Columbia Alvarez, Christine M. 2003-12-31

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^  Genotype-Phenotype Correlations in Hereditary Multiple Exostoses in British Columbia  By Christine M . Alvarez B.Sc, University of Victoria, 1986 M.D., University of British Columbia, 1993 FRCSC, Royal College of Physicians and Surgeons of Canada, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Faculty of Medicine, Department of Surgery) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 2003 © Christine M. Alvarez, 2003  In  presenting  degree freely  at  the  available  copying  of  department publication  this  of  in  partial  University  of  British  for  this or  thesis  reference  thesis by  this  for  his thesis  and  for  her  Department  of  The University of British C o l u m b i a Vancouver, Canada  Date  DE-6  (2/88)  I further  the  representatives. gain  shall  requirements  agree  that  agree  purposes may  financial  permission.  of  Columbia, I  study.  scholarly  or  fulfilment  be  It not  that  the  be  Library  an  advanced  shall make  permission for  granted  is  for  by  understood allowed  the  extensive  head  that  without  it  of  copying my  my or  written  Abstract Hereditary Multiple Exostosis is an autosomal dominant condition in which multiple benign cartilage-capped tumours grow in relation to the growth plates of long and flat bones. . HME has a wide spectrum of clinical presentations and results in considerable morbidity from lesions due to mass effect causing limb deformity, mal-alignment, and shortening. Mutations in EXT 1 and 2 genes result in multiple exostoses. The presumptive role of the EXT genes is either tumour suppression or growth plate regulation. The purpose of this study was to determine the relationship between the genotype and phenotype in HME. Ten families were identified with HME. Genotyping was completed by linkage analysis of all families and the EXT 1 or 2 gene was sequenced based on these results. Mutation identification and confirmation was performed. Phenotyping consisting of clinical and radiographic examinations generated 89 features for each subject. Thirty-two affected individuals from 10 families participated. Eight of 10 mutations were identified, confirmed and segregation verified. Six of the mutations were unique and 2 previously had been reported in the literature. Three mutations were in EXT 1 and 5 in EXT 2. Two were missense, 3 nonsense, 2 splice site and 1 frameshift. EXT 1 patients were found to have more exostoses, with a higher percentage of flat and pelvic bone involvement. EXT 1 patients had more mal-alignment and were shorter. Males also had a more severe phenotype and modulated the severity of EXT 1 expression. No other genotypic factors were found to influence phenotype. An established genotype phenotype correlation will aid in patient management in terms of surveillance, determining prognosis and mangement.  In conclusion a genotype  phenotype correlation exists where EXT 1 is linked to a more severe phenotype.  ii  Table of Contents  Abstract Table of Contents ListofTables List of Figures Acknowledgements  ii iii viii ix xi  Chapter I  Background  1  1.1 Osteochondroma (Exostosis) 1.1.1 definition 1.1.2 features 1.1.1.1 radiology 1.1.1.2 gross pathology 1.1.1.3 microscopic pathology... 1.1.1.4 clinical 1.2 Hereditary Multiple Exostoses 1.2.1 definition 1.2.2 demographic features 1.2.3 genetics and molecular biology 1.2.3.1 general information 1.2.3.2 physiologic function 1.2.3.3 EXT gene products 1.2.4 Mutations 1.2.4.1 EXT 1 mutation summary 1.2.4.2 EXT 2 mutation summary 1.2.5 Phenotyping 1.2.5.1 Schmale's findings 1.2.5.2 Porter's findings 1.2.5.3 Genotype-phenotype correlations 1.2.5.3.1 Carroll 1.2.5.3.2 Francannet.... 1.3 Proj ect rationale 1.4 Hypothesis 1.5 Objective  1 1 1 1 2 4 5 8 8 8 10 10 14 20 22  Methods and Materials  40  2.1 Ethical Approval  40  2.2 Protocol Overview  41  2.3 Subject Recruitment 2.3.1 Subject identification 2.3.2 Pedigree accumulation  42 42 42  Chapter II  iii  29 29 30 30 32 33 33 35 36 39 39  2.4 Genotype 2.4.1 Sample collection 2.4.2 DNA extraction 2.4.3 Gene Assignment - Highly Polymorphic Repeats 2.4.3.1 Marker Selection 2.4.3.2 PCR 2.4.3 3 PAGE 2.4.3.4 Hybridization 2.4.3.5 Visualization 2.4.3.6 Exclusion Analysis 2.4.4 EXT 1 and EXT 2 amplification 2.4.5 DNA sequencing 2.4.6 Mutation identification 2.4.7 Segregation Analysis 2.5 Phenotype  Chapter III  42 42 43 44 44 46 46 47 48 48 48 51 52 54  54 2.5.1 Clinical 55 2.5.1.1 Demographics 55 2.5.1.2 Lesion count 55 2.5.1.3 Limb alignment 55 2.5.1.4 Limb segments 56 2.5.1.5 Range of Motion 56 2.5.2 Radiographic 57 2.5.2.1 Lesion quality 57 2.5.2.1.1 Count 57 2.5.2.1.2 Size 57 2.5.2.1.3 Side 58 2.5.2.1.4 Location 58 2.5.2.1.5 Complexity 58 2.5.2.1.6 Flaring 58 2.5.2.1.7 Type 58 2.5.2.2 Limb alignment and deformity... 58  2.6 Data Analysis 2.6.1. Genotype 2.6.2 Phenotype 2.6.3 Genotype-phenotype correlation  63 63 63 64  Results  66  3.1 Subject recruitment 3.1.1. Subject identification 3.1.2 Family pedigrees  66 66 68  3.2 Genotype  68 68 81 82  3.2.1 Highly Polymorphic Repeats 3.2.2 Mutation identification 3.3 Phenotype iv  3.3.1 Phenotype data 3.3.2 Range of motion 3.3.3 Pearson Correlation matrix 3.4 Genotype-Phenotype Correlations 3.4.1 Gene versus phenotype 3.4.2 Gene and gender versus phenotype 3.4.3 Gene and mutation type versus phenotype... 3.4.4 Gene and severity versus phenotype 3.4.5 Gene and mutation location versus phenotype 3.4.6 Gender versus phenotype 3.4.7 Mutation type versus phenotype 3.4.8 Mutation severity versus phenotype 3.4.9 Mutation location versus phenotype 3.4.10 Gender and severity versus phenotype 3.4.11 Gender and mutation type versus phenotype Chapter IV  82 82 83 83 88 89 90 91 91 92 93 93 93 94 95  Discussion  96  4.1 Subject recruitment 4.2 Genotpye 4.3 Phenotype 4.3.1 Lesion Quality 4.3.2 Limb Alignment 4.3.3 Limb segments and percentile height 4.3.4 Intra-family variability 4.4 Genotype-phenotype correlation  96 96 103 103 107 108 110 Ill  Chapter V  Summary  118  Chapter VI  Conclusion  119  Chapter VII  Future Work  120  Bibliography  123  Appendices  136  8.1 Ethics Approval 136 8.1.1 Children's and Women's Hospital of British Columbia 137 8.1.2 University of British Columbia 138 8.2 Letter of information and consent forms 139 8.3 EXT 1 142 8.3.1 EXT 1 map (cDNA, primers, and positions). 142 8.3.2 EXT 1 translation 144 8.4 EXT 2 147 v  8.4.1 EXT 2 map (cDNA, primers, and positions). 8.4.2 EXT 2 translation 8.5 Genotyping 8.5.1 HPR markers 8.5.2 HPR sequences 8.6 Data 8.6.1 Family pedigrees 8.6.2 STR gels 8.6.3 Phenotype data 8.6.3.1 Core data 8.6.4.1.1 Lesion quality 8.6.4.1.2 Limb alignment 8.6.4.1.3 Limb segments and percentile height 8.6.3.2 Pearson Correlation matrix 8.7 Genotype-Phenotype Correlation tables 8.7.1. Gene 8.7.1.1 Lesion Quality 8.7.1.2 Limb alignment 8.7.1.3 Limb segments and percentile height 8.7.2 Gender 8.7.2.1 Lesion Quality 8.7.2.2 Limb alignment 8.7.2.3 Limb segments and percentile height 8.7.3 Mutation Type 8.7.3.1 Lesion Quality 8.7.3.2 Limb alignment 8.7.3.3 Limb segments and percentile height 8.7.4 Mutation severity 8.7.4.1 Lesion Quality 8.7.4.2 Limb alignment 8.7.4.3 Limb segments and percentile height 8.7.5 Mutation location 8.7.5.1 Lesion Quality 8.7.5.2 Limb alignment 8.7.5.3 Limb segments and percentile height 8.7.6 Gene and gender 8.7.6.1 Lesion Quality 8.7.6.2 Limb alignment 8.7.6.3 Limb segments and percentile height 8.7.7 Gene and mutation type vi  147 158 161 161 162 163 163 173 179 179 179 181 186 188 202 202 202 203 204 205 205 206 207 208 208 208 210 211 211 212 213 214 214 215 216 217 217 219 220 221  8.7.7.1 Lesion Quality 8.7.7.2 Limb alignment 8.7.7.3 Limb segments and percentile height 8.7.8 Gene and severity 8.7.8.1 Lesion Quality 8.7.8.2 Limb alignment 8.7.8.3 Limb segments and percentile height 8.7.9 Gender and Severity 8.7.9.1 Lesion Quality 8.7.9.2 Limb alignment 8.7.9.3 Limb segments and percentile height 8.7.10 Gender and Mutation Type 8.7.10.1 Lesion Quality 8.7.10.2 Limb alignment 8.7.10.3 Limb segments and percentile height 8.7.11 Gene and Location 8.7.11.1 Lesion Quality 8.7.11.2 Limb alignment 8.7.11.3 Limb segments and percentile height  vii  221 225 229 231 231 233 235 236 236 238 240 241 241 243 245 249 249 251 253  List of Tables  Table 1.1 Table 1.2 Table 1.3 Table 1.4  Summary of Family Mutations Summary of Mutations Identified in the EXT 1 Gene Summary of Mutations Identified in the EXT 2 Gene Modified Functional Assessment Scale of the Musculoskeletal Tumour Society (as per Schmale 1994)  Table 2.1 Table 2.2  Primer pair sequences used for EXT 1 Primer pair sequences used for EXT 2  Table 3.1 Table 3.2  Subject Recruitment Summary of STR Markers as per family and EXT gene assignment for mutations identified in EXT 1 and EXT 2 Mutations identified in each proband Breakdown of Genotype Features Summary of Results for Comparison between EXT 1 and EXT 2 Genes Summary of Results for remaining unvariant data Summary of Results for Comparison between Males and Females Covariant Data  Table 3.3 Table 3.4 Table 3.5 Table 3.6 Table 3.7  viii  11 24-25 26 32 50 51 67 69 81 84 88 92 94  List of Figures  Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 1.10 Figure 1.11 Figure 1.12 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Figure 2.10 Figure 2.11 Figure 2.12 Figure 2.13 Figure 3.1a Figure 3.1b Figure 3.2a Figure 3.2b Figure 3.3a Figure 3.3b Figure 3.4a(i) Figure 3.4a (ii) Figure 3.5a  X-rays showing presence of an exostoses at the (a) distal femur (Wold 1990) and (b) proximal humerus X-ray and CT scan showing location of an exostosis in relation to the parent bone Gross pathology of with the xray of the same lesion in situ (Wold 1990) X-rays showing a (a) pedunculated exostosis, (b) sessile exostosis (solitary osteochondroma subject) and (c) a lesion causing metaphyseal flaring (a) Epiphyseal growth plate (Wheater 1987); (b) An osteochondroma at low magnification and (c) at high magnification (Wold 1990) (a) X-rays showing exostosis tethering the growth plate in an affected ankle (b) A normal ankle is shown for comparison (a) X-ray showing an exostosis causing deformity in the foream; (b) a normal forearm is shown for comparison X-ray showing exostosis causing growth impedance Alignment of EXT 1 and EXT 2 genes Distribution of Mutations in the EXT 1 Gene Distribution of Mutations in the EXT 2 Gene Anatomical Distribution of Lesions (Schmale 1994). Overview of materials and methods HPR marker locations in relation to EXT 1, 2, and 3 Calculation of Lesion Size and Rank Measurement of carpal slip Measurement of radial inclination and ulnar shortening Measurement of radial bowing Radial head subluxation / dislocation Measurement of the elbow joint angle Measurement of the femoro-tibial anatomic angle Measurement of the weight bearing axis, the femoral neck/shaft angle, and the femoral anatomic angle Measurement of Sharp's acetabular angle Measurement of fibular height. Measurement of ankle j oint angle EXT 1 and EXT 2 STR Markers for Family 1 Sequencher output for segregation analysis for Family 1 EXT 1 and EXT 2 STR Markers for Family 16 Sequencher output for segregation analysis for Family 16 EXT 1 and EXT 2 STR Markers for Family 18 Sequencher output for segregation analysis for Family 18 EXT 1 STR Markers Family 6. EXT 2 STR Markers Family 6  1 2 3 4 5 7 7 8 13 27 28 31 41 45 58 59 59 59 60 60 60 61 62 62 62 70 70 71 71 72 72 73 73  EXT 1 and EXT 2 STR Markers Family 2 ix  74  Figure 3.5b Figure 3.6a Figure 3.6b Figure 3.7a Figure 3.7b Figure 3.8a Figure 3.8b Figure 3.9a(i) Figure 3.9a (ii) Figure 3.10a Figure 3.10b  Sequencher output for segregation analysis for Family 2 EXT 1 and EXT 2 STR Markers Family 5 Sequencher output for segregation analysis for Family 5 EXT 1 and EXT 2 STR Markers Family 17 Sequencher output for segregation analysis for Family 17 EXT 1 and EXT 2 STR Markers Family 8 Sequencher output for segregation analysis for Family 8 EXT 1 STR Markers for Family 4 EXT 12 STR Markers for Family 4  74 75 75 76 76 77 77 78 78  EXT 1 and EXT 2 STR Markers for Family 3  79  Sequencher output for segregation analysis for Family 3  80  x  Acknowledgements  Dr. Michael Hayden Dr. Stephen Tredwell Dr. Peter O'Brien Odell Loubser - Lab support for genotyping Mary De Vera - Study research assistant Heather MacDonald - Study research assistant Kirsten Roomp - Genotyping technical support Kathryn Duff - Phenotyping Susie Clee - Genotyping technical support Keith Fichter - Genotyping technical support Angie Brooks-Wilson - Genotyping technical support Jennifer Collins - Genotyping technical support Dr. Bonita Sawatzky - Phenotyping BCCH Department of Orthopaedics - Patient recruitment  xi  Chapter I: Background 1.1 Osteochondroma (Exostosis) 1.1.1 Definition (osteochondroma, exostosis)  An exostosis or osteochondroma is a benign, cartilage-capped, bone tumour. These lesions can grow adjacent to the physis of all bones (Solomon 1963). They have a propensity to grow at the ends of the long bones, in particular around the knee and shoulder, which account for 57% of lesions (Wold 1990,). They can also occur on flat bones and on vertebrae.  Figure 1.1 X-rays showing presence of an exostoses at the (a) distal femur (Wold 1990) and (b) proximal humerus  1.1.2 Features 1.1.1.1 R a d i o l o g i c  Radiographically, these lesions appear as bony projections which are contiguous with the parent bone (Figure 1.2). The cartilage caps are radiolucent and not appreciated on plane xray when there is no mineralization in the cap. With maturity of the patient, mineralization is seen in the cartilaginous component of the tumor. The continuity of the 1  cortical and cancellous bone with the parent bone is reliably demonstrated using computed tomography (CT scan). Visualization of the unmineralized cartilage cap is only possible by magnetic resonance imaging (MRI) (Pierz et al. 2001).  Figure 1.2  X-ray and C T scan showing location of an exostosis in relation to the parent bone  1.1.1.2 Gross Pathology  The pathology of osteochondromas was described in detail by Jaffe in 1943. The gross pathology of these lesions shows that a layer of smooth translucent, bluish cartilage is evident on the cut surface (Figure 1.3).  2  Figure 1.3 Gross pathology of an exostosis with the xray of the same lesion in situ. (Wold 1990)  The thickness of the cartilage cap varies with the activity of the lesion. Lesions typically develop and grow during childhood when the cartilage cap can be up to two centimetres thick. In contrast, adults do not develop new lesions and those that are present are quiescent with cartilage caps that are less than one centimeter in thickness. If an adult's lesion continues to grow, the cap is greater than two centimeter thick, and there is mineralization in the cap, transformation from a benign to a malignant process may have occurred (Pierz et al. 2001). The morphology of the lesion may be sessile or pedunculated (Figure 1.4a and b). In some cases, particularly in cases of multiple exostoses, the metaphysis may be globally involved by the lesion resulting in metaphyseal flaring (Figure 1.4c).  3  Figure 1.4 X-rays showing a (a) pedunculated exostosis (Wold 1990), (b) sessile Exostosis (solitary osteochondroma subject) and (c) a lesion causing metaphyseal flaring  1.1.1.3 M i c r o s c o p i c Pathology  Microscopically, the cartilaginous cap mimics the appearance of an epiphyhseal plate (physis) with the maturation architecture seen in enchondral bone formation (Figure 1.5). The chondrocytes exhibit a lack of pleomorphism, nuclear hyperchromasia, and binucleation. The underlying cancellous bone shows intertrabecular spaces filled with fatty or hematopoietic marrow (Wold 1990, 53).  4  Figure 1.5 (a) Epiphyseal growth plate (Wheater 1987); (b) An osteochondroma at low magnification and (c) at high magnification (Wold 1990) 1.1.1.4 Clinical  Clinically these lesions are identified as a palpable lump; however, they cause a plethora of secondary symptoms. By mass effect alone they can compress local nerves leading to pain, paraesthesia, and paralysis. The lesion can interfere with local tendons causing locking, pain, or erosions leading to ruptures. Compression of the surrounding vasculature can also result in pain, pseudoaneurysm formation, or downstream thrombus generation. Osteochondromas depending on size and or location may also cause unacceptable cosmetic disfigurement (Mirra 1989). Depending on their relationship with the adjacent growth plate, they may be separate and innocuous or may tether the growth plate resulting in limb malalignment (Figure 1.6), bony deformity (Figure 1.7), or growth impedance (Figure 1.8).  These three effects are usually seen in patients with multiple exostoses as  opposed to patients with solitary lesions. The mechanical effect of an exostosis relates to the number of lesions present in the area, how big the lesion is, and when it develops. If a lesion were to develop in isolation (especially seen in solitary osteochondromas), they tend to simply result in an innocuous bump with respect to bony deformity or joint malalignment. Often with these solitary cases, a lesion that develops early in life (before  5  the pubertal growth spurt) matures into a tumour that is not in contact with the growth plate and then migrates away from it as the patient grows. This is illustrated in Figure 1.4b where one sees a large solitary osteochondroma which is remote from the physis and the joint, causing no malalignment and minimal bony deformity save for the bump itself. This is in contrast to an osteochondroma that gets caught up in the growth plate and by its maturation, the stalk causes a bony bar which bridges the local physis, thereby preventing growth in that location (Figure 1.7a). This causes the growth plate and epiphysis to tilt, ultimately resulting in joint malalignment. The secondary longitudinal deformity can occur by this joint line tether or can also result from disruption of the bony architecture of the limb itself. Specifically, when looking at the forearm (Figure 1.7a), if the distal ulnar physis becomes tethered, the ulna will become shortened with respect to the radius (with which it shares an intimate relationship with respect to length), the radius will continue to grow but will become bowed because of its connections with the ulna. Another cause of deformity as well as shortening is the large lesion contained within the medullary space resulting in metaphyseal flaring (Figure 1.8a). These lesions affect the entire physis, causing severe distortion of the metaphyseal region and can cause global shortening of that limb due to the interference of the majority of that particular growth plate. Figures 1.6, 1,7, and 1.8 demonstrate all these effects.  6  Figure 1.6 (a) X-rays showing exostosis tethering the growth plate in an affected ankle (b) A normal ankle is shown for comparison  Figure 1.7 (a) X-ray showing an exostosis causing deformity in the foream; (b) a normal forearm is shown for comparison  Normal Proximal Fibula Height Growth imneHflnPi  1  Figure 1.8 X-ray showing exostosis causing growth impedance.  1.2 Hereditary Multiple Exostoses 1.2.1 Definition  Exostoses occur either as solitary lesions or in multiples. When multiple lesions exist, they can be the result of an inherited trait called Hereditary Multiple Exostoses (HME) which accounts for two-tnirds (66%) of the multiple exostoses cases (Boyer 1814) or represent sporadic cases called Spontaneous Multiple Exostoses (SME for the purposes of this thesis) which account for the remaining one-third (33%) of multiple exostoses cases. The latter case is then inherited as a dominant trait in the offspring with a 50% chance of transmitting the trait. The hereditary form of the disease, HME is the subject of this thesis. 1.2.2 Demographic features  The prevalence of HME is estimated at 1 in 50,000 (Wicklund et al. 1995; Pierz et al. 2001) with a male to female ratio of 1.5 (Schmale et al. 1994; Legeai-Mallet et al. 1997). The male to female distribution varies between 53% male and 46% female (Pierz et 8  al., 2002) to 49.5% male and 50.5% female (Solomon 1963). The differences between male and female distribution may be explained by a 95% penetrance rate in females. Exostoses are typically detected as palpable lumps by the age of five in most patients (65%) and by twelve in all (100%) patients (Legeai-Mallet 1997; Solomon 1963). The proportion of individuals with HME who have clinical findings, increases from 5% at birth to 96% by age twelve (Chansky and Raskind 2002; Schmale et al. 1994; Wuyts et al. 1996). The bony distribution of exostoses found in HME patients is as follows; 50% humerus, 50% forearm, 70% knee, 25% ankle, 50% scapula (Schmale et al. 1994). The exostoses in HME cause similar symptoms to those mentioned above; however, the problem is multiplied by the number of lesions present. Their numbers also increase their potential to alter the growth of bones. Common symptoms include limb deformity (39%), limb malalignment (8%), limb length discrepancy (10%), and short stature (which is intrinsic to this disease) defined as a height two standard deviations below the mean on standard growth charts or a height less than the third percentile. (Wicklund et al. 1995). The clinical impact on these patients is significant.  Limb deformity for the  purposes of this thesis and in keeping with orthopaedic opinion includes distortion of any part of a bone resulting in abnormal longitudinal or cross-sectional anatomy. Examples are abnormal bowing of the forearm or abnormal angulation of the femoral neck. Malalignment relates to the joints and longitudinal alignment of a limb. Examples are knee joint varus or increased radial inclination. Seventy-four percent of patients have removal of at least one lesion, and the average patient has three surgeries over the course of treatment (Schmale et al. 1994). The indications for surgery generally include pain, growth disturbance, angular deformity, decreased joint range of motion, degenerative arthritis, pressure on neural and vascular structures, or unacceptable appearance (Pierz et al. 2002). Furthermore, in a low percentage of HME patients (<1%), one lesion can degenerate into a 9  chondrosarcoma (Wicklund et al. 1995; Legeai-Mallet et al. 1997; Pierz et al. 2001) or other sarcoma (Schmale et al. 1994). The range of transformation rates in HME is reported from 0.5% to 25%. This broad range is influenced by referral bias (Pierz et al. 2001). Signs of sarcomatous degeneration of an exostosis include rapid growth and or pain in a skeletally mature individual (Lange and Rao 1984). CT and MRI imaging reveal a bulky cartilaginous cap of greater than 2 centimetres (Hudson et al. 1984) and a bone scan usually shows increased radionucleotide uptake (Bouvier et al. 1986). It is usually a low grade chodrosarcoma that develops in a pre-existing benign osteochondroma. Treatment involves wide surgical excision to reduce the local recurrence rate (Wusman 1997; Young et al. 1990).  1.2.3 Genetics and molecular biology of HME 1.2.3.1 General Information  HME is inherited as an autosomal dominant trait with a penetrance rate of 95% (Wicklund 1995; Schmale 1994) to 100% (Pierz 2002). Incomplete penetrance has been reported in female patients (Legeai-Mallet 1997). HME is a genetically heterogeneous disease as evidenced by linkage analysis (Hecht et al., 1995; 1997; Bovee et al., 1999; Phillipe et al., 1997; Wuyts et al., 1998). Two different genes, EXT 1 and EXT 2, have been associated with this disease. The exostoses genes represent a family of homologous genes consisting of six genes. EXT 1 is located on chromosome 8 (8q23-24) (Cook 1993) and EXT 2 on chromosome 11 (1 lpl 1-12) (Wuyts et al. 1996; (Wu et al. 1994). Other genes that were originally thought be associated with exostosis occurrence are EXT 3 on chromosome 19 (19pl 1-13) (Le Merrer et al. 1994), EXTL 1 on chromosome 1 (lp36) (Wise et al. 1997), EXTL 2 on chromosome 1 (lpl 112) (Wuyts et al. 1997), and EXTL 3 on chromosome 8 (8pl2-p22) (Van Hul et al 1998). To  10  date, no mutations causing exostoses have been identified in these genes. EXT 3 is has recently been excluded as an EXT gene causing exostoses (Wuyts 2002). The majority of cases (80%) of HME are accounted for by mutations in EXT 1 or EXT 2 (Cook et al. 1993; Blanton et al. 1996; Legeai-Mallet et al. 2000; Wuyts et al. 1996). Many authors, as noted in Table 1.1 have looked at the distribution of mutations between EXT 1 and EXT 2. It is most likely that there is an even distribution among EXT 1 (36%), EXT 2 (27%), and those remaining unidentified (36%) are most likely either EXT 1 or EXT 2 mutations. Table 1.1 Summary of Family Mutations Author  Philippe et al., 1997 Wuyts et al., 1998 X u et al, 1998 Seki et al., 2001 Francannet et al., 2001 Gigante et al., 2001  Ancestry  Number of families studied  EXT1 Mutation  #  (%)  MS or nontruncating mutations  #  EXT 2 Mutations  #  (%)  #MS or nontruncating mutations  (%)  #  (%) 8.3  #of Unidentified Mutations  Mixed  17  12  71  2  16.7  5  29.4  1  Mixed  26  10  38.5  2  7.9  10  38.5  ~  Chinese  36  5  13.9  2  5.6  12  33.3  1  2.8  19  Japanese  43  17  39.5  ~  6  13.9  1  2.3  20  French  42  27  64.3  —  9  21.4  1  2.4  6  Italian  9  4  44.4  ~  3  33.3  ~  0 6  2  Abbreviations used: MS - missense mutation  EXT 1 and EXT 2 genes have been isolated (Stickens et al. 1996; Wuyts et al. 1996). Both genes lack sequence similarity to any known gene and represent a new family of genes (Ahn et al. 1998, Stickens et al. 1996). These genes are ubiquitously expressed, with the highest expression in the liver (Stickens et al. 1996), however mutations in the EXT genes only affect growing bone (Hecht et al. 1997). EXT 1 and 2 encode homologous proteins of 746 (Ahn et al. 1998) and 718 (Stickens et al. 1996; Wuyts et al. 1996) amino 11  acids respectively. Thirty-one percent identity exists at the amino acid level with significant sequence similarity throughout the entire protein as can be seen in Figure 1.9 (Stickens et al. 1996). This is particularly noted in the 260 carboxy terminus tail. EXT 1 and 2 are large genes. EXT 1 has a genomic size of over 250 kilobases, with a cDNA of 3304 base pairs comprising eleven exons. The EXT 2 gene is also over 250 kilobases and has a cDNA of 3781 base pairs encoding sixteen exons. Characterization of the EXT 1 and 2 genes including the intron and exon boundaries and the translation of each gene can be found in Appendices 8.3 and 8.4.  12  EXT 1: FWPRFPEPLRPFVPWDQLENEDSSVHISPRQKRDANSSIYK—GKKCRMESCFDFTLC— 109 FWP E EXT 2: FWPHSIESSND  W+ WNV  E S+ P + A+S I + CRM +CFD C EKRSIRDVPWRLPADSPIPERGDLSCRMHTCFDVYRCGF 98  EXT  1:-KKNGFKVYVYPQQK GEKIAESYQNILAAIEGSRFYTSDPSQACLFVLSLD KN KVY+Y +K 1+ Y +L A l S +YT D ++ACLFV S+D EXT 2:NPKNKIKVYIYALKKYVDDFGVSVSNTISREYNELLMAISDSDYYTDDINRACLFVPSID  159  EXT  219  1: TLDRDQLSPQYVHNLRSKVQSLHLWNNGRNHLIFNLYSGTWPDYTEDVGFDIGQAMLAKA L+++ L + + L W+ G NHL+FN+ G PDY + +A+LA EXT 2: VLNQNTLR IKETAQAMAQLSRWDRGTNHLLFNMLPGGPPDYNTALDVPRDRALLAGG  158  215  EXT  1: SISTENFRPNFDVSIPLFSK DHPRTGGERGFLKFNTIPPLRKYMLVFKGKRYLTG 274 ST +R +DVSIP++S DP G P R+Y L+ G EXT 2: GFSTWTYRQGYDVSIPVYSPLSAEVDLPEKG PGPRQYFLLSSQ VG 260 EXT  1: IGSDTRNAL—YHVHNGEDWLLTTCKHGKDWQKHKDSRCDRDNTEYEKYDYREMLHNAT 332 + + R L V +GE V++L C + + RC + ++ +DY ++L AT EXT 2: LHPEYREDLEALQVKHGESVLVLDKCTNLSEGVLSVRKRCHK HQVFDYPQVLQEAT 316 EXT  1: FCLVPRGRRLGSFRFLEALQAACVPVMLSNGWELPFSEVINWNQAAVIGDERLLLQIPST 392 FC+V RG RLG + LQA CVPV++++ + LPFSEV++W +A+V+ E + + S EXT 2: FCWLRGARLGQAVLSDVLQAGCVPWIADSYILPFSEVLDWKRASWVPEEKMSDVYSI 37 6 EXT  1: IRSIHQDKILALRQQTQFLWEAYFSSVEKIV1TTLEIIQDRIFKHISRNSLIWNKHPGGL 452 ++SI Q +1 +++Q ++ WEAYF S++ I L TL+II DRI+ + + + WN P EXT 2: LQSIPQRQIEEMQRQARWFWEAYFQSIKAIALATLQIINDRIYPYAAISYEEWNDPPA— 434 EXT  1: FVLPQYSSYLGDFPYYYANLGLKPPSK—FTAVIHAVTPLVSQSQPVLKLLVAAAKSQYC 510 ++ S P + L L PP FTA++ + S + +++ +K EXT 2: VKWGSVSN—PLF LPLIPPQSQGFTAIVLTYDRVES LFRVITEVSKVPSL 482 EXT 1: AQIIVLWNC-DKPLPAKHRWPATAVPVWIEGESKVMSSRFLPYDNIITDAVLSLDEDTV 569 ++++V+WN +K P WP VP+ V+ +S+RF PYD I T+AVL++D+D + EXT 2: SKLLWWNNQNKNPPEDSLWPKIRVPLKWRTAENKLSNRFFPYDEIETEAVLAIDDDII 542 EXT  1: LSTT-EVDFAFTVWQSFPERIVGYPARSHFWDNSKERWGYTSKWTNDYSMVLTGAAIXXX + T+ E+ F + VW+ FP+R+VGYP R H WD+ +W Y S+WTN+ SMVLTGAA EXT 2: MLTSDELQFGYEVWREFPDRLVGYPGRLHLWDHEMNKWKYESEWTNEVSMVLTGAAFYHK  628  EXT  1: XXXXXXXXXXPASLKNMVDQLANCEDILMNFLVSAVTKLPPIKVTQKKQYKETMMGQTSR P +KN VD NCEDI MNFLV+ VT IKVT +K++K EXT 2: YFNYLYTYKMPGDIKNWVDAHMNCEDIAMNFLVANVTGKAVIKVTPRKKFKCPECTAIDG  688  EXT  74 6  1: ASRWADPDHFAQRQSCMNTFASWFGYMPLIHSQMRLDPVLFKDQVSILRKKYRDIERL S D H +R C+N FAS FG MPL + R DPVL+KD K + +1 L EXT 2: LS—LDQTHMVERSECINKFASVFGTMPLKWEHRADPVLYKDDFPEKLKSFPN1GSL  602  662  718  Figure 1.9 Alignment of EXT 1 and EXT 2 genes. Identical amino acids are outlined in boxes. EXT 1 sequencefromNCBI database, Accession number NM 000127 and EXT 2 sequence from NCBI database, Accession number NM 000401. Overlapping sequences detected using BLAST searching of NCBI.  13  1.2.3.2 E X T P h y s i o l o g i c F u n c t i o n  The function of the EXT genes remains unclear. Two theories have been brought forward for the EXT genes functioning as either a tumour suppressor gene (Hecht et al. 1995, Raskind et al. 1995, Hecht et al. 1997) or that the EXT genes act in the regulation of bone growth at the physis (Alman et al. 2002, Bornemann et al. 2002, Wuyts et al. 1998). Evidence suggesting a tumour suppressor function played a large role in the early days of EXT gene investigations. Historically, prior to 2000, this was the main theory regarding the function of the EXT genes. This work was based on identification of the genes involved from contiguous gene syndromes and then further support by LOH studies followed by identification of two mutations in a few osteochondromas and then more consistently in chondrosarcomas. From a pathologists view point similarities were drawn between HME and other familial benign multiple tumour conditions. Since 2000 the molecular function of the gene has been further described and it puts the tumour suppressor theory into question. In general as of 2003, it is the cell-to-cell signalling and growth plate regulation roles that are receiving more attention and evidence continues to mount against the tumour suppressor role and grow towards a signalling function via heparan sulfate. The following is a synopsis of the history to the tumour suppressor role. Exostoses were noted to develop in patients with chromosome abnormalities involving chromosome 8 such as Langer-Giedion syndrome (facial dysmorphism, mental retardation, abnormal cone-shaped phalangeal epiphyses, multiple exostoses) where 8q24.11-q24.13 is deleted (Parrish et al. 1991; Ludecke et al. 1997). In Tricho-rhino-phalangeal (TRP) syndrome (thin nails, sparse hair, short metacarpals and tarsals, unusal facies, coned shaped epiphyses of the digits) the deletion was found in the area of 8q24.12 (Buhler and Malik 1984; Fryns and Van Den Berghe 1986). TRP II has a q24.1 deletion and has mental retardation and exostosis development whereas TRP I is a deletion of 8q22.3 to 23.2 and does not develop 14  exostoses. Exostoses also develop in chromosomal abnormalities in chromosome 11 as in Defect 11 syndrome (multiple exostoses, enlarged parietal foramina, craniofacial dysostosis, mental retardation) where there are rearrangements at 1 lpl 1-12 (Bridge et al. 1998; Ligon et al. 1998; Bartsch, Wuyts and Van Hul 1996). These contiguous gene syndromes helped localize where the presumptive EXT genes were located. EXT 1 and EXT 2 genes were then isolated and cloned (Stickens et al. 1996; Wuyts et al. 1996). Germline EXT mutations were then identified as being involved with the development of multiple benign bone tumours seen in HME and SME (Legeai-Mallet 1997, Hecht et al. 1995, Hecht et al. 1997, Raskind et al. 1995, Wuyts et al. 1997, Wuyts et al. 2000, Phillipe et al. 1997). It was this relationship between gene mutation and tumour formation that suggested the putative role of the EXT genes was tumour suppression and therefore the EXT genes were considered as tumour suppressor genes. Osteochondromas were then shown to be true neoplasms by Bovee (Bovee et al. 1999), the presence of loss of heterozygosity in 6 of 14 osteochondromas and aneuploidy in 4 of 10 osteochodnromas. She concluded that this indicated a clonal origin for the cartilaginous tissue of the tumours studied. Other studies were also done looking at the genetic composition of osteochondromas. In some solitary osteochondromas both copies of the EXT gene had been mutated by somatic mutations (Porter and Stickens 1999; Mertens et al. 1994; Bovee et al. 1999, Hecht et al. 195 and 1997; Raskind et al. 1995). In addition, two mutations have been found to exist in the chondrocytes of osteochondromas in HME: one in the germline and the other in the remaining wild type or somatic allele involving EXT 1 or 2 (Bovee et al. 1999; Mertens et al. 1994). Specifically, Bovee found in two patients with HME with mutations in EXT 1, 3 of 4 osteochondromas carried two mutations, the first being the germline mutation and the second a loss of the remaining wild-type allele. The remaining osteochondroma failed to show loss of heterozygosity and 15  it was hypothesized by the authors it may have been a small somatic mutation that was undetected. The conclusion that Bovee proposed is that inactivation of both copies of the EXT gene is required for osteochondroma formation. However, in these mentioned papers investigating the tumours for two mutations only up to 30% (4 to 30%) of the second mutations were found in all the tumours studied (including solitary osteochondromas and those found in HME and chondrosarcomas related and unrelated to pre-existing osteochondromas). At least one mutation was always found in either EXT 1 or 2 but the second mutation was unidentified in 70 to 96% of cases, possibly due to methods used to identify mutations in EXT 1 and 2 (single strand conformaiton polymorphism (SSCP), mutation analysis, sequencing only the coding region) or possibly a different tumour suppressor system is involved, for example p53. However, it is more likely that the cells within the tumour mass are simply at a higher risk of suffering a second mutation. It is these second injuries which may be more responsible for cells that go on to become malignant cells, for example a chondrosarcoma. This then supports the two-hit hypothesis of tumourogenesis proposed by Knudson (1971), in that it takes more than just one mutated allele to result in malignant degeneration. Inactivation of the remaining allele in HME has been seen more consistently in chondrosrcomas (Bovee et al. 1999; Mertens et al. 1994). The loss of function of the EXT genes has been shown in malignant neoplasms originating from osteochondromas, regardless if they are from a spontaneous solitary osteochondroma or found in a lesion in a subject with HME or SME. This also supported the theory that these genes serve as tumour suppressors. Loss of heterozygosity studies revealed loss of genetic markers which flank EXT 1, EXT 2, and EXT 3 loci (we now know EXT 3 has been excluded as an EXT gene) (Porter and Stickens 1999; Hecht et al. 1995; Raskind et al. 1995; Hogue et al. 1996; Hecht et al. 2002). Hogue (1996) traced mutations in an HME patient from constitutional DNA 16  through to osteochondroma and into chondrosarcoma. These results support Vogelstein's theory of stepwise carcinogenesis as it relates to phenotype: specifically, degeneration of a neoplasm (accumulation of mutations) undergoing malignant transformation (1992). In addition, work completed on de novo chondrosarcomas, have also shown mutations in EXT 1 and2(Hogue 1996). Osteochondromas were also discussed by pathologists as having certain neoplastic pathologic behaviours reminiscent of other tumours, for example adenomas in the large bowel, which also supports the premise that EXT genes have a tumour suppressor function. Adenomas like osteochondromas are benign tumours originating in the colon versus ostechondromas which are benign tumours that originate in the proximity of the physis. They can both be solitary and benign. They can also exist in a familial multiple form: familial adenomatous polyposis (ape gene mutation) and hereditary multiple exostoses (EXT gene mutation) (Porter et al. 1999). Specific features common to neoplasms are: random location at sites of predisposition (lesions develop in HME in an asymmetric, random distribution at common juxtaphyseal sites) (Schmale et al. 1994). They demonstrate behavioural or cellular disorder, in that these lesions develop in abnormal positions for this cell type, excessive cartilage volume, and though the architecture is similar to the growth plate the zonal definition is not as succinct. Finally, lesions in HME have the potential to transform into malignancies, representing not only loss of control of cellular growth but also the ability to metastasize (Porter et al., 1999). The underlying mechanism, or final common pathway for the tumour suppressor theory is likely due to a lack of heparan sulphate presentation on the chondrocyte cell surface. EXT genes are believed to be involved in heparan sulphate polymerization and this will be discussed in greater detail in the following paragraphs. Heparan sulphate is also part of the extracellular matrix and is known to be involved with cell mobility 17  adhesiveness, differentiation, and cell-to-cell signalling. Loss of these features in part describes neoplasia or tumour generation. Cell-to-cell signalling is an extracellular matrix activity and mutations involving genes acting in this system resulting in tumours, does not mean the genes are tumour suppressor genes. Tumour suppressor genes normally function as negative regulators of cell proliferation (Griffiths et al. 1996). For example p53, a known tumour suppressor gene, serves as a monitor of DNA damage. Mutations in this gene allow cell division to occur in the absence of DNA repair. There is then an accumulation of mutations, chromosomal rearrangements and aneuploidy, which increases the chances of that further uncontrolled cell proliferation occurs. EXT genes have been shown to be glycosyltransferases (see next section) involved in heparan sulfate polymerization which is not tumour suppressor activity. The alternate and now more popular proposed physiologic function of the EXT genes is growth plate regulation. EXT gene products form a hetero-oligomeric complex involved in the regulation of cell surface heparan sulfate proteoglycan presentation (described further in the molecular function section following). Heparan sulfate is a dominant component of cartilage, which is the matrix of the growth plate. Heparan sulfate is integrally involved in the diffusion of several families of cell signalling molecules including those in the hedgehog, TGF-beta (tumour growth factor), and FGF (fibroblast growth factor) families. Specifically EXT genes are involved in the diffusion of Indian hedge hog by way of their glycosyltransferase activity. Indian hedgehog in humans, invokes osteoblast differentiation in the lower growth plate (by being in low concentration in the distal zones of the growth plate), incites chondrocyte proliferation, inhibits chondrocyte differentiation (in the proximal aspect of the growth plate where Indian hedgehog is in its highest concentration) and stimulates Parathyroid hormone related protein (PTHrP) in the perichondrium to produce chondrocytes in the zone of proliferation 18  of the physis and prevent movement of chondrocytes down the differentiation pathway. Mutations in the EXT genes effect the normal diffusion of Indian hedgehog (from distal to proximal) likely due to the alteration in the extracellular matrix caused by an absence of heparan sulfate. The EXT mutations may then cause a disruption in the negative-feedback loop by inhibiting Indian hedgehog diffusion, which would normally prevent chondrocyte differentiation resulting in abnormal ectopic development of chondrocytes. It remains an abnormality of heparan sulfate polymerisation, which in turn appears to regulate growth and differentiation of the chondrocytes. The end result of EXT mutation is that Indian hedgehog does not diffuse and establish an appropriate concentration gradient in the growth plate. Proximally the concentrations are high, resulting in excessive chondrocyte proliferation without differentiation, which then becomes the nidus for tumour or osteochondroma genesis. If this were truly the case however, one would expect the entire growth plate to be abnormal, with osteochondromas developing throughout the physis, peripherally and intramedullary, resulting predictably in juxtaphyseal/metaphyseal flaring, and multiple osteochondromas at each and every growth plate. It would be unlikely to see well-defined isolated lesions affecting only a few of the growth plates (which is a common pattern of presentation in HME/SME). On the other hand as Hecht has shown by her cross sectional studies of growth plates there are multiple niduses of presumptive osteochondroma nests in the perichondrium all along the physeal and metaphyseal zone (Hecht 2002). In her opinion there are secondary factors in the local and humoral environment affect the survival of specific nests that go on to form the clinical tumours. The different physiologic mechanisms of action of the EXT genes should express themselves as different phenotypes at the clinical level as aluded to above. It is therefore a  19  useful project to determine if the phenotype varies and then how it relates to the potential physiologic role of the EXT genes.  1.2.3.3 E X T gene p r o d u c t s a n d f u n c t i o n  The proteins encoded by the EXT 1 and 2 genes are type II transmembrane glycoproteins situated in the endoplasmic reticulum (ER) (McCormick et al. 1998). The initial work done by McCormick indicated that the function of the protein expressed by EXT 1 was involved in the synthesis and presentation of heparan sulfate (HS) glycosaminoglycan (GAG) on the cell surface (McCormick et al. 1998). Biosynthesis of heparan sulfate chains involves the formation of an initial simple polysaccharide chain composed of alternating D-glucuronic acid (GlcA) and N-acetyl-D-glucuronic acid (GlcNac) units that are joined by 1-4 links. The polymer is then modified through a series of reactions involving partial N-deacetylation and N-sulfation of the GlcNac units, C-5 epimerization of GlcA to L-iduronic acid and O-sulfation at various positions (Salmivirta et al. 1996). EXT1 and 2 both possess the GlcNAc and GlcA transferase activities representative of heparan sulfate polymerase (Lind et al. 1998; Seany et al. 2000; Wei et al. 2000). GAGs, in particular heparan sulfate, are known to function as co-factors in several signal transduction systems (as aluded to above) that affect cellular growth, differentiation, adhesion, and motility (Bernstein and Liotta 1994). GAGs may also play a role in the malignant transformation of cells, tumour adhesiveness, invasiveness, and metastasis. Given the activity of GAGs and that the EXT genes are involved with HS expression lends support that EXT genes may have either a tumor-suppression activity or growth plate regulation function.  20  When McCormick (McCormick et al. 1998) examined the effect of different mutations on the gene product, he found the more severe mutations such as fameshifts, nonsense, and splice sites caused truncated proteins not localized to the ER and there was no heparan sulfate presentation on the cell surface. However, in a single amino acid change, as seen in missense mutations, the protein remained located in the ER with reduced stability and yet HS cell surface display was again absent. McCormick concluded that mutation type does not differentially affect the molecular function of the EXT genes. More recent work has shown that EXT 1 and 2 gene products though endoplasmic reticulum based proteins go on to form a hetero-oligomeric complex that leads to an accumulation of both proteins in the Golgi apparatus which in turn has the catalytic activity of heparan sulfate polymerization (Koboyashi et al. 2000; McCormick et al. 2000). McCormick demonstrates that EXT 2 does not exhibit significant glycosyltransferase activity in the absence of EXT 1 (McCormick et al. 2000). When the EXT1/2 complex exists in the Golgi apparatus, a much higher glycosyltransferase activity results compared to when EXT 1 or 2 present alone. Therefore, it is the complex of the two genes that forms the biologically relevant enzyme.  This situation would explain why patients with  mutations in either EXT 1 or 2 present with the formation of osteochondromas. This would also support the hypothesis that it is irrelevant which of the two genes is effected and that the phenotype would not be influenced by genotype. Gullberg looked further into the activities of EXT 1 and 2 (Gulberg 2002). They are both catalytic enzymes as mentioned above and in both of their absence the heparan sulfate chain fails to elongate. In catalytic assays when EXT 1 alone is preserved it shows higher catalytic activity than when EXT 2 is alone. This then led to the concept that EXT 2 is a 'chaperone' or 'stabilizer' of EXT 1. Given that the two have varying impact on the  21  catalytic activities of heparan sulfate one may deduce that it does matter with respect to phenotype whether it is EXT 1 or EXT 2 that is mutated. In summary, the EXT genes may have one of two physiologic functions; tumour suppressors via heparan sulfate extracellular matrix function (not tumour suppressor genes), or growth plate regulation via Indian hedgehog signalling, both contingent upon the existence of heparan sulfate presentation/presence in the physeal zone. The function of the EXT genes is to catalyze heparan sulfate polymerization. There is recent evidence that the two genes contribute differing amounts of activity whereby EXT 1 catalytic function is greater than that of EXT 2. There is also evidence showing mutation type, truncating versus nontruncating, causes different results with regards to EXT protein location but not in terms of ultimate heparan sulfate presentation. The basic science of the EXT genes suggests there may potentially be a difference in phenotype as a result of which gene is affected and by what type of mutation.  1.2.4  Mutations  Several groups have been working to identify the mutations in HME (Seki et al. 2001; Xu et al. 1998; Park et al. 2001; Raskind et al. 1998; Wells et al. 1997; Hecht et al. 1995; Wuyts et al. 1998; Philippe et al. 1997; Ahn et al. 1995).  Table 1.2 and 1.3 list the  known mutations in a variety of ethnic backgrounds. Figures 1.10 and 1.11 show the location of the mutations in relation to their distribution in EXT 1 and 2; more mutations have been located in EXT 1 than in EXT 2 (85 EXT 1 versus 44 EXT 2). The most common type of mutation identified in both EXT 1 and EXT 2 is a frameshift mutation, which truncates the protein and significantly changes the portion of the protein coded for. In addition, the majority of mutations occur early in the gene. Both 22  genes are approximately 3300 base pairs long; in EXT 1, sixty-eight of eighty-five occur prior to base pair 1500, while in EXT 2, forty-two of forty-four occur prior to base pair 1500. Most of the above-mentioned studies have an average of 20% percent unidentified mutations. In general, the 5' and 3' UTRs and the promoter regions were not screened and very large mutations involving one or more exons may be missed. Furthermore, EXT 3 was not studied and the missing mutations could be present in these regions. However, no mutations in EXT 3 have been found in cases of any form of exostoses and EXT 3 is now considered not to be involved with exostosis formation (Wuyts 2002). Also, not all intronic regions were investigated and these may be sites of unidentified mutations as well.  23  Table 1.2 Summary of Mutations Identified in the EXT 1 Gene cDNA  Exon  change"  Protein  Type  Reference  Change  42delG 79C^A  1  G15  FS  2  1  Q27K  MS  D . Zaletayev, unpublished  3  118delC  1  FSH40  FS  Raskind et al., 1998  1  Francannet et al., 2001  4  174-176delC  1  F S P59  FS  Philippe et al., 1997  5  204G-»A  1  W68X  NS  Wuyts e t a l . , 1998  6  242-247insC  1  FSR83  FS  Wells e t a l . , 1997  7  248insC  1  R83  FS  Francannet et al., 2001  8 9  248-249delG 250C^T  1  F S Q84  FS  Wells e t a l . , 1997  1  Q84X  NS  Francannet et al., 2001  10  331A-»T  1  K110X  NS  X u et al., 1999  11  352insC  1  V118  FS  Francannet et al., 2001  12  357C-»A  1  Y199X  NS  Raskind, et al., 1998  13  357C->G  1  NS  A l v a r e z et a l . , 2003  14  388delAG  1  FS  D . Zaletayev, unpublished  15  420ins4  1  F A S130 FSS141  FS  Hecht e t a l . , 1997  16  456delC  1  FSL153  FS  D . Zaletayev, unpublished  17  458delTC  1  FS  Francannet et al., 2001  18  460del2T  1  L153 F154  FS  Francannet et a l . , 2001  19  477delTA  1  D160  FS  Francannet et a l . , 2001  20  490G->C  1  D146H  MS  Bovee etal., 1999  21  515delA  1  H I 72  FS  Francannet et a l . , 2001  22  527del8  1  FS K177  FS  Hecht e t a l . , 1997  23  549delGT  1  S180  FS  Francannet et al., 2001  24  590-59 l d e l C  1  F S S197  FS  X u e t a l . , 1999  25  599G->A  1  W200X  NS  Wuyts e t a l . , 1998  26  600G->A  1  W200X  NS  Wuyts e t a l . , 1998  27  624ins5  1  F S F209  FS  Wuyts e t a l . , 1998  28  651-664dell4  1  FSL216  FS  Seki e t a l . , 2001  29  679delC  1  R227  FS  Francannet et a l . , 2001  30  679C-»T  1  R227X  NS  31 32  703dell5  1  PLFSKdel  5 AAdel  712delT  1  S238  FS  33  713delC  1  F S S238  FS  Hecht et al., 1997 (2 families)  34  742insTT  1  F S R248  FS  D . Zaletayev, unpublished  35  820-82 l d e l G G  1  FS G274  FS  Seki e t a l . , 2001  36  838A-»G  1  R280G  MS  Wuyts et al., 1998, Raskind et al., 1998  37  840G->C  1  R280S  MS  Raskind et al., 1998  38  876-877insT  1  FS V292  FS  D. Zaletayev, unpublished  39  943delGA  1  F S D315  MS  D . Zaletayev, unpublished  40  947A->G  1  N316S  MS  Bovee etal., 1999  41  1016G-»A  2  G339D  MS  Philippe et al., 1997  42  1018C->T  2  R340C  MS  Philippe et al., 1997  43  1018C^A  2  R340S  MS  Wuyts e t a l . , 1998  44  1019G^T  2  R340L  MS  Hecht et al., 1997; Seki et al., 2001  Seki et al., 2001 Bovee e t a l . , 1999 Francannet et a l . , 2001  " A l l mutations were uniformly numbered with the adenosine o f the start codon nucleotide position +1. Abbreviations used to indicate mutation types: M S - missense, N S - nonsense, F S - frameshift, S S - splice site Blue font indicate missense or non-truncating mutations.  24  Table 1.2 (continued) Summary of Mutations Identified in the E X T 1 Gene c D N A change*  Exon  Protein Change  Type  45  1019G-»A  2  R340H  MS  46  10351056+2del24  2  F S F345  SS  47 48 49 50 51 52 53 53 54 55  1056+G-»A 1091-1093delG 1122G-»A 1157T-KJ 1198-1199insA 1203-1204deIC 1213-1216del4 1215del4 1215-1218del4 1370delT  56 57 58 59 60 61 62 63 64 65 66  1320insT 1333-1334insG 1376C->G 1409dell0 1417+lG^A 1417+2del6 1426-1431insC 1431insT 1457C->T 1468-1469insC 1469delT  67 68 69 70 71 72 73  1474-1475delTC 1487C->T 1568delT 1642delA 1642delA 1679-1680insC 1723G->C 1745G-»A 1744G-»A 1773delG I776C-»A 1784delGC 1797G->A 1817G->A 1878del3  6 6 7 8 8 8 8 9 9 9 9 9 9 9 9  1883+2T-»G 1980delG 2053C->T 2101C-»T  9 10 10 11  74 75 76 77 78 79 80 81 82 83 84 85  Intron 2 3 3 3 4 4 4 4 4 4 5 5 5 5 Intron 5 Intron 5 6 6 6 6 6  F S E365 W374X L386X F S D339 FS L402 423STOP F S R405 F S R405 T424 441 S T O P FSN446 S459X  F S S478 F S S478 A486V FSL490 FS L490  FS L492 P496L L523 621 S T O P S548 F S V561 W582X W582X G591 Y592X R595 W559X W606X H627del  664STOP Q685X R701X  SS FS NS NS FS FS FS FS FS FS FS FS NS SS SS SS FS FS MS FS FS  FS MS FS FS FS FS SS NS NS FS NS FS NS NS 1AA deletion SS FS NS NS  Reference Raskind et al., 1998 (2 families); Sekit et al., 2001; A l v a r e z et a l . , 2003 Seki et al., 2001 Wells et al., 1997 Raskind et al., 1998 Philippe et al., 1997 Seki et al., 2001 Seki et al., 2001 Raskind et al., 1998 Gigante et al., 2001 Raskind et al., 1998 (2 families)? S e k i et al., 2001 Francannet et al., 2001 Gigante et al., 2001 Seki etal., 2001 Wuyts etal., 1998 Park etal., 1999 Philippe et al., 1997 Wuyts etal., 1998 Hecht et al., 1997, Raskind et al., 1998 Wells etal., 1997 X u e t a l . , 1999 Seki et al., 2001 Wuyts et al., 1998, A h n et al., 1995 (2 families) Wells et al., 1997, Philippe et al., 1997, X u e t a l . , 1999 Seki etal.. 2001 X u etal.. 1999 Francannet et al., 2001 Gigante et al., 2001 Francannet et al., 2001 Wuyts etal., 1998 Alvarez et al., 2003 Francannet et al., 2001 Francannet et al., 2001 Francannet et al., 2001 Francannet et al., 2001 Francannet et al., 2001 Seki et al, 2001 Wells etal., 1997 Raskind et al., 1998 Seki etal., 2001 Gigante et al., 2001 Raskind et al., 1998 Seki e t a l . , 2001  " A l l mutations were uniformly numbered with the adenosine o f the start codon nucleotide position +1. Abbreviations used to indicate mutation types: M S - missense, NS - nonsense, F S - frameshift, SS - splice site Blue font indicate missense or non-truncating mutations.  2 5  Table 1.3 Summary of Mutations Identified in the E X T 2 Gene c D N A change*  1 2 3 4 5 6 7 8 9 10 11 12  67C->T 77-78insT 233delC 239-244insG 253T->C 302del56 313A-»T 315-316insG 319insGT 374-443del70 449del4 455T-»G  Exon  Protein Change  Type  2 2 2 2 2 2 2 2 2 2 2 2  Q23X FSY26 FS P78 FS G81 C85R FS K101 K105X FS VI06 FSC107 FS 1126 FS A150 L152R  NS FS FS FS MS FS NS FS FS FS FS MS  Wuyts et al., 1998 Philippe et al., 1997 Sekietal., 2001 Raskind et al., unpublished Parketal., 1999 Raskind et al., unpublished Xuetal., 1999 Xu et al., 1999 Xuetal., 1999 Seki et al., 2001 Stickens etal, 1996 Xuetal., 1999  Reference  13  455del4  2  FS  Alvarez et al., 2003  14 15  495delG 514C-»T  2 2  FSL165 Q172X  FS NS  16 17 18 19 20 21 22 23 24 25  537G->C 537-lG->A 580G^T 605C^T 624deIC 627-2A^G 629-63 linsC 649-652delT 666C-»G 679G->A  2 Intron 2 3 3 3 Intron 3 4 4 4 4  R180T  MS SS NS MS FS 3' Splice Junction FS FS NS MS  Xuetal., 1999 Wuyts et al., 1998; Wuyts etal., 1996; Xuetal., 1999 Francannet et al., 2001 Seki et al., 2001 Francannet et al., 2001 Sekietal., 2001 Francannet et al., 2001 Gigante et al., 2001 Xuetal., 1999 Wuyts et al., 1998 Philippe et al., 1997 Philippet et al., 1997 (2 families);  26 27  730G->T 751C->T  28 29 30 31 32 33 34 35  772C->T 812-814delC 1079+G->T 1079+G^C 1104insGA 1132C-»T 1139T-»C 1173+G-»A  G193X A202V D208 FSL211 FSS218 Y222X D227N  Alvarez et al., 2003  NS NS NS FS SS SS FS NS  4 5  5 5 Intron 6 Intron 6 7 7 7 Intron 7  Q258X FS A271 FS Q313  FS R360  36  1173+G->T  Intron 7  37  1174G->A  Intron 7  38 39 40 41 42 43 44  1188G->A 1201C->T 1234C-»T 1257T-»A 1263 ins AT 1669delC 1726G-»A  8 8 8 8 8 11 11  E368 Q378X I380T FS R360  SS SS SS NS NS NS NS FS FS  W396X Q401X Q412X Y419X FS A422 FS R557 E576K  Alvarez et al., 2003 Alvarez et aL, 2003  Francannet et al., 2001 Wuyts etal., 1998 Wolf et al., 1998 Sekietal, 2001 Francannet et al., 2001 Raskind et al., unpublished Gigante et al., 2001 Wuyts et al., 1998 (2 families); Wuyts etal., 1996 Wuyts etal., 1998 Alvarez et al., 2003  Xuetal., 1999 Philippe et al., 1997; Xu et al., 1999  Xu et al., (3 families) Francannet et al., 2001 Wuyts etal., 1998 Seki et al., 2001 Gigante et al., 2001  "All mutations were uniformly numbered with the adenosine of the start codon nucleotide position +1. Abbreviations used to indicate mutation types: M S - missense, NS - nonsense, FS - frameshift, SS - splice site; Blue font indicate missense or non-truncating mutations.  26  K-OKHZ  EX-±Z+e88t  o o  w  H 3 OSU!69tl--99t'l  R I — **  a  J 5 CO  a  3<-09Zei  .2 2 OJ  a u CO 09  11  V<-9ZZU  Mr 0 6 101  V0I°PCT6  1 9«-V8E9  1SUI/28-9Z9 0<-DC*9  99ISPL28-0Z9  0I3PEUsuapeoz—I  maPS99-l99 V<-O009  V<-0669OI6P16S-06S-  ±9PP 6*9VI»P 919—1  1Z l»P 09*  01I8P 99*  *su|0Z*  VW88E  W9Q*  V1I3P i i *  0«-306»  w<-ozse  3SU3SU0N  i«-viee  1  9|Bp6*Z-9*i  0«U! 9 t z  Osui.£t-z-z*z  0|8P9Zl-*Zt 0I8P8LI  OiepZ* —•  V<-EI*0Z  27  W-0 09Z  .  aiisaonds  o  a  _LVSU!£92i  CO  V^SPIU  H  fc  ' i  w  •=  i  •a 2 c  o o *•» c  W  O  2«  B> co CU (/) C CO CO  CD  asuasuoN  asuasuoN  CO O  -S c  it-sou  0^-0999  ±|3PZ99"6t'9  " e « ,o  3 <u OJD J 3  S  oisps6t> SJ  O^ISS*  ±<-099*  I— OZiaPEW^ZE  0<-lE9Z  osuitt-z-eez 3ieP££3 " I  ~l  28  13SU!6t£  3  1.2.4.1 E X T 1 Mutations Summary  Eighty-five different mutations in EXT 1 have been identified to date including the results of this study. Table 1.2 summarizes all known mutations. Some of the mutations have been found in more than one unrelated family (Table 2.1: 25, 33, 34, 53) but most are unique to each family. Of the eighty-five mutations thirteen (15%) are missense, seventeen (20%) are nonsense, forty-eight (56%) are frameshift and seven (8%) are splice site mutations. Forty of eighty-five (47%) are located in exon 1. One mutation has been identified in exon 7, and three mutations have been found in introns 2 and 5.  1.2.4.2  E X T 2 Mutations Summary  In comparison, only forty-four mutations have been identified in EXT 2. Table 1.3 summarizes all the previously published mutations plus those discovered in this study. As in EXT1, some overlap is seen in terms of unrelated families carrying the same mutation (from Table 1.2: 16, 22, 29, and 33). Of these forty-four mutations four are missense (9%), twelve (27%) are nonsense, eighteen (41%) are frameshift, and seven (16%) are splice site. Exon 1 of EXT 2 encodes the 5'UTR, and mutation analysis has not been done in this region by any of the authors. Currently, there are no identified mutations in exons 6, 9, 10, 12, 13, or 14. Exon 2 mutations (17 of 44) account for most of EXT 2 mutations.  1.2.5 Phenotyping 1.2.5.1 Schmale's Findings  Several studies and case reviews involving the phenotype of patients with HME are available. In 1994, Schmale (1994) assessed 113 individuals from forty-six families, and mapped their clinical expression. Features examined in this study included anatomical 29  locations, age at onset, orthopaedic operations, family pedigrees, number and location of palpable bumps, tenderness, range of motion, deformity, and limb lengths. The subject's overall functional status was evaluated using a modified version of the Musculoskeletal Tumor Society classification system (Enneking 1987, Table 2.3). Schmale's study was a review of all patients in the state of Washington known to have HME. The prevalence in this state was estimated at 1 in 50,000; however, Schmale does admit to a variety of potential biases and therefore expects the overall frequency may be lower. The summary of their results show 49% of females at risk of having the disease were affected and 57% of males (p>.l), mean onset (no difference was found between genders) was 4 +/- 1 years, all cases were identified by 12 years, 4 percent of persons who carried the gene mutation did not express the disease (Schmale's coauthor Raskind had studied 34 of these families and identified the mutations and it is from this data the 96% penetrance rate was established for this population (Raskind et al. 1998)), 1% had chondrosarcoma. Figure 1.12 shows the anatomical distribution of lesions over the skeleton. With respect to the functional rating scale, 42% of males and 67% of females were rated as mild with good or excellent function; the remaining 58% of males and 33% of females were rated as severe with fair or poor function. Seventy-four percent of subjects had surgery, and on average each patient had 3 procedures.  30  Figure 1.12 Anatomical Distribution of Lesions (Schmale 1994)  31  Table 1.4 Modified Functional Assessment Scale of the Musculoskeletal Tumour Society (as per Schmale 1994) Rating  Motion  Strength  Pain  Activity  Deformity  (% Total Motion  Bowing of forearm  of Normal Joint)  Shortening of Forearm  (cm)  Excel.  >90  5/5  Good  60-90  4/5  Fair  30 - <60  3/5  Poor  <30  1-2/5  N o n e (no medication) M i d (medication occasionally) Mod. (medication weekly) Severe (narcotics or other medication daily)  Varus Valgus Angulat of Knee  Shortening of limb  (cm)  n N o restrict  None  None  0-5  None  Restric. i n recreational activities Partial disability  Mild  <1  6-10  <1  Mod.  1-2  11-20  1-3  Total disability  Severe  >2  >20  >3  1.2.5.2 P o r t e r ' s F i n d i n g s  Porter's objective was "to assess the evidence that the presence of local osteochondromas might be the major criterion affecting local bone growth" (2000). The essence behind this work was to re-define Hereditary Multiple Exostoses as a result of local bone growth interference caused by an osteochondroma rather than a dysplasia of bone (global skeletal growth disturbance). Porter based his work on sixteen of twenty-seven individuals who had forearm xrays available to examine. Comparison of palpable lesions versus radiographically present lesions revealed that on average there were twice as many radiographic lesions as there were palpable ones; therefore, radiographic data was relied upon entirely. Results showed that the greater number of lesions present the shorter the forearm. Further, the ulna was proportionately shorter than the radius in eight of ten patients, and when an osteochondroma was present near a physis, the growth of the bone as compared to normal was inhibited by as much as 80%. The forearm is a paired-bone construct, and Porter found the relative lengths of the bones correlated inversely with the 32  relative size of their osteochondromas. That is to say, the physical presence of the lesions results in local same-bone deformity and growth inhibition. This then leads to bony deformity, joint malalignment, and length discrepancies of a two bone system or the limb itself. If HME were a skeletal dysplasia, simple excision would not arrest the development of new, or further growth in this case, of excised lesions. Porter concluded that it is the local affect of the lesion (number of lesions, proximity to the physis and two bone systems) causing the pathology.  1.2.5.3 G e n o t y p e - P h e n o t y p e C o r r e l a t i o n s  In the past few years, despite the discoveries made in the molecular biology and genetics of exostoses, only a few papers have been published looking at the phenotype as it relates to the genotype in HME (Carroll et al. 1999; Francannet et al. 2002; Pierz et al. 2002). 1.2.5.3.1 Carroll's Findings  In 1999, Carroll assessed nine families (twenty-eight patients) with genetic mapping and evaluated the patients to determine if "genetic variations" correlated with clinical manifestations (Carroll et al. 1999). Linkage analysis was done using 6 highly polymorphic repeat (HPR) markers that flanked EXT 1. Families were assigned to either EXT 1 or not by calculating a two-point likelihood of difference using a MLINK subroutine of the computer program LINKAGE . Provisonal groupings developedfromthe linkage resulted in Group A representing the EXT 1 linked patients and Groups B and C representing not EXT 1 related which were clinically distinct. Clinical evaluation included range of motion of the joints, angular and limb length discrepancies, radiographs of the spine, pelvis, forearm and humeri, and hips to ankle standing films of the lower extremities. Features  33  evaluated were location, type and number of lesions, spine assessed for scoliosis, femur neck shaft angle, Sharp's acetabular index, Reimer migration index, radial bowing, carpal slip, radial articular angle, radial head subluxation/dislocation, ulnar shortening, femoral and tibial anatomic angle, ankle angle, and mechanical axis. Three clinical groups were identified based on the number of sessile lesions which appeared to correlate with severity of deformity and limb-length inequalities: Group A, EXT 1 linked, (87% sessile lesions) were moderately involved, Group B (95% sessile lesions) were severely involved, and Group C (72% sessile lesions) were mildly involved. Group C was ultimately deduced to be linked to EXT 2 based on the findings that chondrosarcomas were to that point only associated with chromosomes 8 and 11 and in this series of patients chondrosarcoma was identified in one patient each from Group A and C. This paper concludes that there are 3 distinct clinical groups where it was felt they represented mutations in EXT 1 (moderate phenotype, chromosome 8), EXT 2 (mild phenotype, chromosome 11) and EXT 3 (severe phenotype, chromosome 19). The weaknesses in this paper includes first the lack of mutation identification, i.e. the true genotype, second, the inclusion of EXT 3 as one of the clinical types since EXT 3 mutations have never been shown to cause osteochondromas (though this was not known at the time of this Carroll's publication) and third, basing severity on whether lesions are sessile or pedunculated alone to categorize the patients. The strength of this paper is the extensive phenotype characterization. The main conclusion to be drawn from this work is that EXT 1 is worse that EXT 2 in this group of patients  34  1.2.5.3.2 Francannet's Findings  In 2001 Francannet reported on a clinical survey and mutation analysis of 42 French families. This study identified that 27 of 42 (64%) cases were accounted for by EXT 1 mutations. Of these, four were nonsense, nineteen frameshift, three missense, and one splice site. EXT 2 mutations accounted for 21% of the mutations and of these four were nonsense, 2 frameshift, two missense and one splice site. The phenotypic features assessed included a questionnaire given to the patients (the contents of the questionnaire was not included in the paper), clinical notes and xrays reviewed, the Musculoskeletal Tumor Society score for functional assessment (Enneking 1987) and development of chondrosarcoma. Severity was described as severe or moderate and based on the following, age of onset (3 or less was severe), number of exostoses (10 or more was severe), vertebral location (presence of vertebral lesions was severe), stature (less than the 10 percentile was th  severe) and functional rating (fair or poor was severe). The conclusion of this study was that EXT 1 caused the most severe forms of the disease and degeneration of exostosis into chondrosarcoma only occurred in EXT 1 (in clear opposition to Carroll's (1999) deductions andfromthe basic science literature (see section 1.2.3.2), where chondrosarcomas were found in both EXT 1 and 2). The strength of this paper is identification of the genetic cause and its comparison with phenotypic features. An important feature that was included is the Musculoskeletal Tumour Society score (Table 1.4), which is a direct reflection of quality of life and ultimately what is the clinically relevant outcome. The phenotyping however in general is weak, not only in terms of only a few features being interpreted but also how they were applied. The 5 features were helpful in describing a portion of the phenotype, however they may not contribute to severity. For example, simply the presence of an exostosis in the 35  spine does not necessarily cause a problem as in pain, or deformity, in particular scoliosis. Also, as the number of lesions increase they may cause more secondary problems as in joint malalignment or bony deformity but this is also contributed to by the size, location and morphology (sessile or pedunculated) of the lesions not simply the presence or absence of lesions. Age of onset is also difficult to determine precisely and is influenced by many factors (family concerns, referral, diagnosis), which may blur the true onset date. In addition, this study identified chondrosarcoma only in patients with EXT 1 mutations whereas other authors have found these mutations in EXT 2 and 3 as well (Kivioja et al. 2000; Porter and Stickens 1999; Hecht et al. 1995; Hecht et al. 1997; Raskind et al. 1995; Hogue et al. 1996; Carroll et al. 1999). Of note once again however is that the patients with the EXT 1 mutations were phenotypically worse than the EXT 2 patients.  1.3  Project Rationale Hereditary Multiple Exostosis (HME) is a relatively uncommon problem with a  high clinical burden seen by Orthopaedic surgeons at British Columbia's Children's Hospital (BCCH). Most patients affected by this disease require surgical intervention an average of three times in their lifetime and usually as a child. The morbidity and complication rates of these surgeries are significant, including pain and disability, and problems implicit to surgery as a whole. Work on the genetics and molecular biology of Exostosis (EXT) genes has opened up the opportunity to further describe and examine this condition from the genotype perspective. Phenotypic features important to function and appearance are now better appreciated and readily investigated. It is the interplay between the genotype and the phenotype which has been incompletely explored. In which gene the mutation exists, what type of mutation it is, its location, and its severity can be established. McCormick (1998; 2000) has shown that examples of both 36  truncating and non-truncating result in a non-functional protein and thereby in osteochondroma growth due to presumed interference in the tumour suppressor system. His original work suggested it is irrelevant where the mutation is (which gene), its type (truncating or non) or its location, the phenotype will be the same. However, missense mutations still produce a protein that localizes to the endoplasmic reticulum. So is it true the EXT gene function is completely eradicated? Also Gullberg's (Gullberg 2002) work suggests mutations in EXT 1 and 2 have a different effect in that EXT 1 catalytic activity is greater than that of EXT 2 and this would therefore cause differing phenotypes based on which gene is mutated. How the mutations manifest their effect on the physiologic function of the EXT genes will also then be potentially different. In terms of growth plate regulation problems if EXT 1 activity is preserved somewhat in isolation (when EXT 2 is mutated) then one would expect some preservation of the concentration gradient of Indian Hedgehog as some of the catalytic activity of heparan sulfate polymerization is preserved and thereby heparan sulfate present allowing for Indian hedgehog signalling to be partly working. This may then in turn result in less severe global growth plate changes, but should still be universal throughout the body. If the physiologic function is related to extrcellular matrix behaviour related to heparan sulfate presence then the partly preserved activity of EXT 1 in the EXT 2 mutated subject would lead to fewer chondrocyte nests developing; the fewer the nests, the fewer the lesions, the less the tumour burden and it secondary effects. Regardless of the actual physiologic function, there should be a difference in phenotype based on genotype whether it is due to which gene is affected, what type of mutation exists and possibly due to location or secondary influences such as gender remains unelucidated.  37  These differences based on genotype variability will then be reflected in the patient's phenotype. As we do not have the exact answer from the basic science work done on the EXT genes we may corroborate the possible mechanisms of function by looking at the phenotype. Clinically based authors suggest that the phenotype does depend on which gene is affected. For example, Carroll (Carroll et al.1999) lead us to the conclusion that if there is a mutation in EXT 1 the disease process in those individuals will be moderate versus EXT 2 which has a more mild presentation. Francennet (Francennet et al. 2001) came to the same conclusion, more specifically saying EXT 1 is worse than EXT 2. Further some authors have noted males have more severe disease and females may have incomplete penetrance, yet this is purely anecdotal (Schmale et al. 1994, Solomon et al. 1963). Neither Carroll's or Francennet's papers (Carroll et al.1999; Francennet et al. 2001) were thorough in one of the two aspects of the genotype phenotype assessment leaving their conclusions needing further exploration, but nonetheless reassuringly consistent. There is obviously tremendous controversy about how genotype influences the phenotype. But to date researchers have worked in isolation in either the basic science or pure clinical arena except for the two above mentioned authors. This project was designed to bridge this gap by defining the genotype and the phenotype thoroughly from both aspects and then exploring the relationships. The rationale behind this study was to determine the genotype of HME: which gene is mutated, with what type of mutation, and its location, in conjunction with defining each affected individual from clinical parameters, which represent a given phenotype. The analysis of this data determines if genotype truly correlates with phenotype such that specific mutations or affected genes cause a predictable pattern of presentation, symptoms, and signs.  38  The results have many implications. If a correlation exists between genotype and phenotype in HME, a complete natural history for each mutation type and gene affected can be charted; this will directly influence day-to-day management of patients. For example, should particular lesions be excised early or later in its course. By knowing a patient's genotype it may be possible to determine which individuals, based on mutation type and location, are at increased risk for growth disturbance, lesion growth potential, and transformation to chondrosarcoma. It will also be possible, based on a patient's phenotype, to determine either the mutation location or type, and from that information, the individual's treatment can be managed accordingly.  1.4  Hypothesis  There is a genotype phenotype correlation in HME such that the major genotypic expressions, for example, EXT 1 versus EXT 2, will present with different phenotypic manifestations, for example, limb alignment or stature.  1.5 Objective  The objective of this study was to explore if a correlation exists between genotype and phenotype in Hereditary Multiple Exostosis in ten British Columbian families.  39  Chapter II: Materials and Methods  2.1 Ethical approval The proposed study was reviewed by the Ethics Review Board of both Children's & Women's Hospital of British Columbia (C&W) and the University of British Columbia. Both boards approved of the study and its design in the fall of 1998; the projects ethical approval extended to 2004. Ethical Approval forms are found in Appendix 8.1.  40  2.2 Study protocol overview  Subject Identification  • Pedigree delineated I  • Informed consent obtained  I  • Recruitment of family members  I  Genotype  Phenotype  •  f  • D N A extraction from blood samples from all participants Gene Assignment  • H i g h l y polymorphic repeats •Family members run together with controls •Assignment criteria (see section 2.4.3.1)  _L  Clinical  Radiographic  •Lesion count • L i m b alignment • L i m b segments •Height (percentile) •Weight  •Lesion quality • L i m b alignment  •Range o f motion M u t a t i o n Identification  • E X T 1 or 2 amplified for each proband •EXT 1 18 primer pairs (11 exons) •EXT 2 16 primer pairs (14 exons)  DNA  Sequencing  •Mutation identification •Confirmation o f true mutation (literature review, controls, type o f mutation, change i n mRNA) f Segregation Analysis  •Sequence exons o f all affected and available family members Genotype Defined  Phenotype Defined  Genotype - Phenotype Analysis  • Gene vs. Phenotype • Gender vs. Phenotype • M u t a t i o n type vs. Phenotype • Mutation severity vs. Phenotype • Mutation location vs. Phenotype C o v a r y i n g analysis  •Gene + Gender vs. Phenotype •Gene + Mutation Type vs. Phenotype •Gene + Severity vs. Phenotype  I S u m m a r y of Significant A : P Correlations  Figure 2.1 Overview of materials and methods  41  2.3 Subject Recruitment 2.3.1 Subject Identification  All subjects involved in this study were identified as patients of British Columbia's Children's Hospital. Patients and their families known to the paediatric orthopaedic department were approached by their respective surgeons, informed of the study, and asked if they would like to become involved. If they agreed, the principal investigator (Dr. C. Alvarez) was introduced to the family. All potential subjects were then informed of the study's rationale, purpose, and protocol. Consent was obtained from all individuals willing to participate in this study; minors consented with parental approval. The Letter of Information and Consents forms are found in Appendix 8.2. Individuals who did not wish to participate in the study continued with their regular care. 2.3.2 Pedigree Accumulation  A pedigree was designed (Cyrillic™ software) from the family history using as many corroborating family members as possible. Many extended family members did become involved in the study; however, a significant number of families had no extended members available. 2.4 Genotype 2.4.1 Sample Collection  Approximately 15 ml blood samples were collected from all participants in EDTA preserved, heparin loaded, 8 ml vacutainer tubes. Blood samples were drawn primarily by the principal investigator using universal precautions or by BCCH's laboratory accessioning personnel in the young subjects (less than 5 years of age). Blood was stored at  42  4°C until DNA extraction was performed. On average blood was not stored more than 1 week prior to extraction. 2.4.2 DNA Extraction 2.4.2.1 F r o m b l o o d  DNA extraction from patients' blood was carried out according to the NH4CI lysis and salt/chloroform protocol set forth by Mullenbach (1989). Red blood cell lysis solution was added: up to 45 ml per 10-15cc of sample in a 50ml falcon tube. The tube was inverted to mix and incubated at 37°C for 20 minutes with frequent mixing. The sample was then centrifuged for 5 minutes at 2000 rpm, and the supernatant was aspirated off. For the final rinse, 10-15ml of isotonic saline was added and the pellet was gently resuspended; this solution was centrifuged for an additional 5 minutes at 2000 rpm. The supernatant was removed down to the pellet and 10ml of saline + 500pl 20% SDS + lOOpl 20mg/ml proteinase - K were added. The lysate was incubated overnight at 37°C and stored at 4°C until ready for extraction. DNA extraction from the lysate was done using a salt/chloroform protocol (Mullenbach 1989). 3.3ml of 6M NaCl was first added to the lysate to yield a final concentration of 1.5M. The solution was mixed gently and an equal volume of chloroform was added followed by a gentle rotation for 30-60 minutes. The solution was centrifuged for 10 minutes at 2000 rpm, and the supernatant containing the DNA was transferred to a new tube. The DNA was precipitated out of the supernatant with 2x volume of 95% ETOH at room temperature. The DNA was spooled out of the liquid and re-suspended in TrisEDTA to 2000pl. The integrity of the sample was checked on a 2% agarose gel and visualized under UV light.  DNA concentration was measured using an UV/visible  spectrophotometer (Ultrospec® 3000, Pharmacia Biotech).  43  2.4.3 Gene Assignment - Highly Polymorphic Repeats  Short Tandem Repeats (STR) were used to help trace the likelihood of the mutation being in EXT 1, 2, or 3. Initially only one marker for each of EXT 1 and 2 was used to direct which gene should be investigated primarily. Some families were too small for any meaningful segregation to occur, (families 1 and 6) and others were determined with only 2 PCRs, AO 1/2 and A03/4 (Families 2,3,5,16,17,18). Families 4 and 6 were assessed by all 8 markers due to lack of mutation identification when both EXT 1 and 2 were sequenced.  2.4.3.1 Marker Selection  Highly polymorphic repeat (HPR) markers were custom selected for the purposes of this project. Using the NCBI database microsatellite markers were identified for EXT 1, EXT 2, and EXT 3. Many of the markers used were the same as those used by Raskind (1995) in the project "Loss of Heterozygosity in Chondrosarcomas for Markers Linked to Hereditary Multiple Exostoses Loci on Chromosome 8 and 11" (Figure 2.2).  44  8  11  19 EXT3  13.3 D11S905  1 3  -  D11S1355 D11S903 D11S5547 12 D11S1319 D11S1313  2  D19S216 D19S221 D19S226  Figure 2.2 HPR marker locations in relation to EXT 1,2, and 3. Ideograms for Chromosomes 8,11, and 19 showing approximate locations of the E X T genes. Locations of the polymorphic microsatellite markers (CA repeats) used to determine L O H are also shown. (Raskind et al. 1995).  All markers were within a 5.4 cM span of the EXT 1 gene, for EXT 2 this was a 9 cM span and for EXT 3 it constituted a 25 cM span. Care was taken to select markers with greater than 71% heterozygosity frequency, fewer than ten alleles, and acceptable denaturation and reannealing temperatures (Table 2.1). Not all markers were required to assign the likelihood of a family carrying the mutation in one gene over another gene; however, two families did require all eight markers to help determine the likelihood of mutation location. Highly Polymorphic (HPR) markers and their features are described in appendix 8.5.1. The HPR primer pairs are named and defined in appendix 8.5.2.  45  2.4.3.2 P C R ( w i t h C A repeats)  PCRs were performed in a 25pl reaction volume with a final MgCL; concentration of 1.5mM, 200pM dNTP, and 0.5uM of each primer (Table 8.5.2), and lul Taq Polymerase (GibcoBRL) (Gene Amp-PCR system 9700, PE Applied Biosystems). Initial denaturation was done for 4 minutes at 96°C, followed by 25-30 cycles of 30 seconds at 94°C, 30 seconds at the determined temperature for each primer (see table), and 45 seconds at 72°C. Extension was performed at 72°C for 5 minutes.  2.4.3.3 P A G E ( p o l y a c r y l a m i d e gel electrophoresis)  Following the PCR, 5ul of PCR product was aliquoted into a microdish (Nunc, Intermed) well containing 5ul of denaturing loading buffer: 40% sucrose, 0.025% xylene cyanol, 0.025% bromophenol blue. Samples aliquoted in this way could be stored at -20° C for several weeks. The sample was denatured by placing the micro-dish on a heat block at 94° C for three minutes then immediately placed on ice. 4pl of the sample was loaded on a 6% denaturing polyacrylamide gel (60ml gelmix: 100ml 30% PAA, 240g urea, 50ml 10(x) TBE, 100ml dH 0, 500pl ammonium perphospate(APS), 50pl Temed). The 0.4mm 2  thick gel was run at 1650V with 1 x TBE running buffer for 1-2 hours on a sequencing apparatus (BRL, model S2, Life Technologies Inc.). The smaller plate used in the gel apparatus was treated with Wynn's Rain Away (Canadian Tire). Approximately 30 minutes before the end of the running period, 500-1000ml of 0.5xTBE was prepared. A Hybond N+ membrane (positively charged nylon membrane, Amersham Life Science, UK) was trimmed to the exact size of the gel. The membrane was placed in a container with 0.5xTBE and cooled in a fridge for at 15 minutes. Five pieces of gel blotting paper (grade 238 cotton cellulose gel blot paper, Island Scientific, WA, USA) were cut slightly larger than the dimensions of the gel. When the run was completed, a 46  piece of blotting paper was placed over the gel, and subsequently peeled off to remove the gel from the glassplate. The wet Hybond N+ membrane was put over the gel in order to make a "gel sandwich" which was placed in a transfer apparatus (Semi-dry blotter, C.B.S. Scientific Co) with the membrane side facing down. The transfer was allowed to continue for 45 minutes at 15 volts. After the transfer was complete, the membrane was rinsed in 0.5 x TBE and dried for 1 hour at 80° C. 2.4.3.4  H y b r i d i z a t i o n a n d chemiluminescent  detection  The following solutions were prepared for hybridization of one membrane. Stock solutions (10 x buffer, component A, component B) bought from Lifecodes Corp. (Stamford, CT, USA) were used in the "Quick-Light" hybridization protocol. Two wash solutions were prepared; Wash 1, 3ml of component A, 3.75ml of component B and 68.25ml of double distilled water, and Wash 2, 0.2ml component A, 2.5ml component B and 45.5 ml of double distilled water and 100ml lxbuffer. The wash solutions, as well as the Quick-Light hybridization solution (15ml per membrane), were preheated at 55° C. The membrane was then soaked in 25ml of the heated Wash 1 in a hybridization tube. 4pl of a (CA)n Quick-Light research probe (Lifecodes Corp) was added to 15ml of heated hybridization solution in a 50ml Falcon tube and mixed well. The probe used was an alkaline phosphatase conjugated oligo that is vialed at 5 units per lOOpl. One unit can was used in 75ml of hybridization solution when the Lifecodes Quick-Light hybridization procedure is followed. Twenty-five millilitres (25ml) of Wash I was poured out from the hybridization tube and the probe solution was added into the tube with the membrane. Hybridization was performed at 55 degrees Celsius for 30 minutes in a hybridization oven (Hybaid). The membrane was washed twice for lOminutes each with Wash 1 at 55 degrees Celsius, after which it was washed twice for 10 minutes each with Wash 2 at 55 degrees Celsius. Then the membrane was twice washed briefly at room temperature with the lx 47  Quick-Light Buffer to adjust the pH of membrane to the Quick-Light chemical detection procedure. 2.4.3.5 V i s u a l i z a t i o n  The membrane was then soaked in CDP-star solution (Roche Diagnostics Corporation, IN, USA) for 5min, drained and wrapped in plastic wrap. The membrane was then exposed to Kodak XAR film at room temperature for 1 hour and developed in a Kodak M35A XOOmat Processor machine. Exposure of the film to the membranes required customization for each membrane to give optimal visualization of the bands. Family members were run next to each other and each gel had control samples. Bands were labelled according to each family to aid in segregation determination (Appendix 8.5).  2.4.3.6 E x c l u s i o n A n a l y s i s  All available family members were run in adjacent lanes with two controls for each family. EXT 1 bands resulting from amplification of markers were assigned numbers (1,2,3) and EXT 2 markers were assigned letters (a,b,c).  The bands were assigned  numbers/letters from the top of the gel to the botton. Each member was assigned with EXT 1 numbers and EXT 2 letters. These assignments were then traced amongst the family members. Cosegregation was deemed to implicate the particular gene involved. Lack of segregation, i.e in an affected or unaffected, resulted in exclusion of that gene and proceeding to the next. From this, EXT 1 or 2 was excluded as being the source of the mutation.  2.4.4 EXT1 and EXT2 amplification  Exon 1 of EXT 1 and exon 2 and 14 of EXT2 were split into overlapping fragments to obtain amplification products of less than 350 base pairs in length. Amplifications of the exons of EXT1 and EXT2 were performed in a 50pl reaction volume in 1.5mM MgCl 48  (except primer pair 1-9 which uses 2.5mM MgCl ), 200pM dNTP, 0.5uM primer (see 2  Table 2.2) and lul Taq Polymerase. 2pl of DNA (80ng/pl) was used for each sample. Samples were heated (Gene Amp PCR system 9700, PE Applied Biosystems) to 96°C for 4 minutes, and then cycled (30 times) through the following temperatures: 94°C for 30 seconds, annealing temp (Table 2) for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes. The PCR product (5ul) was combined with 5ul of sucrose loading dye and run on a 2% agarose gel containing ethidium bromide (lug/ml) at constant voltage (BioRad system) for one hour in lxTBE buffer. A lOObp DNA ladder (50ul ladder, 50ul xylene cyanol, 400ul TE) was run alongside the samples.  DNA was visualized under UV light and  photographed using Polaroid film.  49  Table 2.1 Primer pair sequences used for E X T 1 Primer Pair 1-1 1-2  Primer Name E X T 1-ex l a EXTl-exlb EXTl-exlc EXTl-exld  Exon 1  1  1-3  EXTl-exle E X T 1-ex If  1-4  EXTl-exlg EXTl-exlh  1-5  EXTl-exli EXTl-exlf2  1-6  EXTl-exlk E X T 1-ex 11  1-7  EXTl-exlm EXTl-exlj  1-8  EXTl-ex2a EXTl-ex2b EXTl-ex3a EXTl-ex3b EXTl-ex4a EXTl-ex4b EXTl-ex5a EXTl-ex5b EXTl-ex5a EXTl-ex5c EXTl-ex6a EXTl-ex6b EXTl-ex7a EXTl-ex7b EXTl-ex8a EXTl-ex8b EXTl-ex9a EXTl-ex9b E X T 1-ex 10a E X T 1-ex 10b EXTl-exlla EXTl-exllb  1-9 1-10 1-11 1-12 1-13 1-14 1-15 1-16 1-17 1-18  1  1  1  1  Sequence CAGGCGGGAAGATGGCGGACTGGC7/CCGGCTG7/GGCT CCTCGATGCCC TGCTCTCAGCTGGCTCTTGTCTCGGAATCCTCGT TTTCCAATTGATCCC CGGAGCCTCTGCGCCCCTTCGTTCCCraG^4rG7T TTGGTAACTTTCGGCG CGTATACCCACAGCAAAAAGGGGC47TG7TCC4C AAGTGGAGACTCTCG CCAGTTGTCACCTCAGTATGTGCGGC7T7UGCC4 GCATCGCCAGG CCTGACTACACCG AGG ACGGGTGTCTGA TCCTA T CCCTG ACggaccaaggCCgg  Length (bp) 212  Temp (°C) 58  201  55  232  55  209  55  168  55  237  55  231  55  1  GGTATTCAAGGGGAAGAGGT  2  ccccacattcgcaatgagtcgagaggtgataatgttaaaccc  225  55  3  cgatAXggaacagcttcgXcXggacgggggcagcaataatctgc  224  55  4  gtgcattctctttgttttacagctgagagaagtgtataaagg  239  55  5  cctttccaaatatcatcaggcatcttcagggtaaacaagggc  237  55  5  cctttccaaatatcatcaggccattttgcaatgctctgctctg  237  55  6  gcmccagcgcttcattaggcctggagctggagcaggcagggg  210  55  7  ggcgtacataaatacatcctaccccccaaggctccacagtggttcc  189  56  8  caagactctgaagttacctctttcccggtgactgcctgaacagcccaacc  204  58  9  cattgttgattgcttgtttggccgtaaagtctgtaagagacatgtcc  235  55  10  cttgtcatcatgigataatggcccgagtgaagcaaggaagaggg  259  55  11  ccttgcacttctctcatattatccCCTCAAAGTCGCTCAATGTCTC GG  230  55  cagagccc  NOTE: Primer names designated by "ex" followed by exon number; italics designate primers in the 3'-5'direction; lower case indicate primers located in introns; all primers used a final concentration of 1.5mM MgCb, with the exception of primer pair 1-9 which used 2.5mM MgCl ; Accession Number: U67356-U67368 (Wuyts 1998) 2  50  Table 2.2 Primer pair sequences used for E X T 2 Primer Pair 2-1  Primer Name EXT2-ex2a  Exon  Length  Sequence  2  C t c t c c c c t g g t g a c c C 4 C 4 G C G ^ TAGACA  TCAAAACACG  Temp  (bp) 338  56  CQ  EXT2-ex2A8 2-2  EXT2-ex2A26 EXT2-ex2A25  2  GACAGTCCCATCCCAGAGCGGGGAGGGAACAA AACAGACAGG  249  56  2-3  EXT2-ex2A4  2  ACTACACTGATGACAlCAACCGccctttagttCCCtg agggcc  176  55  EXT2-ex2b 2-4  EXT2-ex3a EXT2-ex3b  3  gttgacacatt.aatt.ctcccgaacaaaaatgatcttgaaccc  184  51  2-5  EXT2-ex4a EXT2-ex4b  4  gaataaagtccUtctttctcatcgcagtaaaggcacacctggc  205  55  2-6  EXT2-ex5a EXT2-ex5b  5  gcaattttccaatcacctgcctgagcctttgcgagagg  267  51  2-7  EXT2-ex6a EXT2-ex6b  6  ctagtttgtaatctcttgcctctacgcagaaccactaatgtagag  222  55  2-8  EXT2-ex7a EXT2-ex7b  7  gggatgtggggctgaaggaggctcctgtccctctgtatccagtc  293  57  2-9  EXT2-ex8a EXT2-ex8b  8  gcttgctcacttaaaacagcgcctcatgtggctagcac  200  56  2-10  EXT2-ex8a EXT2-ex8c  8  gcttgctcacttaaaacagcttatgctgcccttatcaggccc  200  56  2-11  EXT2-ex9a EXT2-ex9b  9  cagctgcttttctgacccggatccagctgagagaggcac  263  55  2-12  EXT2-exl0a EXT2-exl0b  10  cctcacaaaagttaggagaaacacactgtgtaaaacc  240  51  2-13  EXT2-exlla EXT2-exllb  11  gaatggttgctgtctgaattgggctcagttttgtcaccttgcc  235  55  2-14  EXT2-exl2a EXT2-exl2b  12  ccccttatttatcagctaaagggcaagtgagtggcagagcc  220  55  2-15  EXT2-exl3a EXT2-exl3b  13  gtccttgacactgacagccaggtagagatcagaggctaaggcgc  175  55  2-16  EXT2-exl4a EXT2-exl4b  14  caaacccctcctccccacctcctcGTGGGTTAGGTGGG TGCATGCC  318  58  NOTE: Primer names designated by "ex" followed by exon number; italics designate primers in the 3'-5'direction; lower case indicate primers located in introns; all primers used a final concentration of 1.5mM MgCb; Accession Number: U67356-U67368 (Wuyts 1998) 2.4.5 DNA Sequencing  DNA was prepared for sequencing using the polyethylene glycol 8000 precipitation protocol (Rosenthal, Coutelle and Craxton 1993).  Several modifications were made  including using 1.5ml Eppendorf tubes in place of 500pl tubes and allowing the sample to sit at room temperature for 20 to 30 minutes following the addition of the PEG solution to 25pi of PCR product. After re-suspending the precipitate in 11 pi of H2O, 2pl of the sample was analyzed on a 2% agarose gel (1 hour at 125V) and visualized under UV light. 51  Once the integrity of the product was confirmed, DNA sequencing was performed using the ABI 3100™ Sequencer (PE Biosystems, Foster City, CA, USA). This system employs capillary electrophoresis-based automated sequencing. Primer concentrations were made to 3.2 pmol. 2.4.6 Mutation Identification  PEG purified and cleaned PCR products were amplified with the ABI Prism Big Dye Terminator Cycle Sequencing Ready kit (version 2, Applied Biosystems, Foster City, CA, USA). Five ng PCR template was mixed with 3.2 pmol of sequencing primer (not nested), 2.4ul BigDye Terminator Ready reaction solution (Applied Biosystems, contains the dye terminators, dNTP's, AmpliTaq DNA polymerase FS etc.), 3ul of 5 x buffer (Applied Biosystems) to make a total volume of 20ul. (BigBye Terminator Ready reaction was diluted 1 to 4 with 5x buffer). Amplification was done in a 96 well microamp plate at 96° C for 10 seconds, 50° C for 5 seconds, 60° C for 25 cycles in a GeneAmp PCR system 9700 thermal cycler. Precipitation of PCR products and removal of unincorporated dye terminators was done in the 96 well plate after the PCR plate was removed and spun in a table top centrifuge capable of centrifuging 96 well plates, 20pl double distilled water and 60pl of 100% isopropanol were added to each well. The plate was sealed with strips of lids or foil, inverted to mix, and left at room temperature for 15 minutes after which it was centrifuged at 1200 rpm for 5 min. Without disturbing the precipitate, the foil was removed and the supernatant discarded. A volume of 70% isopropanol was added and the plate recentrifuged. After removing the supernatant by gently inverting the plate onto a paper towel, the samples were re-suspended in 2 pi of ultrapure formamide (Applied Biosystems). Samples were denatured by putting the plate into a thermal cycler and by 52  rurining a denaturing program at 94° C for 3 minutes and then put on ice. The plate, containing the fluorescent-labelled extension products, was loaded in the sample tray of an ABI Prism 3100 automatic sequencer (Applied Biosystems). POP-6 polymer and a 50 cm capillary array were used (both from Applied Biosystems). Data was analysed using the ABI Sequencing analysis software, version 3.2™. Nucleotide sequences were assembled and aligned using programs in the Sequencher 3.0™ software package (Gene Codes, Ann Arbor, MI, USA). Two programs were used to analyze the DNA sequences: SEQUENCHER™ software (Gene Codes Corporation, Ann Arbor, MI, USA), and Consed (University of Washington Genome Center, Seattle, WA, USA). Sequence chromatograms for EXT1 and EXT2 were aligned into "contigs" and viewed using Phred, Phrap and Consed (version 6.0) (Ewing et al. 1998; Ewing  and  Green  1998;  Gordon,  Abajian  and  Green  1998).  (http://www.genome.washington.edu.) Identified mutations using these programs were confirmed using both the 5'-3' and the 3'-5' reads. Heterozygosity on both reads was required to confirm a true mutation. All probands plus the genbank sequence were compared to each other to ensure this was a true mutation versus a polymorphism. The identified mutation was compared to previously described mutations to determine whether it was novel. The translation of the gene with the new mutation was examined to determine the nature of the mutation, that is, was the mutation a missense, nonsense, frameshift (insertion, deletion), or splice site. If it was a missense, the new amino acid was interpreted in relation to whether it caused a change in the nature of the amino acid, i.e., basic vs. acidic and uncharged polar versus non-polar (hydrophilic vs. hydrophobic).  53  2.4.7 Segregation Analysis  Once a proband's mutation was confirmed, the available family member's DNA's were sequenced as described using the primer pair representing the location of the mutation. Contigs designed in Sequencher™ were developed using the primer pair in both 5'-3' and 3'-5' read for each family member plus the GenbBank sequence. Sequences were aligned and the identical mutation was looked for in all clinically affected family members and absent in unaffected members. Care was taken to identify subjects not affected clinically but carrying the genetic mutation.  2.5 Phenotype All subjects identified as having at least one exostosis underwent thorough physical examinations. Xrays taken as part of the patient's care were examined. Phenotyping was divided into two categories: clinical and radiographic. Clinical features included demographics, percentile weight, percentile height, percentile limb segment lengths as well as total limb lengths, limb alignment, and range of motion. All affected patients had range of motion measured at the shoulder, elbow, wrist, ankle, knee, and hip. Method of data collection and standardization (for age and gender) is listed below. Radiographic features were obtainedfromavailable films; the data collected included lesion quality (count, size sidedness, complexity, location, and metaphysealflaring)and angular alignments (carpal slip, radial inclination, ulnar shortening, radial head subluxation/dislocation, radial bow, elbow joint angle, femoral/tibial anatomic and mechanical angles, weight bearing axis, femoral neck-shaft angle, Sharp's Acetabular angle, fibular height, and ankle joint angle).  54  2.5.1 Clinical features  All physical examinations were performed by the author who is a Pediatric Orthopaedic Surgeon and a member of the Royal College of Physicians and Surgeons of Canada in Orthopaedic Surgery. 2.5.1.1  Demographics  Each affected subject's age, weight, height, ethnic background, and address were collected. The subjects' weight and height were converted to a percentile figure to standardize for age and gender to allow for comparison amongst groups. Height and weight were standardized using updated Green Anderson Charts (Hamill et al. 1979). Clinically palpable lesions were recorded, and surgically excised lesions were accounted for. All extremities and accessible flat bone were examined for exostoses. 2.5.1.2  Lesion count  All accessible aspects of the long bones, hands, fingers, feet, toes, scapulae, clavicles, ribs, sternum, spinous processes, and ilia were palpated for lesions. Any significant local deformity was also recorded (that is some lesions are so large they expand the entire local bone). All palpated lesions were recorded as present and specific location noted: for example, right distal radial radius or left proximal medial tibia. If more than one discrete lesion was palpable in a location, each was counted separately. All lesions were correlated with xray visualization; however, not all lesion areas were radiographically imaged, in particular, the hands and feet. 2.5.1.3  L i m b alignment  Clinically the overall alignment of the elbow and knee were measured using a large, hand-held goniometer. The hinge of the goniometer was centred over the elbow joint which was held in full supination and extension. Each limb of the goniometer was placed along the long central axis of the upper and lower arm, and the subtended angle was measured. 55  Knee joint measurements were taken in a standing position. The goniometer was centred on the middle of the anterior knee joint, and each limb of the goniometer was lined up against the centre of the long axis of the femur and tibia. Again, the subtended angle was recorded. In both cases, valgus or varus alignment was denoted. 2.5.1.4  Segment a n d L i m b L e n g t h s  Segments and limb lengths were measured in centimetres and used surface landmarks as follows: 1. upper extremity total length - top of humeral head to ulnar styloid. 2. upper arm - top of humeral head to capitellum. 3. lower arm - tip of olecrenon to ulnar styloid. 4. lower extremity total length - anterior superior iliac spine to medial malleolus 5. upper leg - ASIS to medial condylar joint surface 6. lower leg - medial tibial joint line to tip of medial malleolus. A conversion for femoral length was required to subtract the distance from the ASIS to the top of the femoral head. Using Caffey's method, 5% of the lower extremity length was subtracted from the total leg measurement and 10% from the upper leg length (Silverman 1985). Again using Caffey's radiologic text, each segment and total length was standardized for age and gender reduced to a percentile to allow for direct comparisons between subjects. 2.5.1.5  Range o f motion  A large, hand-held goniometer was used to measure a joint's range of motion. Range of motion was measured for the shoulder (abduction, adduction, internal rotation, external rotation, and forward elevation), elbow (flexion, extension, supination, and pronation), wrist (flexion, extension, and radial and ulnar deviation), hip (flexion, extension, internal and external rotation, and abduction and adduction), knee (flexion and 56  extension) and ankle (flexion and extension). If no restriction in motion was identified, full ROM was indicated, if there was any reduction in the normal range, precise measurements were recorded. 2.5.2  Radiographic features  Alignment and deformity measurements from the radiographs were made. Specific details covering how each measurement was calculated is outlined below. A complete radiographic record for the purposes of this project included standard images of the upper and lower extremities as well as chest and pelvis: anteroposterior (AP) proximal humerus to wrist inclusive with the elbow fully extended and forearm fully supinated, AP chest, AP standing pelvis if not incorporated into the hips to ankle film, AP standing hips to ankles inclusive. As they are not part of a patient's routine care, films of the head, hands and feet were not universally available. Lesions in areas not xrayed that were easily palpable were recorded as a clinical lesion. When orthogonal views were available from the same date, data from the two views were generally used. However, usually only one AP view was used; therefore, the size of some of the lesions may be underestimated. 2.5.2.1 L e s i o n q u a l i t y 2.5.2.1.1  Count - all visible lesions were accounted for.  2.5.2.1.2  Size - to account for magnification and patient-size variations a standardized size calculation was obtained for every lesion. L e s i o n size was calculated and ranked. First the protrusion ratio ( A ) was obtained b y dividing the protrusion distance o f the lesion (bony stalk) (a) b y the native bone width (b). The height ratio ( B ) was obtained b y d i v i d i n g lesion height (bony cap long axis) (c) b y (b). The average o f the two ratios ( D ) was expressed as a percentage. This average percentage was ranked as follows: < 2 5 % (1), 26-49% (2), 50-74% (3), > 7 5 % (4). The lesion ranks were also categorized as small (1), medium (2 and 3) and large (4).  57  Calculation of Lesion Rank  a / b = profusion = A c/b = height = B A / B = D% D values Lesion Rank  < 25% 26 - 50% 51-75% >75%  1 2 3 4  Size  small medium medium large  Figure 2.3 Calculation of Lesion Size and Rank  2.5.2.1.3  Side - left or right total count  2.5.2.1.4  Location -  distal, proximal, metaphyseal, or flat bone (includes any o f the pelvic bones,  sternum, scapula, or ribs)  2.5.2.1.5  Complexity - i f the lesion was multilobulated and too complex to obtain any o f the three measurements it was deemed complex. In general a l l these lesions were also large (category 4).  2.5.2.1.6  Metaphyseal flaring - i f the metaphysis o f the long bone showed aneurysmal dilatation and abnormal expansion o f the metaphysis globally.  2.5.2.1.7  Type - sessile versus pedunculated. I f a lesions stalk is narrower than its cap, it was called pedunculated. I f the stalk was equal to or larger than the cap, it was called sessile.  2.5.2.2  L i m b alignment  Measurements taken are defined below and referenced accordingly. This study also introduces new measurements and these are thoroughly described in the following pages.  58  2.5.2.2.1  C a r p a l slip - normal value = 5 +/- 2 m m (Keats 1990) The ulnar displacement i n milimetres o f the ulnar edge o f the lunate with respect to the ulnar border o f the distal radius.  F i g u r e 2.4 Measurement o f carpal slip  2.5.2.2.2  R a d i a l inclination - normal value equals 21 +/- 2 ° (Green 1993) The angle between the perpendicular o f the radius long axis ( A - B ) and a line j o i n i n g the radial and ulnar distal edges o f the radius ( A - C ) .  2.5.2.2.3  U l n a r shortening - normal value equals 0 +/- 1mm (Green 1993)  F i g u r e 2.5 Measurement o f radial inclination and ulnar shortening  The distance between the distal surface o f the ulna and the radius.  2.5.2.2.4  R a d i a l b o w i n g - expected normal value equals 10 +/- 5 ° (Green 1993) The angle subtended between the long mid-axis o f the forearm ( A - B ) and the m a x i m a l radial deviated point o f the radius' diaphysis ( C - D ) .  F i g u r e 2.6 Measurement o f radial b o w i n g  59  2.5.2.2.5  R a d i a l head subluxation/dislocation - normal value equals no subluxation or dislocation. The radial head is either subluxated/dislocated ( B ) or not ( A ) .  F i g u r e 2.7 Radial head subluxation / dislocation  2.5.2.2.6  E l b o w joint angle - normal range equals females 10+/-2 valgus, males 8+/-2 valgus 0  0  (Keats 1961) The angle subtended between a line drawn through the long axis o f the humerus ( A - B ) and forearm ( C - D ) .  F i g u r e 2.8 Measurement o f the elbow joint angle  2.5.2.2.7  F e m o r o - t i b i a l a n a t o m i c angle - normal value equals 7 +/- 5° valgus (Hsu et al. 1990) The angle subtended by a line drawn between the long axis o f the femur ( A - B ) and the tibia ( C - D ) .  F i g u r e 2.9 Measurement o f the femoro-tibial anatomic angle.  60  2.5.2.2.8  F e m o r o - t i b i a l m e c h a n i c a l angle - normal value  equals 0 +/- 5 ° ( H s u et al. 1990) The angle subtended b y a line drawn from the centre o f the femoral head to the centre o f the knee joint ( H - K ) and a line from the centre o f the knee to the centre o f the ankle joint (K-A).  2.5.2.2.9  W e i g h t - b e a r i n g axis - normal equals 50 +/- 10% ( H s u e t a l . 1990) A line is drawn from the centre o f the femoral head (left leg H - A ) to the centre o f the ankle joint. The weightbearing axis is where this line crosses the knee joint and is expressed as a percentage o f the total tibial joint surface. The distance i n millimetres from the lateral tibial-joint-line border to the weight-bearing line is divided b y the total joint width and expressed as a percentage. Numbers greater than 5 0 % are i n varus and those less than 5 0 % are i n valgus.  2.5.2.2.10  F e m o r a l neck/shaft angle - normal equals 135 +/- 5 ° (Pettersson and Ringertz 1991) The angle subtended b y a line drawn between the long axis o f the femoral neck (right leg B - H ) and the long axis o f the femoral diaphysis (right leg B - K ) .  61  F i g u r e 2.10 Measurement o f the weight bearing axis, the femoral neck/shaft angle, and the femoral anatomic angle.  2.5.2.2.11  S h a r p ' s A c e t a b u l a r angle equals 40 +/- 5 ° (Pettersson and Ringertz 1991) The angle subtended by a line drawn between the base o f the right and left acetabular teardrops ( C - E ) and a line j o i n i n g the tip to the lateral edge o f the F i g u r e 2.11 Measurement o f  acetabulum ( A - B ) .  2.5.2.2.12  Sharp's Acetabular angle.  F i b u l a r height - 50 +/- 10% (described in this study) Expressed as a percentage o f the distance from the p r o x i m a l tibial j o i n t line to the p r o x i m a l tip o f the fibula ( A ) over the distance from the p r o x i m a l tibial joint line to the p r o x i m a l fibular physis or physeal scar ( B ) .  F i g u r e 2.12 Measurement o f fibular height.  2.5.2.2.13  A n k l e j o i n t a n g l e - n o r m a l range equals 0 + / - 5 ° valgus (Hsu e t a l . 1990) The angle subtended by the lines drawn between the talar dome ( A - C ) and the perpendicular line to the long axis o f the tibia ( A - B ) .  F i g u r e 2.13 Measurement o f ankle joint angle.  62  2.6 Data Analysis Data was compiled as genotype and phenotype, and analysis was run on comparison groups as outlined below. 2.6.1 Genotype  Each affected individual was classified according to the following: 1.  Gene affected - EXT 1 or EXT 2  2. Type of mutation - missense (MS), nonsense (NS), frameshift (FS), or splice site (SS). 3. Severity of mutation - severe or mild; severe included NS, FS, and SS, and mild included MS. 4. Location of mutation - early or late; early mutation found prior to the 1500  th  base pair or late after the 1500 basepair in either EXT 1 or 2. th  5. Gender - male or female 2.6.2 Phenotype  Data were tabulated as clinical or radiographic for each affected individual. In total, 89 phenotypic parameters were collected. These were divided into three categories; lesion quality (38), limb alignment (26), limb segments (12 (x2 for left and right)) plus percentile height. Due to the large number of phenotypic features, a Pearson's correlation matrix (STATVIEW™ software) was run on the averaged data of all twenty-nine affected members to test association between any variables and to determine if any of the features were duplicated. If so, one of the variables would be eliminated as it could introduce potential statistical errors. 2.6.3 Genotype-phenotype correlation.  The genotype phenotype correlation analysis was based on comparison of the genotypic features versus the phenotypic features. For ease of presentation phenotype 63  features are grouped, called phenotype and represent the thirty-eight lesion quality parameters, the twenty-six limb alignment parameters, and the twenty-six limb segment parameters plus percentile height. All eighty-nine phenotypic features were evaluated versus the genotype. In the results, features showing significant differences are dissected out of the groups and discussed individually. The data analysis groupings are as follows; 1. Gene (EXT 1 vs. EXT 2) versus phenotype 2. Gene and gender versus phenotype 3. Gene and mutation type versus phenotype 4. Gene and severity versus phenotype 5. Gene and mutation location versus phenotype 6. Gender (male vs. female) versus phenotype 7. Mutation type (nonsense, missense, frameshift, splice site) versus phenotype 8. Mutation severity (severe{FS, NS, SS) vs. mild {MS}) versus phenotype 9. Mutation location (early{<1500bp} vs. late {>1500bp}) versus phenotype 10. Gender and mutation type vs. phenotype 11. Gender and severity vs. phenotype An unpaired t-test was calculated on all 2-way analyses, and an ANOVA was calculated when the analysis was greater than 2-way. Power was calculated for every comparison because of the large variation in sample size. In many instances, sample size was too small to warrant any statistical analysis. Statistical significance was set a priori at 0.05 and power of0.8. As this project was designed to determine if any correlation exists between the various parameters, the data was scrutinized in terms of looking for patterns. Statistical testing was therefore done on all comparisons in an attempt to dissect out a relationship between the different categories of comparisons. This project is meant to be a descriptive 64  study especially since the sample sizes are small in many comparisons and therefore the power not substantial. The significant correlations gleaned from this approach will then be isolated as parameters of interest for future prospective study.  65  Chapter III: Results 3.1 Subject Recruitment 3.1.1 subject identification Eleven probands and their families were provisionally diagnosed with HME. All interested members were informed of the study protocol and gave informed consent. All minors were consented for by their parents (a summary of all seventy-five study subjects follows in Table 3.1). Thirty-four individuals were found to have at least one exostosis and were deemed affected. However, proband 7-1 was later discovered to be the founder because her mother did not carry the mutation found in 7-1; and therefore, family 7 has been excluded. The final study sample includes ten families, ten probands, sixty-nine subjects, thirty-two affected individuals and 37 unaffected family members.  66  Table 3.1 Subject Recruitment ID  Position  Affected  Blood  Family 1  ID  Position  Affected  Blood  Family S  1-1  P  yes  yes  5-1  F  yes  yes  1-2  M  no  yes  5-2  M  no  yes  1-3  F  yes  yes  5-3  P  yes  yes  1-4  GM  no  yes  5-4  S  no  yes  5-5  GM  2-1  P  yes  yes  2-2  B  yes  yes  2-3  S  no  yes  6-1  2-4  F  yes  yes  6-2  2-5  M  no  yes  2-6  GM  no  yes  2-7  GF  no  yes  Family 2  Family 3  no  yes  no  yes  P  yes  yes  step B  yes  yes  6-3  M  yes  yes  6-4  F6-2  no  yes  6-5  F6-1  no  5-6 Family 6  Family 7  3-1  GM  yes  yes  7-1  P  yes  3-2  P  yes  yes  7-2  M  yes  yes  3-3  S  no  yes  7-3  GF  no  yes  3-4  F  yes  yes  7-4  S  no  yes  3-5  M  no  yes  7-5  s  no  yes  3-6  B  yes  yes  7-6  GM  no  yes  3-7  step S  no  yes  3-8  M  yes  yes  8-1  P  yes  yes  3-9  F  no  yes  8-2  M  yes  yes  3-10  S  yes  yes  8-3  B  no  yes  3-11  B  no  yes  8-4  F  no  yes  3-12  B  no  yes  3-13  F  yes  yes  16-1  P  yes  yes  3-14  M  no  yes  16-2  F  yes  yes  3-15  S  yes  yes  16-3  M  no  yes  3-16  B  no  yes  16-4  S  no  yes  3-17  S  no  yes  16-5  GM  yes  yes  3-18  M  yes  yes  3-19  P  yes  yes  17-1  P  yes  yes  3-20  S  no  yes  17-2  M  yes  yes  3-21  Aunt  no  yes  17-3  B  no  yes  3-22  M  no  no  17-4  B  no  yes  3-23  P  yes  yes  17-5  GF  yes  yes  3-24  S  no  no  17-6  GM  no  yes  Family 4  yes  Family 8  Family 16  Family 17  Family 18  4-1  M  yes  yes  18-1  P  yes  yes  4-2  F  no  yes  18-2  F  yes  yes  4-3  S  yes  yes  18-3  M  no  yes  4-4  P  yes  yes  18-4  B  no  yes  Abbreviations used: GM-grandmother; GF-grandfather; P-Proband; M-mother; F-father; Bbrother; S-sister  67  3.1.2 Family pedigrees  The extended family pedigrees are located in appendix 8.6.1  3.2 Genotype Results  3.2.1 Highly Polymorphic Repeats  Eight markers were designed to assist in assigning the most likely site of mutation in either EXT 1, 2 or 3. Initially A03/04 for EXT 1 and AO 1/02 for EXT 2 were used on all families. Enough information was gleaned from these two markers alone to assign EXT status to families 2, 3, 5, 8, 16, 17, and 18. Additional marker information (EXT 1, 85, and 547; EXT 2, 13 and 905; EXT 3,216 and 221) was required to further evaluate Families 4 and 6. The DNA from Families 4 and 6 were sent to Dr. Jacqueline Hecht M.D., Professor of Pediatrics at the University of Texas Medical School in Houston, Texas for more extensive linkage analysis. The results of the additional marker analysis are included.  68  Table 3.2  Family 1 16 18 6  Summary of STR Markers as per family and E X T gene assignment for mutations identified in EXT 1 and EXT 2  Exclusion Analysis EXT1 EXT 2 NI NI NI NI No Yes No No  Mutation Found Yes Yes Yes No  2 5 17 8 4  Yes Yes Yes No Yes  No No No No No  Yes Yes Yes Yes No  3  NI  NI  Yes  69  Gene Sequenced EXT 1 EXT 1 EXT 1 EXT 1 and EXT 2 EXT 2 EXT 2 EXT 2 EXT 2 EXT 1 and EXT 2 EXT 1 and EXT 2  Location of Mutation EXT 1 exon 2 EXT 1 exon 8 EXT 1 exon 1 None found EXT 2 exon 4 EXT 2 exon 4 EXT 2 exon 2 EXT 2 exon 7 None found EXT 2 exon 5  o ID: 1-2  ID: 1-3  EXT1 D8S555 1, 3 EXT 2 D11S903 c, c  2, a,  4 b  Exclusion analysis: not informative (NI)  ID: 1-1  EXT1 D8S555 1, 4 EXT 2 D11S903 c, a  Figure 3.1a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 1  62.1-1 • 1.SB #34 of 170 -  1-1 proband affected 1-2 mother unaffected 1-3 father affected  Figure 3.1b - Sequencer output for segregation analysis for Family 1. Mutation location: EXT 1 exon 2.  70  ID: 16-5  EXT1 EXT 2  D8S555 D11S903  1, 3 b, c  ID: 16-2  EXT 1 EXT 2  D8S555 D11S90  ID: 16-3  o  2, c,  1, 4 c, a  ID: 16.4  0  ID: 16-1  EXT1 EXT 2  Exclusion analysis: not informative (NI)  D8S555 D11S90  2, 1 c,  1, ? c,  Figure 3.2a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 16.  C A  C  16-1 proband affected  - 3 3 . 1 6 - 1 - 1 . 1 S A 0 1 3 O o f 161 « G A G f B l T A A G  A  C  A  R  C  R  A  A  C  C  A  A C  A  C  A  C"  R  G  B  T  A  A"  G  C  16-1 control unaffected A  A  i  H C H_ R C R  C  A  A  C  A  G  A  R  C L R  fd  T  T l  A  A  G  A  A C  C  fl B  G  A  A C  C  C  C l  16-4 sister unaffected A  A C  A  A  C  • 0 8 . 1 6 S - 1 . 1 5 A #85 o f 2G8 A G A R B I T W A G A  A R C A R C R G A R  N  A  C  C  C A  I UJ R G H fl C C C R  16-5  Figure 3.2b - Sequencer output for segregation analysis for Family 16. Mutation location: EXT 1 exon 8 71  o ID: 18-3  ID: 18-2  6  EXT1 EXT 2  D8S555 D11S90  I, 2 a, d  2, 3 c, c  Exclude EXT 2 because marker a was passed to an affected and an unaffected child.  IDID848-1  EXTl EXT2  D8S555 D11S90  Cannot exclude EXT 1 because affected father passed marker 2 to the affected child and marker 1 to the unaffected child.  2, 3 a, c  1, 2 a, c  Figure 3.3a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 18. 28.18.1 -MAKWofM  18-1 proband affected  18-2 father affected  I C T T C A A A 6 T C T A C 6 T A T A S C HA C A G C A A AA AG GG GAGAA A E C T ICflH H E T E T R C B T H TBS C C f l C H C C f i R HH RC C C C H G H H H  fCAAAGT C I i  i n  CTAC  GTATAC CHACAE CAAAAAGGGGA  cue  { I I II  HfitC  l  SMM-UHBMln  18-4 brother unaffected  ' - - •  18-3 mother unaffected  C A A A G T CTAC  .  "  Cy A[  •_  57.18-3-mm of 1 «  .i  T  GT A TA C C• A C A G CAAAAAGGGGA  t i n 1111 f t J T l 1 1 1 f I 11  11  i i 111  Figure 3.3b- Sequencer output for segregation analysis for Family 18. Mutation location: EXT 1 exon 1  72  ID: 6-3  ID: 6-4  D8S85  1,  4  3  4  D8S547  1  2  1  3  D8S555  3  2  3  1  D8S592  2  5  5  4  65CA  3  10  7  8  46  1,  3  3,  3  D8S522  4,  9  4,  9  ID: 6-1  Exclusion analysis: not informative (NI)  ID: 6-2  D8S85  4,  1  ?  ?  D8S547  3  1  1  2  D8S555  1  3  3  2  D8S592  5  2  5  J  65CA  7  10  1  10  46  3,  3  3,  3  D8S522  4,  9  4,  9  Figure 3.4a(i) EXT 1 STR Markers. Pedigree for Family 6. ID: 6-3  ID: 6-4  D11S903 2, D11S905 1, D11S903 3,  Exclusion analysis: ID: 6-1  D11S903 2, D11S905 1, D11S903 3,  ID: 6-2  4 2 3  1, 3, 2,  4 3 3  Figure 3.4 a(ii) EXT 2 STR Markers. Pedigree for Family 6.  73  not informative (NI)  ID: 2 - 5  ID: 2-4  EXT1 EXT 2  D8S555 1, 3 D11S903 a, b  I, c,  ID: 2-1  ID: 2-3  EXT 1 D8S555 EXT 2  Exclude EXT 1 because marker 3 from the affected father was passed to an affected and an unaffected child.  3,  2  D11S903 a,  d  ID: 2-2  1, b,  Cannot exclude EXT 2 because marker b from the affected father was only passed onto both affected children.  2 c  Figure 3.5a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 2.  2-3 sister unaffected  2-1 proband  A G R C  - 2-SA #133 of 163 • T C r A B l A E A A A G C C f l T R G f l f l R G  G T G G A T C T G I C G H C  G A C C A G G 6 T A A C R C C R G G G T R R  2-4 father affected  2-2 brother affected  I T ~  G G  G G  A R  T T  C T TT^f  T T  • 47.2-2 - 2.5A #132 of 163 • C f ' f A G A A A G G A C C C R ! f f G R H fl G G R C  C A C~R  G G  G G  2-5 mother unaffected  Figure 3.5b - Sequencer output for segregation analysis for Family 2. Mutation location: EXT 2 exon 4. 74  I i  IE): 5-1  EXT 1 EXT 2  ID: 5-2  D8S555 2, 2 D11S903 a, d  Can exclude EXT 1 because the affected mother passed marker 2 to an unaffected and an affected child.  I,  a-  ID: 5-4  ID: 5-3  EXT1 EXT 2  Cannot exclude EXT 2 because the affected mother passed an undistinguishable marker a to an unaffected and an affected child.  D8S555 2, 1 D11S903 d, a  2, a.  Figure 3.6a - EXT 1 and EXT 2 STR Marker. Pedigree for Family 5. 40 5-2 2 5B #81 of 181 T s T• • AT  5-2 mother unaffected 5-3 proband affected  5-1 father affected  5-3 proband affected reverse read  G C A A G G  • 37.5-1 - 2.5A #71 of 155 « C TA C M A T G T  C A G C A T  • 43.5.3 - 2-5A #133 of 215 > C G G C A A G G r / T A r B I * T(JT CA G C A T  CG  G C R R  G C C T H C N  R T G T Cfl  TC  G C R T T C I  5-4 sister unaffected  Figure 3.6b - Sequencer output for segregation analysis for Family 5. Mutation location: EXT 2 exon 4.  75  ID: 17-6  ID: 17-5  o EXT 1 D8S555 1, 4 EXT 2 D11S903 d, d  1, b,  2 d  ID: 17-2  EXT 1 D8S555 4, 1 EXT 2 D11S903 d, b  ID: 17-4  ID: 17-1 ,—I—, ID: 17-3  EXT1  D8S555  3, 1  EXT 2  D11S903 a, d  1, c,  4 b  1, c,  Can exclude EXT 1 because the affected mother passed marker 1 to both an affected and an unaffected child and marker 4 to an unaffected child. Cannot exclude EXT 2 because the affected mother passed marker d only to an affected child and marker b only to unaffected children. She also received marker d from her affected father.  1 b  Figure 3.7a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 17.  17-2 mother affected 17-1 proband affected 17-5 grandfather affected 17-3 brother unaffected 17-4 brother unaffected 17-6 grandmother unaffected Figure 3.7b - Sequencer output for segregation analysis for Family 17. Mutation location: EXT 2 exon 2.  76  II): 8-4  I D : 8-2  Exclusion analysis: EXT 1 D8S555 EXT 2  not informative (NI)  I, 2  D11S903 b, b  ID: 8-3  ID: 8-1  EXT1 EXT 2  D8S555 1, 1 DUS903 b, a  2, b.  Figure 3.8a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 8. •33.8-1 - 2 . 8 A # 1 9 1 of 251 •  A  T G C A G A  G A C A G B T A A G A G G  8-1 proband affected  36.8-3-2.8B #124 of 156  C  A G  A G  A C  A G P l T A  A G  A G  8-3 brother unaffected  • 34.8-2 - 2 . 8 B #106 o f 144 •  AC  A G l M T  A  A G  A G  8-2 mother affected  8-4 father unaffected  Figure 3.8b - Sequencer output for segregation analysis for Family 8. Mutation location: EXT 2 exon 7. 77  ID: 4-2  ID: 4-1  D8S85  2  1  1  3  D8S547  1  1  1  2 3  D8S555  1  3  2  D8S592  1  5  4  6  65CA  1  5  1  8  46  1  3  1  2  D8S522  9  8  6  4  Can exclude EXT 1 because D8S85 marker 1 from the affected mother was passed to both an affected and an unaffected child. Cannot exclude EXT 2 because ID: 4-4  ID: 4-3  o  passed to an unaffected child.  D8S547 D8S555 D8S592 65CA 46 D8S522  EXT 1 STR Markers. Pedigree for Family 4. ID: 4-2  D11S903  ID: 4-1  2  D11S905  1  D11S1313  1  D11S903  2  ID: 4-4  ID: 4-3  D11S903  Figure 3.9a (ii)  from the affected mother was passed to an affected child  while marker 3 was  D8S85  Figure 3.9a (i)  D11S903 marker 1  2  D11S905  1  D11S1313  1  D11S903  2  EXT 2 STR Markers. Pedigree for Family 4.  78  •  o  o f  ro  • -o . N  —  "a -a  • •a l-H  I  "O  Vi  f-i 0  o  •a —  u  H CN  •o  ro  "a  X W  T3  •  H  X  •O  o  H  W  «  OS  s-  bo  •  <N  bo  s 00 00  Q  ro xo wo 00 00  Q  wo wo CO 00  On  ~, ~-i  Q  Q  79  ro Ov Co ~»  Q  3-21 24af»o>232  3-21 Aunt unaffected • 3 * 2-6» »13 ot2*0  H H W W f f f H f i H iii 11 3-8 Mother affected  3-3 cousin unaffected  H I t Hf £1 Iff t f H H 111  til  HH 1  • 3-6 2-6a *28 of 233 •  3-6 proband affected  tt 1 i Hit i*ttl  • 3-15 2 « a »17 of 221 •  ' T 6 T A6 T C N C  3-15 cousin affected  t i l 1 til  GBl A A T A t T T C C T C  iii  11 ill  T G T C A T C T C A  T M R t I C s C if S B R I R C 1 1 CC I C t C t C f i t C t C R  3-7 cousin unaffected  Figure 3.10b Sequencher output for segregation analysis for Family 3. Mutation location: EXT 2 exon 5.  80  3.2 Mutation Identification and Segregation Table 3.3 Mutations identified in each proband. Family  EXT gene  Mutation  Exon  1  1  G1019A  2  2  2  G730T  4  3  2  C751T  5  4 5  2 2  ?  -  G679A  4  6 8 16 17 18  1 2 1 2 1  9  -  -  -  G1174A G1723C 455del4 C357G  7 8 2 1  -  Splice Site Splice Site Frameshift Nonsense  Amino Acid Change  RtoH Arginine to Histidine Basic to Basic EtoX Glutamic acid to Stop QtoX Glutamine to Stop -  Type  Missense  No  Nonsense  Yes  Nonsense  Yes  -  D to N Missense Aspartic acid to Asparagines Acidic to uncharged polar  Premature Stop at 1293 YtoX Tyrosine to Stop  Unique  -  No Yes Yes Yes Yes Yes  Once the mutations were identified in the probands confirmation of segregation was done as described in the Methods section. The Sequencher files can be reviewed in the previous section. These files confirm the appropriate identification of mutations in affected family members and the lack of mutation in the unaffected members. All family members plus controls and the GenBank sequence were tested in the same contigs. The summary of genotyping is as follows and can be reviewed in table 4.3; All 10 families were assessed for linkage to either EXT 1 or 2 (4 EXT 1, 6 EXT 2). Eight of these 10 families had their mutation identified. Six of these eight mutations are novel and all mutations were unique to each family. Two mutations have been previously reported in the literature (Family 1 and 5). There were three nonsense, two missense, two splice site and one frameshift mutation. All mutations segregated appropriately in that those with exostoses carried the mutation and were heterozygotes at that location and those who were unaffected did not carry the mutation and had no sequence varience at that location 81  consistent with the Genbank sequences. Mutations in family 4 and 6 could not be identified despite sequencing both genes for two affected family members. Intronic and promoter sequences however were not sequenced. As well, very large deletions, for example an entire exon may have also been missed as the software would not pick up a heterozygozity if an entire reading frame was missing.  3.3 Phenotype Results  In the ten families represented in this study there were 32 affected individuals. Two families (4 and 6) with 6 subjects, did not have their mutation identified and therefore their data is not included in the genotype-phenotype analysis.  3.3.1 Phenotype data  Every affected individual that participated in the study including those members from families 4 and 6 completed the clinical and radiographic examinations. Save for a few data points the phenotype files were complete for every affected participant. The core data files are located in Appendix 8.6.4.1. The data includes 38 lesion quality items (8.6.4.1.1), 26 limb alignment items (8.6.4.1.2) and 25 limb segment items (8.6.4.1.3) for a total of 89 items per subject.  3.3.2 Range of Motion  Range of motion at the shoulder, elbow, forearm, wrist, hip, knee and ankle were essentially within normal limits for all subjects. In the cases of radial head dislocations in one family member of family 3 and one of family 18 there was reduction in forearm pronation and supination but the functional range was preserved (arc of 90 degrees). As 82  there was little effect on the clinical examination or functional range of motion, range of motion is therefore not included in this thesis, nor is this data analyzed in relation to genotype. 3.3.2 Pearson correlation matrix  All eighty-nine phenotypic parameters were placed on the x and the y-axis of the correlation matrix. Some of the limb segments correlated well but as there were so few correlations that were deemed duplicate (R> 0.8) all features were treated as separate items and therefore interpreted independently including sidedness. Appendix 8.6.4.1 contains the matrix in its entirety.  3.4 Genotype-phenotype Correlations  The data sets are based on 26 affected individuals who had both complete genotype and phenotype data. Table 3.4 outlines the breakdown of the number of subjects per category as well as the age distribution.  83  Table 3 . 4 Breakdown of Genotype Features Genotype  N u m b e r of  Feature  subjects  EXT 1 EXT 2  7 9  Male  14  Female  12 4 14  MS NS  Severe  3 5 4 22  Early  19  Late  7 4 3 2 5 2 3 2 10 9 17  FS SS Mild  E X T 1 Male E X T 1 Female E X T 1 Severe E X T 1 Mild EXT 1 MS E X T 1 SS E X T 1 NS E X T 2 Male E X T 2 Female E X T 2 Severe  E X T 2 NS  2 2 2 12  M a l e s severe  12  Males mild  2 2 8 2 2 10 2 2 6 3 1  E X T 2 Mild EXT 2MS E X T 2 SS  Males M S Males N S Males SS Males F S F e m a l e s severe Females mild Females M S Females N S Females SS Females F S  D i s t r i b u t i o n o f ages at time o f s t u d y  Family 4  Family 6  9, 14, 14,44,47, 55, 72 7, 7, 10, 11, 14, 14, 15, 15, 16,31,36, 38, 39, 44, 45,46,47, 70, 74 10, 11, 14, 14, 14, 15, 15, 39,44,44, 45, 47, 55, 73 7, 7, 9, 14, 16, 31, 36, 38, 46,47, 70, 72 7, 9, 39, 47 7, 10, 14, 14, 14, 15, 15, 36, 38, 44,46, 47, 55, 70 16,45, 73 11, 14,31,44, 72 7, 9, 39, 47 7, 10, 11, 14, 14, 14, 14, 15, 15, 16,31, 36, 38, 44, 44,45, 46, 47, 55, 70, 72, 73 7, 7, 10, 14, 14, 14, 15, 15, 16, 36, 38, 39, 44, 45, 46, 47, 55, 70, 73 9, 11, 14,31,44,47, 72 14,44, 47, 55 9, 14, 72 14, 14, 44, 55, 72 9, 47 9, 47 14, 44, 72 14, 55 10, 11, 14, 14, 15, 15,39,44,45, 73 7, 7, 16,31,36,38,46, 47, 70, 7, 10, 11, 14, 14, 15, 15, 16,31,36,38, 44, 45, 46, 47, 70, 73 7,39 7,39 11,31 7, 10, 14, 14, 14, 15, 15, 36, 38, 46, 47, 70 10, 11, 14, 14, 14, 15, 15, 44, 44, 45, 55, 73 39, 47 39, 47 10, 14, 14, 14, 15, 15,44,55 11,44 45, 73 7, 14, 16, 31, 36, 38, 46,47, 70,72 7,9 7,9 7, 36, 38, 46,47, 70 14,31,72 16  -  3  84  3 -  2  3  1  Phenotype parameters were grouped into lesion quality, limb alignment and limb segments. To simplify the presentation of the data they are dubbed "phenotype". From the literature review and the author's clinical experience numerous possible genotypic factors could potentially influence phenotype. Foremost was whether the EXT 1 or the EXT 2 gene mutations had a more severe clinical presentation. EXT genes were evaluated separately and then combined with other factors that were thought to potentially influence or modify the phenotype. These relationships dictated the 5 first comparisons as listed below. Mutation type (missense, frameshift, splice site and nonsense) was looked at independently and as severity of mutation (truncating (ns, ss, fs) = severe and nontruncating (ms) = mild). Different types of mutations are often found to have different influences on the gene product and therefore the gene's function. As noted in the introduction, truncating mutations prevent localization of the EXT gene product to the endoplasmic reticulum (ER) whereas missense or nontruncating mutations result in the gene product being present in the ER. However both mutation types prevent heparan sulfate presentation on the cell surface. The question remains whether there is some preservation of EXT gene function when the product still localizes to the ER, which would then possibly result in differing phenotypes. Gender was also analyzed as there is an anecdotal opinion that males have more severe disease (Solomon et al. 1963). This may be explained by the 100% penetrance in males and 96% in females (Schamle et al. 1998, Raskind et al. 1998), or that other growth factors are influencing tumour growth. This parameter was therefore tested as well to corroborate this unfounded opinion. The last factor looked at was the location of the mutation. The last 780 base pairs of both EXT 1 and 2 genes is the carboxy terminus, which is highly conserved in EXT 1 and 2 and also the EXTL genes. Wuyts (Wuyts et al. 2000) believes given the conservation 85  of such an area, and given the ubiquitous presence of the EXT genes in human tissue, it is possible that other sources (specifically the EXTL genes (Wuyts etal. 2000)) may back up the function of the carboxy terminus thereby resulting in a milder phenotype. This suggestion is both highly speculative and paradoxical, as most highly conserved regions are crucial to function. Interestingly the fewest mutations are found in the last 780 bps (2/44 in EXT 1 and none in EXT 2). Attempts were made to look at the mutation from an early and late aspect based on most of the mutations being located prior to the 1500 base pair (bp). However given that it is only the last 780 bps that are involved in the highly conserved area and none of the mutations in this study were located so late in the gene one would expect to see no difference in these mutations. At the same time few mutations are seen beyond exon 8 (approximately at base pair 1500 for EXT 1 and 2), as can be confirmed by reviewing Figures 1.10 and 1.11 for either gene, that possibly a difference in phenotype would occur. All the following comparisons were tabulated and are found in the indexed Appendix. Gene versus phenotype  Appendix 8.7.1.1-3  Gene and gender versus phenotype  Appendix 8.7.6.1 -3  Gene and mutation type versus phenotype  Appendix 8.7.7.1 -3  Gene and severity versus phenotype  Appendix 8.7.8.1 -3  Gene and mutation location versus phenotype  Appendix 8.7.11.1-3  Gender versus phenotype  Appendix 8.7.2.1 -3  Mutations type versus phenotype  Appendix 8.7.3.1 -3  Mutation severity versus phenotype  Appendix 8.7.4.1 -3  Mutation location versus phenotype  Appendix 8.7.5.1 -3  Gender and severity versus phenotype  Appendix 8.7.9.1 -3  Gender and mutation type versus phenotype  Appendix 8.7.10.1-3  86  After observation of the data set lesion quality, certain features consistently showed tendencies towards differences in the various comparisons. Specifically, the average number of lesions influenced all other features and therefore percentages were looked at to standardize the data. In the lesion quality category certain items that were observed to have specific interest, or noted in the literature review were highlighted. These included: average number of lesions, size (small, medium, large), percent pedunculated lesions, percent sessile lesions, percent pelvic lesions, percent metaphyseal flaring and percent flat bone involvement. These items are highlighted below. With regards to limb alignment, there were twenty-six items recorded for every subject. An item was categorized as abnormal if the value measured by xray analysis was greater than one standard deviation outside the published norm. The data is presented as the number of abnormal measurements (the average of each comparison group was used) out of twenty-six possible parameters. Limb segment results were influenced by the percentile height, such that the shorter the subject was overall, the shorter the separate segment length. Tables 3.5, 3.6 and 3.7 summarize the patterns of phenotype versus genotype. Table 3.5 summarizes the gene comparison analysis and covariant analysis data. Tables 3.6 and 3.7 summarize the mutation type, severity and location analysis and the gender covariant analysis. Only the data showing a trend or significance is included in these tables for clarity sake. Complete analysis of the data can be found in Appendices 8.7.1.1 through 8.7.11. Specific details of all comparisons is included in the text following.  87  Table 3.5 Summary of Results for Comparison between E X T 1 and E X T 2 Genes E X T 1 vs. E X T 2 Comparisons E X T 1 vs. E X T 2 (Appendix 8.7.1) Gene and Gender (Appendix 8.7.6)  Gene and Mutation Type (Appendix 8.7.7)  # lesions  % Pelvic  1>2 p < 0.01 power .82 1M> 1F> 2M>2F p < 0.01 gene and gender  1>2 p < 0.01 power .68 1M>1F> 2M>2F p<0.01 gene  INS > ISS > 1NS> 2NS > 2SS >2MS> 2FS n/s  1NS> 1MS> 1SS>2SS >2NS> 2MS> 2FS n/s  % Flatbone 1 >2 p<0.01 power .91 1M>2M> 1F>2F n/s  % Flared n/s  1M>2M> 1F>2F p < 0.02 gender  1NS> IMS > ISS >2SS> 2NS> 2 M S > 2FS n/s  L i m b Alignment 1>2 (17 vs 10) n/s 1 M > 2 M > 1F>2F (16 > 13 > 10 > 8)  % Height 1<2 p < 0.01 power .8 1F< 1M < 2M<2F p<0.01 gene  1NS> 1 S S > 2 M S > (15) (14) (14) 2FS > I M S > 2NS > (12) (11) (9) 2SS  1NS> 1MS>2 F S > ISS >2SS> 2MS> 2NS n/s  (9) Gene and Severity (Appendix 8.7.8)  Gene and Mutation location (Appendix 8.7.11)  1S> 1M> 2S>2M n/s  1S> 1M >2S> 2M n/s  1S> 1M> 2S>2M n/s  1S>2S> 1M>2M (15 > 14 > 11 > 7)  1E>2E p< 0.0021 power .95 1 E > 1L  1E>2E p < 0.001 power .99 1 E > 1L 2E<2L  1E>2E p < 0.001 power .99 1 E > 1L 2E<2L  1E= 1 L > 2 E > 2 L (15 = 15 > 13 > 10)  1M< IS < 2M<2S n/s  1 E > 1L  1E< 1L< 2L< 2E n/s  Abbreviations used: For gene comparison 1 - EXT1 and 2 - EXT2; for gender M - males and F females; for mutation type, MS - missense mutation, NS - nonsense mutation, FS - frameshift mutation, and SS - splice site; for mutation severity, S - severe mutation and M mild mutation, E early, L late n/s Difference seen but not statistically significant; — no difference seen  3.4.1 Gene versus phenotype (Appendix 8.7.1.1-.3)  Subjects with EXT 1 mutations had more lesions than those with EXT 2 mutations, 32.7, versus 19.1 (p-value 0.0036). EXT 1 subjects have more percent pelvic lesions, 9.6 versus 2.3 (p-value 0.012) and more involvement of the flat bones, 11.8% versus 30 .%> (pvalue 0.0019). There were no differences noted between EXT 1 and 2 in terms of size, percent pedunculated versus percent sessile, percent complex versus percent simple or 88  percent metaphyseal flaring. EXT 1 subjects had more mal-alignment than subjects with EXT 2 mutations, 17 versus 10 of 26 possible parameters. EXT 1 subjects were shorter than EXT 2 subjects, 9.3 percentile versus 42.5 percentile (P-value .0081) and the overall upper extremity length (right and left) was shorter for EXT 1 patients (p-value 0.026, right and 0.027 left). Even though there were no significant differences in the remaining 10 segments measured, EXT 1 subject's measurements were always less than those of EXT 2 subjects.  3.4.2 Gene and gender versus phenotype (Appendix 8.7.6.1)  In general (not exclusively nor statistically significant in all cases) the following were noted; EXT 1 was worse than EXT 2 (See table 3.5), when further subdivided males were worse than females, nonsense mutations were worse than splice site which were worse than frameshift which were worse than missense; and severe mutations were worse than mild ones. In detail, EXT 1 males have more lesions, 37.3, than EXT 1 females, 26.7, who had more than EXT 2 males, 24.0, who had more than EXT 2 females, 13.6. This is significant with regards to both gender (p-value 0.0032) and gene (p-value 0.0011). The same pattern exists when looking at percent pelvic lesions and percent flat bone involvement but it is only significant with regard to gene (p-value 0.015 and 0.0026) and not gender (p-value 0.51 and 0.52); %flared, EXT 1 male, 54.6, EXT 2 male, 40.6, Ext 2 female, 18.9, EXT 1 female, 17.3; % pelvic, EXT 1 males, 11.3, EXT 1 females, 7.3, EXT 2 males, 2.5, EXT 2 females, 2.1. EXT 1 and 2 males have more metaphyseal flaring than females and by gender the p-value is 0.0097. The pattern of mal-alignment also reflects males being worse than females with EXT 1 males having 16 of 26 parameters abnormal, EXT 2 males 13, EXT 1 females 10 and EXT 2 females, 8. Percentile height showed EXT 1 females to be 89  the shortest, 5 percentile, then EXT 1 males, 12.5 percentile, followed by EXT 2 males, 40 percentile and EXT 2 females at the 45 percentile. This was significant for gene (pth  th  value 0.011) but not gender (p-value 0.79). If you were to cross reference to number of lesions it is as follows respectively; 26.7, 37.3, 24, 13.6.  3.4.3  Gene and mutation type versus phenotype (Appendix 8.7.7.1-. 3)  EXT 1 missense had more lesions, 27.0 than EXT 2 missense, 15.0 (p-value 0.013), and EXT 1 nonsense also had more lesions, 43.5, than EXT 2 nonsense, 19.4, (p-value 0.0071) but the splice site mutation numbers between EXT 1 and 2 were similar. Further EXT 1 nonsense (43.5) mutations followed by EXT 1 splice site (29.3) had more lesions than EXT 1 missense (27). Similarly, in its series EXT 2 splice site (25.5) then nonsense (19.4) and then missense (15) followed by frameshift (11). This relationship (EXT 1 worse than 2) also held true for percent pelvic lesions and percent flat bone involvement. That is to say EXT 1 is significantly more involved than EXT 2. But again no difference was noted in the EXT 1 and 2, splice site subjects. More specifically when looking within a group for % flat bone the data is for EXT 1; nonsense 19.3, missense, 9.2 then splice site, 8.4. For EXT 2; splice site 6.1, nonsense 3.3 and missense and splice site 0. With regards to limb alignment EXT 1 missense mutation subjects had more abnormal values, 15, than EXT 1 splice site, 14, which had more than EXT 2 missense, 14, than EXT 2 frameshift, 12, followed by EXT 1 missense, 11, and EXT 2 nonsense and splice site at 9 each. When evaluating percentile height, EXT 1 was always shorter than EXT 2 with respect to the same mutation type. This was statistically significant only with respect to nonsense mutations (p-value 0.026). EXT 1 missense, nonsense, and splice site  90  were shorter than any of the EXT 2 mutation types except for the only frameshift identified in EXT 2.  3.4.4  Gene and severity versus phenotype (Appendix 8.7.8.1-.3)  EXT 1 severe, 35, and mild, 27, mutations had more lesions than their EXT 2 counterparts, 20.9 and 15 respectively (p-value 0.012 and 0.014). EXT 1 severe, 11.3, and mild, 5.4, mutations had more involvement of the pelvic bones than EXT 2, 3.2 and 0.0 (pvalues 0.017 and 0.42). Similarly EXT 1 severe, 12.9, and mild, 9.2, mutations involved the flat bones more than the EXT 2 mutations, 3.9 and 0.0 respectively (p-value 0.0081 and 0.026). Alignment data showed EXT 1 severe had more abnormal values than EXT 2 severe, 15 versus ?, but EXT 2 mild had more mal-alignment than EXT 1 mild mutation subjects, 14 versus 11. However the EXT 2 mild data was only from one individual for most parameters. As for percentile height EXT 1 severe, 11.4 and mild, 4.0, were shorter than EXT 2 severe, 42.9 and mild, 39.0 (p-values 0.035 and 0.28 respectively).  3.4.5 Gene and Mutation location versus phenotype (Appendix 8.7.11.1-.3)  When comparing EXT 1 early versus EXT 2 early, EXT 1 early had more lesions, 43.5 vs. 18.3 (p-value O.0021) more pelvic bone involvement, 18.4 vs. 1.8 (p-value < 0.001) and more flat bone involvement, 19.3 versus 2.6 (p-value O.001). There were no differences or even trends towards differences between EXT 1 and EXT 2 late mutations. When looking at EXT 1 independently early mutations tended to have more lesions, 43.5 vs. 28.4, more pelvis involvement, 18.4 vs. 6.1, more flat bone involvement, 19.3 vs. 8.8 and more metaphyseal flaring, 61.3 vs. 29.6. This is in contrast to EXT 2 where the early mutations had fewer pelvis lesions, 1.8 vs. 6.1 and less flat bone involvement, 33.5 vs. 73.  91  Limb alignment data showed EXT 1 early and late mutations to have the most malalignment with 15 abnormal parameters each followed by EXT 2 early mutations and then EXT 2 late mutations. Limb segment abnormalities were confined to percentile height where EXT 1 early subjects were the shortest at the 3 percentile, followed by EXT 1 late mutations at the 11.8 rd  th  percentile, then EXT 2 late mutations at the 25 percentile and finally EXT 2 early mutations at th  the 44.6 percentile. th  Table 3.6 Summary of Results for remaining unvariant data Comparisons  # lesions  %  Pelvic  %  Flatbone  %  L i m b Alignment  %  Height  Flared  M a l e s vs. Females (Appendix 8.7.2)  M >F  F >M (12 vs 9)  p < 0.01  n/s M S vs. N S vs. S S vs. F S (Appendix 8.7.3)  M S > SS = F S > NS (13 > 1 2 = 1 2 > 11)  F S < M S < SS < NS  p<0.01  n/s Severe vs. M i l d (Appendix 8.7.4)  Severe = M i l d (11 vs 11)  M i l d < Severe  n/s  n/s Early vs. Late (Appendix 8.7.5)  Early = M i l d (11 vs 11)  Late < E a r l y  n/s  n/s  Abbreviations used: n/s - Difference seen but not statistically significant; — no difference seen; MS - missense mutation; NS - nonsense mutation; SS - splice site; FS -frameshift mutation  3.4.6 Gender versus phenotype (Appendix 8.7.2.1-.3)  Male subjects had more lesions than females, 28.1 versus 17.2 (p-value 0.01) and males had more metaphyseal flaring than females, 45% versus 18.5% (p-value 0.01) while females had less flaring than males, 81.5% versus 55% (p-value .0079). No differences were noted in any of the other lesion quality items. Males had nine of twenty-six abnormal alignment parameters and females had 12. There was no difference with respect to percentile height or the 12 segments measured between males and females.  92  3.4.7 Mutation type versus phenotype (Appendix 8.7.3.1-.3)  Missense mutations had the highest percentage of small lesions, 48.5% (p-value 0.045) and splice site mutations had the highest percentage of large lesions, 48.5% but this was not statistically different than the other mutation types. Though not statistically significant, splice site mutation subjects also had the highest percentage of pelvic lesions and flat bone involvement. There were no differences between the four mutation types with respect to mal-alignment. Frameshift subjects, represented by one family, were the shortest at the 9.7 percentile and nonsense mutation subjects, represented by 3 families, were the th  tallest, 51.3 percentile (p-value 0.048). rd  3.4.8 Mutation severity versus phenotype (Appendix 8.7.4.1-.3)  No differences were identified between severe and mild mutations. Both groups had eleven of twenty-six abnormal mal-alignment parameters and there were no significant differences in limb segment features except mild mutation subjects were consistently shorter in all characteristics.  3.4.9 Mutation location versus phenotype (Appendix 8.7.5.1-.3)  There were no differences noted in any of the thirty-eight lesion quality items when comparing early and late mutations. There were the same number of mal-alignment abnormalities between mild and severe mutations, eleven of twenty-six. Subjects with a late mutation were shorter than those with an early mutation. Limb segments and percentile height were not significantly different between the two groups.  93  Table 3.7 Summary of Results for Comparison between Males and Females Covariant data Male and Female  # lesions  Comparisons  %  %  Pelvic  Flatbone  % Flared  Limb  % Height  Alignment  Gender and Severity (Appendix 8.7.9)  M s>M m> F m > Fs p < 0.01  M s> M m> F m > Fs p < 0.01  F m>M m > (12) (11) M s> Fs (10) (9)  M m smaller than the rest n/s  Gender and Mutation Type (Appendix 8.7.10)  M M F M F  M M M F F  F fs > M ss > (16) (14) F ss > M fs > (13) (12) M ms > F ms >  F M F M  ns > F ns > ss > M ns > ss > F fs > fs > F ms > ns  ns > F ns > ns > M ss > ms > M fs > ms > F ns > ss > F fs n/s  fs < M fs < ms < F ss < ms < M ss < ns < F ns n/s  (11) (11) M ns > F ns (9) (9)  Abbreviations used: For gene comparison 1 - EXT1 and 2 - EXT2; for gender M - males and F females; for mutation type, ms- missense mutation, ns - nonsense mutation, fs - frameshift mutation, and ss - splice site; for mutation severity, s - severe mutation and m mild mutation n/s - Difference seen but not statistically significant; — no difference seen  3.4.10 Gender and Severity versus phenotype (Appendix 8.7.9.1-.3)  Males versus females with severe mutations showed a significant difference in regards to lesion number, 29.9 versus 17.1 (p-value 0.0061), but in comparison to males and females with mild mutations there was no difference with 21 lesions each. The only remaining difference noted was again between males and females with severe mutations for percent flared metaphyses, 46.1 and 17.4 (p-value 0.0049) and the converse held true where females with severe mutations had the least flared metaphyses, 81.3 versus 53.9 (p-value 0.0075). Trends existed where males had more sessile lesions and females more pedunculated lesions. Females with a mild mutation had more limb mal-alignment, 12, followed by males with mild mutations, 11, males with severe mutations, 10 and females with severe mutations, 9, last. There was no difference in the overall percentile height for this grouping. Males with missense mutations had the greatest shortening, 10 percentile. th  94  3.4.11 Gender and mutation type versus phenotype (Appendix 8.7.10.1'-.3)  Males with splice site mutations had the most number of lesions at 34.5, versus males with nonsense mutations who had 30.4. The remaining groupings had about the same number of lesions. The only significant difference noted was between male and female nonsense subjects (p-value 0.005). Males for all mutations had more sessile lesions whereas females had more pedunculated lesions, but this was not significant. Males in all categories showed more flaring than females. Mal-alignment was not different between the males and females for each mutation type. Females with frameshift mutations (1 subject) had 16 abnormal parameters, followed by males and females with splice site mutations, 14 and 13 respectively, then males with frameshift mutation, followed by missense where both genders had 11 and for the nonsense subjects, 9. As for height the one female frameshift patient was the shortest at the 8 percentile followed by males with misssense and frameshift mutations at the 10 percentile, the females with splice site, 12 percentile, and th  th  the rest were greater than the 30 percentile. th  95  Chapter IV: Discussion  4.1 Subject Recruitment HME is a relatively rare disorder and this is reflected in the sample size assembled (69 total participants), ten families with thirty-two affected individuals. Access to extended family members is also limited in Canada due to our multinational population and the geography. As such, family members often reside at great distances and are unavailable for recruitment. Despite this, a satisfactory sampling of all the families was obtained for a pilot project designed to determine if a trend exists between genotype and phenotype. Only one family, Family 3, was of sufficient size (36) and subjects available (24) for analysis of intra-familial correlations (the data can be found in appendix 8.6.4.1). Family 1 was the smallest with only 4 participating members. Due to the limited sample size this thesis is designed to explore correlations between phenotype and genotype. Statistical testing, paired t-tests and ANOVA where appropriate, were used to assist the observational analysis of the data. The relations being tested between genotype and phenotype generated a large number of p-values, which were used only to focus the attention on any pattern generation as opposed to determining statistical significance. Consistent patterns were identified to generate hypotheses of association that will be tested in future larger collaborative studies.  4.2 Genotype Previous studies (Cook et al. 1993; Blanton et al. 1996; Legeai-Mallet et al. 1997; Wuyts et al. 1995; Wuyts et al. 1998; Philippe et al. 1997; Xu et al. 1998; Seki et al. 2001) have identified from 30 to 100% of the mutations in the families studied. This study found 80% or eight out of the ten family's mutations. The remaining 2 families had multiple HPR studies done resulting in Family 4 having a high probability of a mutation in EXT 2 and Family 6 with an 96  EXT 1 mutation. However despite sequencing both genes twice for the proband and then once for another affected family member no mutation could be identified. As the purpose of this study was to explore correlations between phenotype and confirmed genotypes these six subjects were excluded. The 8 families included had their mutations identified and confirmed as described above in section 3.4 and segregation analysis confirmed only affected family members carried the mutation and were heterozygous at the locus of interest. One silent polymorphism was also discovered in EXT 1 in exon 9 as G1761 A. This was noted in five of eight subjects sequenced at this locus and was compared to the GenBank sequence of GAG. There was no change in the amino acid as both GAG and GAA code for glutamic acid. Thirty percent of the ten families had mutations in EXT 1 and 50% were in EXT 2, while 20% remained unidentified. This is in contrast to the overall reported mutations where 36% are in EXT 1, 27% in EXT 2 and 36% unidentified (section 2.2.4.1 and 2.2.4.2) (Philippe et al. 1997). In this study 3 of 8 mutations (37.5%) were identified in EXT 1 and 5 of 8 (62.5%) were in EXT 2. If the two families with unidentified mutations are included based on their linkage analysis alone then 40% are found in EXT 1 and 60% in EXT 2. The ratio of EXT 1 to EXT 2 in this population is therefore 2:3 in contrast to the literature where the ratio of EXT 1 to EXT 2 mutations is 2:1. It is likely these differences relate to the small sample size available in reported studies as well as this study. It would appear the previously reported ratio is suspect and requires further study. Many of the previous studies have looked at primarily one race. Seki looked at Japanese families where the ratio of EXT 1 to 2 was 3:1 (Seki et al. 2001), Xu looked at Chinese families where this ratio was 7:1 (Xu et al.1998), Wuyts (Wuyts et all998) looked at a variety of nationalities including European and Middle Eastern families and found a ratio of 1:1 and Phillipe (Phillipe et al. 1997) looked at French families and found a ratio of 2.5:1. This study 97  (EXT 1:2, 3:5) includes a number of ethnic groups including, East Indian (EXT 1), Welsh (EXT 2), Austrian (EXT 2), Japanese (EXT 2), German (EXT 1 and 2), and British (EXT 1 and 2). It is likely that once enough races and cultures are evaluated the ratio between EXT 1 and 2 may be 1:1. The mutations identified were in keeping with those found in the literature in terms of the type. The literature suggests frameshift mutations are the most common and yet it was the least common in this study. However this sample size is likely a skewed sampling simply because of the small size. The most common mutation in this series is the nonsense mutation (3), followed by splice site and missense (2 each) and one frameshift. Most mutations quoted in the literature occur in the early half of EXT 1, 80% (Table 2.1), and EXT 2, 93% (Table 2.2). Similarly in this study 67% of EXT 1 mutations occur in the first half of the gene and 100% of the EXT 2 mutations occur in the first half. In summary the mutational profile with respect to gene effected, mutation type, mutation location and mutation severity are in keeping with what is reported in the literature as of January 2003. The mutations identified were not unique in two of eight families. Family 1 carries an EXT 1 G1019A missense mutation and has previously been described by Raskind (1998) and Seki (2001). This base change causes a change in the amino acid from arginine to histidine, which are both basic. However the amino acid change is sufficient to cause a conformational change in the EXT 1 protein thereby precluding its function and ultimately the presentation of heparan sulfate on the cell surface. This was confirmed previously by Raskind (1998). The second previously described mutation was also a missense mutation and was found in Family 5. The base change was in EXT 2 G679A causing an aspartic acid, which is acidic to be replaced by, an asparagine, which is uncharged polar. This mutation has been previously described by Phillipe (1997) and here again this work showed that the amino acid change ultimately caused alteration in the EXT 2 protein sufficient enough to result in exostosis formation. 98  [Phenotypically, these 2 missense mutation families were indistinguishable from the other mutation types other than a slight tendency for them to be more malaligned (13/26 versus 12/26), and slightly shorter (21 versus 27 percentile average for the others). No other features of the st  th  89 were significant of any trend. However when looked at in the context of EXT 1 and 2 missense mutation phenotypes were always milder than nonsense and generally milder than the other truncating mutation types table 3.5 for the highlighted phenotypic features]. The remaining six mutations were unique. Three were nonsense mutations resulting in early stop codons. This, as McCormick (1998) has shown, results in a protein which is truncated and does not localize to the endoplasmic reticulum and therefore no heparan sulfate presentation on the cell surface. Two of the mutations were found in EXT 2 and one in EXT 1. One frameshift mutation was identified in EXT 2 and caused an early stop codon downstream. This would have a similar effect as a nonsense early stop. There were two splice site mutations, one both in each EXT 1 and 2. The one located in EXT 2 (Gil74A) was located in intron 7 at the 5' splice site in the first intronic position. Interestingly this is one base pair further along than the 1173 +1G-»A that Wuyts described in 1998 (1998). Both however cause the first base pair in the intron to be an adenine instead of guanine resulting in splice site malfunction. The mutation found by Wuyts occurred in a Dutch family and in this study the mutation occurred in a German family. Confirmation of this splice site mutation was done by Wuyts, by amplifying the 5' splice site with custom designed primers to flank the region. The wild-type PCR fragment contains an ScrFI restriction site where the mutant allele of this splice site does not. The end result causes a skipping of exon 7 and leads to a truncated protein (Wuyts 1996). It is likely the mutation found in this study has an identical effect.. The EXT 1 splice site mutation occurred in intron eight. It too was located at the first intronic position at the 5' splice site. There have been no other described mutations in the area of 99  this splice site mutation. There is likely a downstream stop codon resulting in a truncated product. It was beyond the scope of the present study to fully describe the actual end result of such a mutation. Suffice is to say, multiple exostoses were still the ultimate outcome. Mutations caused by nonsense, splice site and frameshifts result in truncated proteins. This results in the complete absence of the EXT proteins in the endoplasmic reticulum and ultimately no hetero-oligomeric complex in the Golgi apparatus. On the other hand missense mutations are altered as a result of an amino acid change but the EXT product nonetheless locates to the endoplasmic reticulum. It has not been shown how this affects the heterooligomeric complex in the golgi apparatus, only that there is still no identification of heparan sulfate on the cell surface in in vitro studies. These two findings were identified by McCormick (1998) and are now universally accepted. It is therefore fair to suggest that missense mutations have a mild effect on the localization of the EXT protein whereas nonsense, frameshift and splice site mutations have a severe effect, by there being no EXT protein localized to the endoplasmic reticulum. This then leads one to think there should be a difference in the phenotype caused by a mild versus severe mutation. One then concludes that, phenotype could be influenced by the type of mutation or the severity of the mutation. This appears to be the case in the 26 individuals studied here. In general, though not statistically significant or universally correct, nonsense and truncating mutations had a worse phenotype. This was far from as impressive a negative effect that EXT 1 has on phenotype. When mutation type was looked at in the context of gene mutated missense mutations tended to have the mildest presentation. It is possible then that some function of the EXT genes is preserved when missense mutations occur by the protein being present in the ER. Gullberg (2002) has gone on to show recently that the function of the two EXT gene products in fact do vary in terms of the effect on the elongation of the heparan sulfate chains. EXT 2 is believed to chaperone or modify the activity of EXT 1 and therefore in EXT 2's 100  absence, chain elongation is altered but not negated as it is when EXT 1 is absent. The two genes work in synergy, but given the differential effect of EXT 1 and 2 on heparan sulfate chain elongation it is very possible that the phenotype is truly affected by which of the two genes is mutated into inactivity. Given the dominant role on the enzymatic activity that EXT 1 has one would then assume EXT 1 phenotype would be more negatively influenced. That is to say, subjects with EXT 1 mutations should have a more severe form of the disease. The results of this study support the findings of Gullberg in that subjects with EXT 1 mutations have a more severe expression of the disease. Even though the germ line mutation exists, how does this then translate into disease expression? There are two possible mechanisms. The first is that the germ line mutation acts in a negative dominant way resulting in exostosis formation. However, this should then result in global involvement of the entire skeleton. Specifically, all growth plates, which appear to be the cell of origin source for exostosis formation, should be affected by exostosis formation. Furthermore there should be significant deformity of the entire growth plate. This sort of effect is seen in skeletal dysplasias such as achondroplasia where all growth plates involved with enchondral bone formation are affected. In achondroplasia, phenotypic features are expressed by the entire skeleton, including the skull, spine and appendicular skeleton. And yet HME rarely affects the spine, or head and has a definite propensity for the long bones including hands and feet. But not in all cases are all juxtaphyseal regions involved with exostoses. When looking at the xrays of achondroplasts the entire bone is influenced by the results of the abnormality of the growth plate function. In some cases of HME or SME, there is global effect on the bones. For example the metaphyses particularly about the knee and proximal humerus can been grossly distorted with flaring. But this is not universal in either all the bones in one subject or in all Multiple Exostoses subjects. In many cases there are simply multiple discrete lesions causing only a bump remote to the physis. This is the case in this study's population. Many subjects 101  showed metaphyseal flaring, but no subjects had 100% of the metaphyses distorted in this way and at the same time many subjects had some unaffected physes and metaphyses. In other subjects many discrete remote lesions existed which were completely innocuous. The alternative mechanism is that the germ line mutation in conjunction with local influences causes exostosis formation. Hecht's (2002) work has demonstrated little nest of cells located in the perichondrium of patients with HME. These nests are possibly the result of monoclonal expression from a chondrocyte that carries the germ line mutation and its survival into a tumour is the result of local forces. This would then better explain the lack of global skeletal involvement and the lack of the entire bone being deformed.. It is then ultimately the effect on the local environment that causes the resulting deformity. As Porter (Porter et al. 2000) has shown, the more lesions on one bone and the involvement of highly integrated two bone systems the more deformity occurs. The local effect of the tumour would then be responsive to a variety of influences, including, when the tumour develops (the younger the patient the more potential for it to get bigger, the older the more likely it will migrate less from the growth plate thereby causing growth plate tethering), where in relation to the growth plate it forms (peripherally versus centrally where it can cause metaphysealflaring),or gender given that males and females have different growing patterns and potential. If there is a difference in which gene is affected in terms of potential for exostosis formation one would expect this to influence the phenotype. Given that EXT 1 has potentially more of a role in tumour formation secondary to a higher catalytic function than EXT 2 it would follow that EXT 1 mutations would have a greater potential for tumour formation, which is in turn influenced by the local environment. Once again the data shows EXT 1 patients have more lesions and a more severe expression of the disease.  102  4.3 Phenotype  In general the eighty-nine phenotypic features explored were normally distributed. All subjects, including those without mutation identification (Family 4 and 6 members), were included in the phenotype analysis. All but 18 of 2848 data points were collected from all subjects. The phenotype data were grouped into three categories reflecting significant areas of clinical concern: 1) lesion quality, 2) limb alignment and 3) limb segment lengths plus percentile height. As the goal of this thesis was to identify a genotype phenotype correlation, phenotype alone is briefly discussed here and to greater length in the genotype phenotype section (section 4.4). In addition, the Pearson correlation matrix did not identify any two of the phenotype parameters to be correlating except with respect to limb lengths, therefore, all parameters were treated independently of each other.  4.3.1 Lesion Quality  Lesion quality was determined by radiographic evaluation of the patient. X-rays were more sensitive in detecting lesions, clinical exam underestimated the count by as much as 50% in this study. Furthermore, it was not possible to determine the morphology or the size of the lesions reliably by physical exam. Specific X-rays of hands, feet and spines were not routinely available as these sites are an uncommon source of morbidity in this population. Using items put forward by Francennet (Francennet et al. 2002) and Carroll (Carroll et al. 1999) as a template for assessing lesion quality to reflect severity of disease expression two of the major factors used by these authors are of questionable significance in the context of the present studies results. Francannet et al. (2002) have put a large emphasis on spine lesions and if present, automatically led to the phenotype being classified as severe. Involvement of the spine in the present sample was not specifically assessed with spine x-rays. However, none of the 103  subjects noted any spinal lesions nor were they noted on physical examination, particularly no spinal deformity (scoliosis) was identified on physical examination. Exostoses were noted in the lower spine on some of the pelvis x-rays in this series of subjects but none were noted to be involved in spinal deformity. None of the thirty-two subjects in this study had scoliosis. Reviewing the BCCH scoliosis clinic database (containing 3137 cases) no cases were found where exostoses were the cause of spinal deformity. Similarly, Schmale (1994) reported no spinal lesions and Wold (1990) reported 3% (1% in cervical, thoracic and lumbar each). Spinal exostoses are very rare and unlikely to cause scoliosis. As a result, using the presence of spinal lesions to define severity is of questionable usefulness. Carroll et al. (1999) in part defined the resultant phenotype of EXT 1 and 2 mutations on the basis of pedunculated versus sessile lesions. It was implied in this study that the higher percentage of sessile lesions present the worse phenotype. Presumed EXT 1 mutation subjects had 87% sessile lesions and were moderately effected, presumed EXT 2 subjects had 72% sessile lesions and were felt to have a mild phenotype and presumed EXT 3 subjects (it has since been decided that EXT 3 is not involved with exostosis formation) (Wuyts et al. 1998) were severe with 95% sessile lesions. The morphology of the lesion in isolation does not appear to be significant in terms of phenotype. Rather it is the location and influence on the growth plate, which cause deformity and mal-alignment. The present study with a larger sample size than Carroll (32 versus 29) did not observe as high a percentage of sessile lesions with the typical proportion being 60% sessile to 30% pedunculated. Therefore discussing phenotype severity on the basis of spine lesions and their morphology in isolation may not be helpful. Involvement of flat bones, including the bones of the pelvis, was thought to be reflective of a more severe phenotype because of their increasing propensity to transform into chondrosarcoma. Fifty-six percent of chondrosarcomas occur on the flat bones with twenty-three percent originating from the pelvis (Mirra 1989). There were 14 subjects, representing both EXT 104  1 and 2, with flat bone involvement, and given that there is roughly a 5% transformation rate, one of the included subjects is likely to suffer from a future chondrosarcoma. Because the pelvis is capable of accommodating a large mass without obvious evidence until it is very large, transformed osteochondromas can remain hidden. In this case to ensure clear resection margins often a hemi-pelvectomy is required. In this series 11 subjects, or 35%, had a pelvic lesion identified, which is significantly higher than the 6% reported by Mirra (Mirra et al.1989) and twice that of Schmale's 15% (Schmale et al. 1994). The difference in reported pelvic lesions may reflect the routine use of pelvic x-rays in the current study that was not used in the other studies. Only Family 17 has a known case of chondrosarcoma. It occurred in a male (not a participant), which is more common (64%) (Mirra et al.1989), involved the pelvis and resulted in a partial hemi-pelvectomy to obtain clear resection margins. The number of lesions has also been proposed to be good measure of disease severity. Porter has shown the more lesions present the greater the bony deformity (Porter et al. 2000). In this study 96% of patients had at least one lesion about the knee and 63% of subjects had knee mal-alignment. Similarly wrist, elbow and ankle alignment had a greater chance of being abnormal as the number of lesions in the involved bones increased and when it involves the two bone systems (forearm and lower leg). In this study population there was no obvious relationship between increasing number of lesions and overall mal-alignment. Besides the knee the actual number of lesions per bone was not mapped precisely but it was observed that mal-alignment and deformity occurred only in the presence of exostoses. Additionally, there were two confounding elements, gender and age. Males had more lesions, and the older an individual the more likely joint mal-alignment exists irrespective of exostoses. The more lesions present, the higher the chance one will be on a flat bone and therefore subject to transformation. Therefore not only location of the exostosis is important but also the number, which increases the probability of their being a pelvic lesion. 105  The number of lesions did not correlate with the percentile height. The average number of lesions in the study was twenty-five and the percentile height thirty-three. The fewest number of lesions in one subject (EXT 2) was nine and their percentile height was at the fifty-first percentile and the subject (EXT 1) with the most lesions, fifty-three, was in the third percentile. However, five subjects were on the third percentile but their lesion counts were 34 (EXT 1), 53 (EXT 1), 11 (EXT 2), 28 (EXT 2) and 32 (EXT 1) while two subjects were above the ninetieth percentile and their lesion counts were 12 (EXT 2) and 14 (EXT 2). A relationship may truly exist here but the sample size is too small to pick this up. If however, the effect of the germline mutation is a global effect as in a skeletal dysplasia then the number of lesions should be irrelevant. Metaphyseal flaring has also been considered to be the sign of a severe phenotype as the aneurysmal dilatation of the metaphyses was thought to cause a greater degree of mal-alignment, deformity and shortening. While a significant correlation was not found in general bony deformity, malalignment and limb length discrepancy did occur in the presence of metaphyseal flaring. The subject (EXT 1) with the highest percentage of flaring (80.5%), was on the 39  th  percentile for height (average 33), had 10 mal-aligned joints (average 12) including the knee and the hip on the left, but normal alignment of the right at these two joints, and a significant leg length discrepancy of 2.5 cm. The shorter leg was on the right even though both distal femurs were involved with flaring. Hence it was a combination of shortening and malalignment, which resulted in the net leg length discrepancy. One phenotypic feature, which was overlooked in the inclusion of parameters, was a quality of life questionnaire. The Musculoskeletal tumour society functional assessment has been used in the HME setting by other authors (Schmale et al. 1994 and Francennet et al. 200). It is a validated scale reflecting the quality of life of patients with tumours. Better would be a disease specific quality of life score but no such scale exists. Pain scales have also been used in this 106  patient population (HME coalition in conjunction with Hecht 2002) and the results were unexpected. In particular, there is a physician/surgeon misconception that pain is not a large factor in these people's lives, but in fact the returned pain scales showed a presence of pain in 70%, with 14%> greatly influencing function (personal communication Hecht 2002). Since ultimately the most important component of the clinical profile is quality of life it will be included in all future studies. Using the Musculoskeletal Tumour Society's scale as a global rating score a disease specific quality of life score will be designed concurrently. 4.3.2 Limb Alignment  Limb alignment data though done both by clinical and radiographic examinations was more accurate and complete from the radiographic examinations and therefore it is only this data that is discussed. Care was taken to standardize the data according to age and gender in the few cases where it made a difference. For example, the female carrying angle (elbow joint alignment) is in more valgus than in males. The results are discussed in two sections using different approaches. First, when discussing phenotype alone, each subject's twenty-six alignment parameters were evaluated and classified as either within normal limits or one standard deviation outside the normal limits. The total number of abnormal results out of twenty-six was then calculated for each subject. It gives a global mal-alignment tally for each individual, which was then related to the study population. This method however did not reflect the severity of the mal-alignment. The second way the data was interpreted is more pertinent to the genotype-phenotype discussion. The group's data was collected, for example EXT 1 patients, and the values for each alignment parameter for all the subject in the group was averaged, deemed within or outside normal limits and then the abnormal alignments were tallied up for each group. By doing this only if the group as a whole had significant mal-alignment did the result register outside normal limits, thereby reflecting severity of mal-alignment. Thefirstmethod looks at the datafroma population perspective whereas the 107  second method looks specifically at severity of phenotype particularly in relation to genotypic features (discussed in section 4.4). The femoral anatomic angle (knee joint angle) and the elbow joint angle were the most commonly abnormal alignments where 31 of 32 individuals had at least one side of knee malalignment and 27 of 32 had elbow joint mal-alignment. Ulnar shortening and radial inclination mal-alignment were also quite common with 27 subjects each being outside the normal limits. The least effected parameter was radial bowing where only 5 individuals were affected. Given that the knee consists of the distal femoral, proximal tibial and fibular physes, it is not unexpected to find it the most commonly effected joint. It also involves a two-bone system where balanced bone growth is essential for alignment. The probability of having at least one of the three bones involved at the knee was reported as 94% by Schmale (1994). In this series 96% of the patients had involvement at the knee joint. On average each subject had twelve of twentyfour abnormal alignments with the range between nine and eighteen. The severity of the malalignment however varied considerably. As discussed above these values were obtained by averaging the alignment values for each group.  4.3.3 Limb segments and percentile height  The significance of measuring height and limb segment lengths was in order to evaluate HME as a skeletal dysplasia. Traditionally HME had been classified as a pathologic short stature (skeletal dysplasia). Short stature is defined as an individual less than the third percentile for their age and gender. The average percentile height for this series of HME subjects was the 27  th  percentile (33 if including family 4 and 6). The range however was from the 3 (5 subjects, 3 rd  rd  males and 2 females) to greater than the 85 (3 subjects, 1 male and 2 females) percentile. Short th  stature is defined as less than or equal to the 3 percentile, therefore rather than classifying HME rd  108  as a pathologic short stature or dwarfism it would better be described as having a propensity for stature below the 30 percentile, i.e. growth impedance. th  The effect of the overall growth impedance correlated reliably with the lower extremity segment measurements. However when each subject's data was analyzed separately, the percentile of the upper and lower limb segments were higher than the overall percentile heights. This can be a result of a variety of factors. Firstly, the shortening experienced by these patients is not accounted for exclusively by the lower extremities and is a culmination of shortening in the pelvis, trunk and spine. Secondly, actual bony measurements are more accurately done using computed tomography versus surface landmarks. This however does not include the soft tissue envelope and therefore underestimates the total length. Thirdly, most of these subjects had a degree of mal-alignment and deformity in their limbs which effected the overall height of the patient but when broken down to the measure of each bone less shortening was identified. Of interest was that the lower leg segment of the lower extremity was always shorter than the upper leg segment. This would be consistent with mesomelic shortening. The same pattern was noted in the upper extremity where the proximal segment was longer (based on percentiles) than the distal segment, and both were consistently below the 37 percentile. In the lower th  extremity they were both below the 51 percentile. In this regard, with respect to these 32 st  individuals, they all had mesomelic shortening in both the upper and lower extremities. Yet considering the growth plates, those with the largest growth potential would logically be the ones more significantly effected; hence it should be that the upper leg segments be shorter on a percentile basis than those of the lower leg segment (the distal femoral growth plate contributes 37% of longitudinal growth versus the proximal tibia which contributes 28%), the remaining growth plates are less still) (Morrissy and Weinstein 1996). The same can be said for the upper extremity where the proximal humeral growth plate contributes more to the overall growth of the upper extremity those of the radius and ulna. The reason behind the mesomelic shortening brings 109  us back to Porter's (2000) work where the two bone systems, which are mesomelic in both upper and lower extremity, are more significantly affected possibly because there is twice the chance for growth impedance. This leads us back to the hypothesis that it is partly environment that causes the effect on the phenotype, but the number of lesions controlled by genotype sets the level of severity (EXT 1 worse than 2). Trunk measurements were not done as part of this study. On retrospect it would be worthwhile to determine if the trunk segments were also shortened. If in fact shortening does exist throughout the entire skeleton, HME may need to be reconsidered as a skeletal dysplasia. This would be supported by the observation of mesomelic shortening and that a germline mutation is present in all subjects with HME. 4.3.4 Intra-Family variability  There were three families with three generations of affected individuals with available data. Family 3 was the only family large enough and with sufficient participating subjects to look at intra-family variation. There were twenty-three participating members and nine of these were affected. There were six females and three males. However when looking at the entire Family 3 tree the ratio was closer to 1:1 between males and females for having multiple exostoses. There was one grand parent, three parents and five children studied. Intra-family variability was assessed by gender and generation, as these were the two most obvious variables. In broad terms for the three, three generational families, the grandparents had the least number of lesions. However they were also the shortest in terms of percentile height. This is consistent with the rest of this study's data set in that number of lesions did not influence percentile height. On the other hand it is possible that since this generation is smaller, their growth potential was also less and therefore they simply did not grow as many lesions. The malalignment data for the three grandparents was within keeping with the entire study population.  110  There was no consistent pattern identified in any of the three phenotype categories between the parents and the children in Family 3 or any of the remaining families. These parameters were influenced by gender in some situations, and generation in others but no correlation could be identified. When looking only at Family 3 lesion quality results showed an average of 17.4 lesions with a range between nine and thirty-nine. Males had twenty-six lesions on average and females had thirteen. The percent distribution of the remaining characteristics were not different between males and females except for metaphyseal flaring where all three males showed greater than thirty-five percent flaring and an average of fifty-five percent of their lesions showed metaphyseal flaring versus seventeen percent in females. All the lesion quality parameters otherwise more or lessfita normal distribution. Percentile height was 48.6 for the family with a range between eighteen and ninety (males 43 , females 51 ). Limb segment shortening was in rd  st  proportion to the overall percentile height as discussed earlier in section 5.3.2 and mesomelic shortening was consistent in both the upper and lower extremities. As for alignment data, each individual had an average of 12 mal-alignments, which is also the average for the entire study population. The severity between and males and females and between generations did not show a consistent pattern. In summary, with regards to intra-family variation, the only consistent influence was by the male gender that had more lesions and metaphyseal flaring. This was also the case in the entire study population therefore it is unlikely secondary influences within a family with a given genotype other than gender has an effect.  4.4 Genotype phenotype correlation Phenotype as it relates to genotype was broken down into the three categories, lesion quality, limb alignment and limb segments plus percentile height. Lesion quality features that 111  were felt to be more representative of a worse phenotype were number of exostoses, percent flat bone involvement, percent pelvic bone involvement and percent metaphyseal flaring. The remaining parameters were not found to be indicative of severity in this population. Limb alignment severity was based on the averaged alignment data for the subjects in each group of interest and then the total number of abnormal alignment items were tallied. The more abnormal alignments present for the group being analyzed the worse the phenotype was considered. Finally the shorter the subjects which was reflected as the average for the group of interest, the worse the phenotype. The limb segment lengths were directly related to the degree of shortening of the overall stature, albeit to slightly different degrees. The features, which appeared to represent severity of disease accounted for 38 of the 89 possible parameters explored. When the number of lesions were standardized to a percentage, the lesion quality parameters were reduced from 38 to 21. So in fact there are 72 parameters representing phenotype of which over half describe severity in this small population. However there are many trends existing amongst the other 34 parameters and all data points need to be collected to ensure differences were not missed simply due to sample bias. Those patients with an EXT 1 mutation consistently expressed more severe phenotypic characteristics. These included a greater number of exostoses, a higher percent involvement of flat and pelvic bones, greater mal-alignment and shorter stature with corresponding limb segment shortening showing the typical mesomelic pattern. Gender in isolation appears to be a modifying feature with males tending to have more exostoses and metaphyseal flaring and perhaps a trend towards a greater degree of mal-alignment. No obvious difference in percent pelvic or flat bone involvement was seen between males and females. No evidence of phenotypic variation was observed in the other comparison groups (mutation type, mutation severity or mutation location) in isolation. One may criticize that this was not a fruitful endeavour to look at these factors independently but it does put to rest that in isolation they are not influencing factors on 112  phenotype. In striking contrast however, when paired with gene affected support is given to previous entertained hypotheses that in conjunction with gene affected, mutation type and severity, and mutation location, and possibly gender did have influences causing variation in the phenotype therefore these factors must differentially affect gene function. When the relationship between EXT 1 in conjunction with gender and phenotype was explored the following was observed. In general males were worse than females within a gene; i.e. EXT 1 males were worse than EXT 1 females and EXT 2 males were worse than EXT 2 females. This was the case for number of lesions, and percent pelvic involvement. For the remaining categories showing differences males as a group were worse than females. Specifically, males were more severe than females in the following order; EXT 1 males greater than EXT 2 males greater than EXT 1 females greater than EXT 2 females in severity for degree of limb mal-alignment and percent metaphyseal flaring. Exploration of the relationship between EXT 1 in conjunction with mutation type and phenotype revealed all EXT 1 mutation types were consistently more severe phenotypically than the EXT 2 mutations. Similarly, when EXT 1 was paired with severity EXT 1 severe and mild mutations consistently had a more severe phenotype than both EXT 2 mild and severe mutations. When dissected further for EXT 1 missense mutations were consistently milder than nonsense mutation for lesion number, percent pelvic and flat bone, limb alignment and percentile height. The same can be said for mild versus severe mutations. The EXT 2 trends were similar except for limb alignment and percentile height comparing specific mutation types. One must be reminded here that these are trends and not statistically significant. Nonetheless, this gives support to the hypothesis that mutation type affects protein localizing in the ER and therefore function and ultimately phenotype. When gene was matched with early versus late mutation EXT 1 early was worse phenotypically than EXT 2 early for lesion number, and pelvis and flat bone involvement. This is 113  likely reflecting the more dominant effect the EXT 1 gene has on phenotype. However there were no differences noted in lesion quality parameters when inspecting EXT 1 versus 2 late mutations. Interestingly however when EXT 1 early was compared to EXT 1 late mutations early mutations were considerably worse with lesion number, percent pelvis, flat bone and metaphyseal involvement and percentile height (EXT 1 early mutations are the shortest, then EXT 1 late, EXT 2 late and EXT 2 early). This gives some support to Wuyts idea (Wuyts et al. 2001) idea that late mutations may be milder. This is not supported by the alignment data as EXT 1 early and late both have 15 malalignments and they are both worse than EXT 2 mutations. This again is the gene effect. Similar exploration of gender in conjunction with mutation type and severity produced no consistent pattern on phenotype. This suggests gender may act to modify the influence that EXT 1 exerts on phenotype but in isolation has less of an impact. Determining causation in an association found, such as EXT 1 versus EXT 2 and phenotype, can be supported by different factors. One is consistency of findings suggested above. A second is a reasonable biological rationale. The EXT 1 gene product is believed to have a higher catalytic activity in heparan sulfate chain elongation than EXT 2 "135". The extent of chain elongation may influence cell division to different degrees in EXT 1 versus EXT 2 patients. While loss of growth regulation appears to depend partly on which EXT gene is mutated other external forces likely are modulating the extent of disease expression. Gender may variably influence clinical expression due to the difference in growing patterns in children. Females are known to have a short but rapid pre-pubertal growth spurt that comes to an end two years after menarche while boys grow more slowly over a longer period of time. In general girl's growth interval from puberty to skeletal maturity is about 4 years compared to the 6 years of males  114  (Lovell and Winter 1996). It is possible that gender differences in hormonal expression and duration of expressed growth factors influence this modulation. HME has been included by some under the umbrella of skeletal dysplasia (Lovell and Wintr 1996). However, as dysplasia in general refers to an intrinsic bone disturbance and HME bony disturbance appears to be confined to those exostoses present, it may not be accurate to include HME among the skeletal dysplasias. There is a wide expression of the disease in terms of phenotype and other than each exostosis having a similar appearance at the pathologic level the variability in the skeleton from subject to subject is quite marked. Perhaps the strongest support used to place HME among the skeletal dysplasias is the associated short stature. However while most HME patients have stature less than the 50 percentile, some are greater than the 85 th  th  percentile and none are below the 3 percentile in these studies. On balance HME does not rd  appear to a true skeletal dysplasia. The disease expression is influenced by both the number of tumours present and when they occur. The number of lesions appears related in part to genotype (gene affected, mutation type, severity and location) but genotype does not seem to influence the location of tumour development. If one considers clonal expression as the mechanics of tumour development then it becomes a matter of what is causing the tumours to grow. From this work it appears that EXT mutations and their related type and location and gender predispose one to HME. There is more loss of control of tumour regulation with EXT 1 mutations, and males may have more growth potential over time to allow for more growth both with respect to actual number and to size including metaphyseal impairment. The mutation characteristics (type and location) are likely affecting the gne function at he cellular level promoting tumour development. If the mechanism of exostosis formation was the result of a malfunctioning growth plate then all growth plates should be deformed. But this is not the case and in many cases small pedunculated exostoses are found remote from the growth plates as innocuous little bumps. This 115  implies that a small nest of cells developed at a point of rapid growth and that the nest did not get caught up in the growth plate thereby causing bony deformity and mal-alignment. In contrast, those joints with multiple lesions and significant mal-alignment may have had multiple monoclonal nests develop and then due to yet unknown local factors they all started to grow but the environment was such that the tumours got caught up in the growth plate and could not migrate away. Then the two bone systems would be even more sensitive to this because any disturbance in one of the bone causes significant deformity for the other. In summary, EXT 1 mutations are associated with a more severe phenotype, which appears to be modulated both by mutation type and location and in part by gender. This may be due to the fact that EXT 1 has a more dominant effect on heparan sulfate chain elongation as a result of increased catalytic activity. Tumour suppression activity is sensitive to the heparan sulfate chain morphology, changes in which gene gives rise to varying loss of control over growth. The mechanism for expression of the disease appears to be more on of focal clonal expression dependent on the local and humoral environment rather than a skeletal dysplasia or a "sick" growth plate as there is far too much variability amongst HME patients. What causes the second hit is unknown but in the cases where both genes are mutated chondrosarcomas have been described (Hecht 1995). The phenotype is partly influenced by the location of tumour development, at what point in a child's development do they appear and in what growing milieu they develop. An established genotype phenotype correlation has significant clinical impact. Patients with HME and in particular those with EXT 1 mutations need to be monitored to possibly avoid bony deformity and mal-alignment, which leads to surgery and the associated risks and complications of intervention. Males with HME need to be further assessed as their phenotype tends to be more severe. Phenotype profiling in relation to the gene mutated ( and the mutation characteristics) will be helpful in providing families with the anticipated course of the disease for 116  their offspring and will aid in determining the prognosis. The relationship of chondrosarcoma to EXT 1 and 2 mutations is still unclear but surveillance for transformation of benign osteochondromas is important.  117  Chapter V: Summary Ten HME families from British Columbia with thirty-two (69 total participants) affected members participated in this project. Eight mutations were identified and confirmed. Six mutations have not yet been described in the literature and two have previously been reported. The features of the mutations are in keeping with what is reported in the literature. Phenotyping was exhaustive and allowed for subjects to be described in terms of their lesion quality, limb alignment and deformity and limb segment lengths plus percentile height. A genotype phenotype correlation exists in that subjects with an EXT 1 mutation have a worse phenotype, mutation type and location also influence severity and gender appears to modulate expression of the disease. This correlation supports the hypothesis that EXT 1 has a dominant affect over EXT 2 in tumour development and that HME is unlikely to be the result of a skeletal dysplasia but rather a combination of loss of chondrocyte growth regulation and then growth parameters specific to each subject.  118  Chapter VI: Conclusion There is a genotype phenotype correlation in HME where patients with EXT 1 mutations have a worse phenotype.  119  Chapter VII: Future work This study was designed as a descriptive study to explore whether a genotype phenotype correlation exists in HME. This was a hypothesis-generating manoeuvre in hopes of identifying factors that represent severity of disease expression. The results of this study have provided a template from which future work can expand in a prospective fashion. A large collaborative network has been established secondary to this pilot project. The mutations identified in the literature have been described by a variety of labs worldwide. These labs, through the assistance of the HME coalition (a non-profit support group for people living with HME and their families) and its members will provide consenting subjects whose genotypes have been identified and confirmed for the anticipated prospective project (website: http://www.geocities.com/rrihecoalition/). We anticipate access to up to a minimum of 60 new families. Ethical approval has been obtained for this site and all the collaborating labs have obtained Ethical approval from their centres. Funding has been secured to carry on with this work. We anticipate approximately 200 new affected subjects from elsewhere and 30 from this centre. Our centre will genotype the BCCH new families (plus further work will be done to elucidate mutations for Family 4 and Family 6). Data will be collected as presented in this thesis. Prospective analysis will be done on the features identified as showing a trend in the pilot project. Specific hypotheses to be tested in a prospected fashion include (with regards to phenotype); EXT 1 gene mutations are worse than EXT 2, Within a gene mutated, nonsense mutations and truncating mutations are the most severe, within a gene mutated early mutations are worse than late. Investigations also need to be done to further elucidate the local and humoral factors that are permitting specific tumours from growing from presumptive osteochondroma niduses. One potentially obvious factor is gender and it hormonal differences and growth patterns. But many other possibilities exist that influence growing bone. 120  Another direction this project has taken from the pilot project is to further study the hypothesis of disease expression being a result of clonal expression of perichondrial chondrocyte nests. Tumours are collected when patients undergo resection of exostoses as part of their routine care. We are working in collaboration with another group to investigate the genetic make up of the tumours themselves. Dr. Hecht's work has outlined some second mutations but her work was limited due to lack of material. Our group has access to the original 10 family's material plus the new probands presenting to the HME clinic on a regular basis. Furthermore solitary exostoses are readily available as they are routinely excised from patients. These two main patient populations will be accessed under appropriate consent and ethical approval. Funding has been obtained for this project and ethical approval is pending. The final offshoot of the original project has been the establishment of the HME clinic at BC Children's Hospital run by the author of this thesis. This clinic provides clinical support for the families affected with HME. Disease surveillance is the main goal of the clinic. 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Human Mutation 15:220-227. 135. Xu, Lei, Jiahui Xia, Hujun Jiang, Jiangnan Zhou, Hejun Li, Daping Wang, Qian Pan, Zhigao Long, Chaohong Fan, and Han-Xiang Deng. 1998. Mutation analysis of hereditary multiple exostoses in the Chinese. Human Genetics 105:45-50. 136. Young, CL. Sim FH. Unni KK. et al. Chondrosarcoma of Bone in Children. Cancer 66, 1641-1648. 1990.  Appendix Appendix 8.1 Ethics Approval  8.1.1  Ethics Approval Certificate from Children's and Women's Hospital of British Columbia  C H I L D R E N ' S hr WOMEN'S H E A L T H C E N T R E O F BRITISH C O L U M B I A  January 14,1998  Dr. Christine M . Alvarez  Dear Dr. Alvarez, Your proposed research project, "Establishing the Genetic Profile of Multiple Hereditary Exostoses in Families of British Columbia" was reviewed and A P P R O V E D by the In-Hospital Research Review Coiximittee of Children's Hospital at its January 14,1999 meeting. The In-Hospital Research Review Committee approval is valid until February 15,2001 providing there are no changes i n the research procedures. Sincerely yours,  Nevio Cimoiai, M D , F R C P C A d Hoc Chair, la-Hospital Research Review Committee  BRITISH C O L U M B I A ' S C H I L D R E N ' S HOSPITAL BRITISH C O L U M B I A ' S W O M E N ' S H O S P I T A L A N D H E A L T H C E N T R E S O N N Y H I L L H E A L T H C E N T R E FOR C H I L D R E N •VI,a AM.CAOtSMK HUAITH CKNTM! AFnuMSO AND  r«e  WITH T f . f i t / « W « I T > I V D W r j S M COLUMt.  a.c. moKMKH m s r m v r c rot> cwtxm*ir*<• SVOMIWS  137  W^ITH  Appendix 8.2 Patient Consent Form  British Columbia's *=» u  mm  Children's Hospital Department of Pediatric Orthopedic Surgery Stephen J. Tredwetl. MD, FRCSC, Department Head Bonita J. Sawatzy, Ph.D., Research Richard D. Beachamp, MD. FRCSC Sharon A Secord. BSc.N.. Nursing Associate H. Michael Bail. MD, FRCSC Kenneth L. B. Brown, MD, FRCSC Telephone: (604) 875-3187 Christopher Reilly, MD, FRCSC Facsimile Line: (604) 375-2275 Letter of Information Project: E s t a b l i s h i n g the G e n e t i c Profile of Multiple Hereditary E x o s t o s e s in F a m i l i e s of British C o l u m b i a Investigators: Dr. C . A l v a r e z , Dr. S . Tredwell, Dr. M . H a y d e n , O s t e o c h o n d r o m a s , a l s o k n o w n a s e x o s t o s e s , a r e b e n i g n b o n e tumors, w h i c h a r i s e n e a r growth plates at the e n d of long b o n e s or o n flat b o n e s . T h e y c a n o c c u r a s solitary l e s i o n s a s i n solitary o s t e o c h o n d r o m a s ( S O C ) o r in multiples a s s e e n in Multiple Hereditary E x o s t o s e s ( M H E ) . O s t e o c h o n d r o m a s d o not usually c a u s e s y m p t o m s but o n o c c a s i o n c a n c a u s e m e c h a n i c a l p r o b l e m s d u e to their s i z e a n d o r location b y c a u s i n g p a i n , nerve c o m p r e s s i o n o r deformity. L e s s c o m m o n l y , they c a n c a u s e asymmetrical growth of the long b o n e s resulting in limb malalignment o r limb length discrepancy. A very rare complication of o s t e o c h o n d r o m a s , particularly in M H E is the transformation of the b e n i g n l e s i o n into a malignant o n e . T h i s h o w e v e r is a n exceptionally rare o c c u r r e n c e a n d u s u a l l y o c c u r s after skeletal maturity. It i s k n o w n that M H E i s a n inherited condition 95% of the time but m a y a l s o o c c u r sporadically. O n the other h a n d , solitary o s t e o c h o n d r o m a s are thought to b e r a n d o m o c c u r r e n c e s . In M H E , 3 principal g e n e s h a v e b e e n identified. R e p o r t e d in the literature to date, i s that m o s t f a m i l i e s with M H E h a v e a n abnormality identified in o n e of t h e s e 3 principal g e n e s . N o study h a s b e e n d o n e to confirm w h e t h e r patients with solitary o s t e o c h o n d r o m a s o r patients without a family history of o s t e o c h o n d r o m a s h a v e similar g e n e t i c c h a n g e s . T h e p u r p o s e of this study is to establish the g e n e t i c m a k e - u p of f a m i l i e s with M H E , patients with multiple l e s i o n s but n o family history, a n d patients with solitary o s t e o c h o n d r o m a s . T h i s entails identifying patients with M H E a n d S O C . T h i s will o c c u r a s the patient p r e s e n t s to a regular clinic v i s i t Dr. A l v a r e z will b e introduced to interested patients a n d their parent(s) a n d a brief d i s c u s s i o n about t h e project will occur. If the patient a n d their direct family a r e interested they will b e e n t e r e d into the study. T h i s will involve interviewing the patients a n d their direct family. T h i s interview will take about 1 hour. W e are interested in  British Columbia's Children's Hospital 4 4 8 0 Oak Street, V a n c o u v e r , B C V 6 H 3 V 4  P h o n e : {604) 8 7 5 - 2 3 4 5  A part of Children's & Women's Health Centre of British Columbia An taAtmie htclit, crmrt djillsud Kith the Uniwrriij of BriiW Chhmbh  139  Letter of consent: "Establishing the Genetic Profile of Multiple Hereditary Exostoses in Families of British Columbia'' I,  understand the above  study and hereby give my assent to participate in the study. Signature,  .  Date I,  have read and  understood the letter of information regarding the above study. I hereby give my consent for  (participant)  My  (relationship)  to participate in the above titled study. Signature Date Witness Date  141  Appendix 8.3 EXT 1 8.3.1  EXT 1 - cDNA showing sequence and primer positions (Ref: GenBank Accession: NM_000127)  1  gcgaccgaac gcggcggtcg gcagcgttcg cgcgggggcc tgcgaagcgc tgctcggggc  61 cggcactgcc cgcggggagg acgcgccgcc gccgccaccc agcgccgccg ccgccgccgc 121 ctccagccgg gccgccgcgc gtcccggggg ccggccccgc gagcgcagga gtaaacaccg 181 ccggagtctt ggagccgctg cagaagggaa taaagagaga tgcagggatt tgtgaggtta 241 cggcgcccca gctgcaagat gcactagccg gctgaacccg ggatcggctg acttgttgga 301 accggagtgc tctgcacgga gagtggtgga tgagttgaag ttgccttccc ggggctcatt 361 ttccacgctg ccgagaggaa tccgagaggc aaggcaatca cttcgtcttg ccattgattg 421 ggtatcggga gctttttttt tctcccctct ctctttcttt tcctccgtct tgttgcatgc 481 aagaaaatta cagtccgctg ctcgcccgcc ctgggtgcga gatattcagc cccgctctct 541 cccgtgcatt gtgcaaccca aagatgaaag accgaagggg agaaagttaa agaaatcgcc 601 cacatgcgct ggatcagtcc acggcttggg gaaaggcatc cagagaaggt gggagcggag 661 agtttgaagt cttuj caggc gggaagatgg cggactgg |ag ctgaaagtgt tgattgggaa >  ex 1 a  ^. \Exon 1 Start  721 acttgggtgattcttgtgtttatttacaa |tcctcttgacc caggcag| gac acatgcaggc apr 111  781 caaaaaacgc tatttcatcc tgctctcagc tggctcttgt ctcgjcccttt tgttttattt ^  ex 1c  ^  841 cggaggcttg cagtttsj ggg catcgaggag ccacagccgg ag agaagaac acagcggtag *  ex lb  *  901 gaatggcttg caccacccca gtccggatca tttctggccc cgcttcc egg agcctctgcg ^  ex le  961 c!becttegtt cc tt gggatc aattggaaaa cgaggalttc c agegtgeaca tttccccccg ex Id > > < apr110  1021 gcagaagcgalgatgecaa ct ccagcatcta caaaggcaag aagtgccgca tggagtcctg apr 211  —»  1081 cttcgatttc accctttgca agaaaaaegg cttcaaagtc ta egtatace cacagcaaaa <  ex Ig  1141 agggg agaaa ategecgaaa gttaccaaaa cattctag eg gc catcgagg gctccaggtt ex If J > < 109 ; «/"•( 1201 ctacacctcg gaccccagd c aggegtgect ctttgtcctg agtctggata ctttagacag <  apr 210  142  >J  1261 aga ccagttg tcacctcagt atgtgc acaa tttgagatcc aaagt gcaga gtctccactt ex li  ex li  1321 gtggaacaatg gtaggaatc atttaatttt tad tttatat tccggcactt gg c ctgacta apr107  ex Ik  1381 caccgaggac | tggggtttg acatcggcca ggcgatgctg gccaaagcca gcatcagta <  apr 209  1441 tgaaaacttc cgacccaact ttgatgtttc tattcccctc ttttctaagg atcatcccag  »  1501 gacaggaggg gagagggggt ttttgaag tt caacaccatc cctcctc tca ggaagtacat <  .  apr 108 vw«  >.  i  .  1561 get ggtattc aagg ggaagaggtac ctga c aggge tagga tcagacacq a ggaatgeett ex lm  *  ex 11  apr207-  1621 atatcaegtc cataaegggg aggacgttgt gctcctcacc acctgcaagc atggcaaaga 1681 ctggcaaaag cacaaggatt ctcgctgtga cagagacaac accgagtatg a gaagtatga exon 2  1741 ttatcgggaa atgetgeaca atgccacttt ctgtctggtt cctcgtggtc gcaggcttgg 1801 gtccttcaga ttcctggagg etttgeag gc tgcctgcgtc cctgtgatgc tcagcaatgg exon 3  1861 atgggagttg ccattctctg aagtgattaa ttggaaccaa gctgccgtca taggegatga 1921 gagattgtta ttacag attc cttctacaat caggtctatt catcaggata aaatcctagc exon 4  1981 acttagacag cagacacaat tcttgtggga ggcttatttt tcttcagttg agaagattgt 2041 attaactaca ctagag atta ttcaggacag aatattcaag cacatatcac gtaacagttt exon 5  2101 aatatggaac aaacatcctg gaggattgtt cgtactacca cagtattcat cttatctggg 2161 agattttcct tactactatg ctaatttag g tttaaagece ccctccaaat teactgeagt exon 6  2221 catccatgcg gtgacccccc tggtctctca gtcccagcca gtgttgaagc ttctcgtggc 2281 tgcagccaag tcccagtact gtgcccag at catagttcta tggaattgtg acaagcccct exon 7  2341 accagccaaa caccgctggc ctgccactgc tgtgcctgtc gtcgtcattg aaggagagag 2401 caag gttatg ageagcegtt ttctgcccta cgacaacatc atcacagacg ccgtgctcag exon 8  2461 ccttgacgag gacacggtgc tttcaacaac agag gtggat ttcgccttca cagtgtggca exon 9  2521 2581  gagcttccct gagaggattg tggggtaccc cgcgcgcagc cacttctggg ataactctaa ggagcggtgg ggatacacat caaagtggac gaacgactac tccatggtgt tgacaggagc 143  2641 tgct atttac cacaaatatt atcactacct atactcccat tacctgccag ccagcctgaa exon 10  2701 gaacatggtg gaccaattgg ccaattgtga ggacattctc atgaacttcc tggtgtctgc 2761 tgtgacaaaa ttgcctccaa tcaaagtgac ccagaagaag cagtataagg agacaatgat 2821 gggacag act tctcgggctt cccgttgggc tgaccctgac cactttgccc agcgacagag exon 11  2881 ctgcatgaat acgtttgcca gctggtttgg ctacatgccg ctgatccact ctcagatgag 2941 gctcgacccc gtcctcttta aagaccaggt ctctattttg aggaagaaat a ccgagacat k— pr exllb  3001tgagcgact ttgagg aatccggct gagtgg gggaggggaa gcaagaaggg atgggggtca > apr215  3061 agctgctctc "txttcccagt gcagatcckc tcatcagcag agccagattg tgccaactat 3121 ccaaaaactt agatgagcag aatgacaaaa aaaaaaaagg ccaatgagaa ctcaactcct 3181 ggctcctggg actgcaccag actgctccaa actcacctca ctggcttctg tgtcccaaga Stop  3241 ctaggttggt acagtttaat tatggaacat taaataatta tttttgaaaa aaaaaaaaaa 3301 aaaa 8.3.2 EXT 1 Translation M  1  Q  C  16  L  31  K  R  Y  F  I  L  L  S  A  G  S  A  L  L  F  Y  F  G  G  L  Q  F  R  L  A  L  L  F  Y  F  G  G  L  Q  F  46  H  H  P  s  P  D  H  F  W  P  R  F  R  61  L  R  P  F  V  P  W  D  Q  L  K  R  E  N  P E  76  S  V  H  I  S  P  R  Q  D  A  91  I  Y  K  G  K  K  C  R  M  E  S  C  N F  106  T  L  c  K  K  N  G  F  K  V  Y  V  Y  121  Q  K  G  E  K  I  A  E  S  Y  Q  N  D  P  I  136  A  I  E  G  S  R  F  Y  T  S  C  L  F  V  L  S  L  D  T  L  D  R  S D  181  H  L  w  N  N  G  R  N  H  L  I  F  N  S  G  T  W  P  D  Y  T  E  144  D  V  G  F  150  Q  165 180  L  CTC CAC TTG TGG AAC AAT GGTAGG AAT CAT TTA ATT TTTAAT TTA Y  135  Q  GCG TGC CTC TTT GTC CTG AGTCTG GAT ACT TTA GAC AGAGAC CAG L S P V H N L R Q Y s K V Q S TTG TCA CCT CAG TAT GTG CACAAT TTG AGA TCC AAA GTG CAG AGT L  120  L  GCG GCC ATC GAG GGC TCC AGGTTC TAC ACC TCG GAC CCCAGC CAG A  105  P  CAG CAA AAA GGG GAG AAA ATC GCC GAA AGT TAC CAA AACATT CTA A  90  D  TTC ACC CTT TGC AAG AAA AACGGC TTC AAA GTC TAC GTATAC CCA Q  75  S  AGC ATC TAC AAA GGC AAG AAGTGC CGC ATG GAG TCC TGCTTC GAT F  60  D  TCC AGC GTG CAC ATT TCC CCCCGG CAG AAG CGA GAT GCCAAC TCC S  45  D  CCT CTG CGC CCC TTC GTT CCTTGG GAT CAA TTG GAA AACGAG GAT S  30  A  TTG CAC CAC c c c AGT CCG GATCAT TTC TGG CCC CGC TTCCCG GAG A  15  A  TCG AGG AGC CAC AGC CGG AGAGAA GAA CAC AGC GGT AGGAAT GGC L  166  K  TGT CTC GCC CTT TTG TTT TATTTC GGA GGC TTG CAG TTTAGG GCA C  151  A  ATG CAG GCC AAA AAA CGC TATTTC ATC CTG CTC TCA GCTGGC TCT  D  195  196  t a t t c c g g c a c t t g g c c t g a c t a c a c c g a g g a c g t g ggg t t t  gac  210  a t c ggc c a g gcg a t g c t g g c c a a a g c c agc a t c a g t a c t gaa a a c  225  I  G  211  F  226  Q  R  P  M  N  L  F  A  D  K  V  241  P  R  T  G  G  A  S  t t c cga ccc aac t t t H  S  I  I  S  P  T  L  E  F  256  P  L  R  K  D  gat g t t t c t a t t ccc c t c t t t  E  K  N  S  R  G  F  L  K  F  Y  M  L  V  F  t c t aag gat  240  t t g aag t t c aac a c c  255  N  c a t c c c a g g a c a gga ggg gag agg ggg t t t I P  T  K G  K  R  a t c c c t c c t c t c agg a a g t a c a t g c t g g t a t t c a a g ggg a a g agg Y  L  271  T  G  I  G  S  D  T  R  N  A  L  Y  L  28 6  T  G  I  G  S  D  T  R  N  A  L  Y  K  301  D  W  Q  K  H  K  D  S  R  C  D  316  E  Y  E  K  Y  D  Y  R  E  M  L  H  331  T  F  C  L  V  P  R  G  R  R  L  G  S  34 6  L  E  A  L  Q  A  A  C  V  P  V  M  N  361  G  W  E  L  P  F  S  E  V  I  N  W  A  37 6  A  V  I  G  D  E  R  L  L  L  Q  I  T  391  I  R  S  I  H  Q  D  K  I  L  A  L  Q  4 06  T  Q  F  L  W  E  A  Y  F  S  S  V  I  421  V  L  T  T  L  E  I I  Q  D  R  t c t t c a g t t gag  I  H  4 36  I  S  R  N  S  L  I  W  N  K  H  P  L  451  F  V  L  P  Q  Y  S  S  Y  L  G  D  4 66  Y  Y  Y  A  N  L  G  L  K  P  P  S  K  4 81  A  V  I  H  A  V  T  P  L  V  S  Q  S  V  4 96  L  K  L  L  V  A  A  A  K  S  Q  Y  Q  511  I  I  V  L  W  N  C  D  K  P  L  P  52 6  H  R  W  P  A  T  A  V  P  V  V  V  I  E  541  S  K  V  M  S S R  F  L  P  Y  gga gag agc aag g t t a t g agc agc c g t t t t I  I  55 6  T  D  A  V  L  S  L  D  E  D  T  V  571  T  T  E  V  D  F  A  F  T  V  W  Q  S  58 6  E  R  I  V  G  Y  P  A  R  S  H  F  W  601  S  K  E  R  W  G  Y  T  S  K  W  T  N  616  S  M  V  L  T  G  A  A  I  Y  H  K  Y  Y L  Y S  H  Y  L  P  A  S  L  K  N  630  M  cac t a c c t a t a c t c c c a t t a c c t g c c a gcc agc c t g aag aac a t g 145  615  Y  t a c t c c a t g g t g t t g a c a gga g c t g c t a t t t a c cac a a a t a t t a t H  600  D  a a c t c t a a g gag cgg t g g gga t a c a c a t c a aag t g g a c g a a c gac Y  585  D  c c t gag agg a t t g t g ggg t a c c c c g c g c g c a g c c a c t t c t g g g a t . N  57 0  F  t c a a c a a c a gag g t g g a t t t c gcc t t c a c a g t g t g g c a g agc t t c P  555  L  a t c a t c a c a gac g c c g t g c t c agc c t t gac gag gac a c g g t g c t t S  54 0  N  c t g c c c t a c gac aac  D  525  E  aaa cac cgc tgg c c t gcc a c t g c t g t g c c t g t c g t c g t c a t t gaa G  510  A  gcc cag a t c a t a g t t c t a t g g a a t t g t gac aag c c c c t a c c a gcc K  4 95  C  c c a g t g t t g aag c t t c t c g t g g c t gca gcc aag t c c cag t a c t g t A  4 80  Q  a c t gca g t c a t c c a t gcg g t g acc c c c c t g g t c t c t cag t c c cag P  4 65  F  c c t t a c t a c t a t g c t a a t t t a ggt t t a aag c c c c c c t c c aaa t t c T  4 50  F  gga t t g t t c g t a c t a c c a cag t a t t c a t c t t a t c t g gga g a t t t t P  4 35  G  aag cac a t a t c a c g t aac agt t t a a t a t g g aac aaa c a t c c t gga G  420  F  aag a t t g t a t t a a c t a c a c t a gag a t t a t t c a g gac a g a a t a t t c K  4 05  E  c a g cag a c a c a a t t c t t g t g g gag g c t t a t t t t K  390  R  t c t a c a a t c agg t c t a t t c a t cag gat aaa a t c c t a gca c t t aga Q  375  P  c a a g c t g c c g t c a t a ggc g a t gag a g a t t g t t a t t a c a g a t t c c t S  360  N  a g c a a t gga t g g gag t t g c c a t t c t c t gaa g t g a t t a a t t g g a a c Q  345  L  a g a t t c c t g gag g c t t t g c a g g c t gcc t g c g t c c c t g t g a t g c t c S  330  F  g c c a c t t t c t g t c t g g t t c c t c g t g g t c g c agg c t t ggg t c c t t c R F  315  N  a a c a c c gag t a t gag aag t a t g a t t a t cgg gaa a t g c t g c a c a a t A  300  R D  ggc a a a gac t g g c a a aag c a c a a g g a t t c t c g c t g t gac a g a gac N T  285  H  g t c c a t a a c ggg g a g g a c g t t g t g c t c c t c a c c a c c t g c a a g c a t G  27 0  H  t a c c t g a c a ggg a t a gga t c a gac a c c agg a a t g c c t t a t a t c a c Y  631  A  64 5  V  D  64 6  Q  V  661  S  A  N  C  E  D  I  L  M  N  F  L  A  V  T  K  L  P  P  I  K  V  T  Q  67 6  Q  Y  K  E  T  M  M  G  Q  T  S R  691  W  A  D  P D  H  F  A  Q  R  Q  S  C  706  T  F  A  S  W  F  G  Y  M  P  L  I  H  721  M  R  L  D  P  V  L  F  K  D  Q  V S  R  K  K  Y  R  D  I  E  R  L  TTG AGG AAG AAA TAC CGA GAC ATT GAG CGA CTT  146  720  I  CAG ATG AGG CTC GAC CCC GTC CTC TTT AAA GAC CAG GTC TCT ATT L  7 05  S  AAT ACG TTT GCC AGC TGG TTT GGC TAC ATG CCG CTG ATC CAC TCT Q  690  M  CGT TGG GCT GAC CCT GAC CAC TTT GCC CAG CGA CAG AGC TGC ATG N  675  A S  AAG CAG TAT AAG GAG ACA ATG ATG GGA CAG ACT TCT CGG GCT TCC R  660  K  GTG TCT GCT GTG ACA AAA TTG CCT CCA ATC AAA GTG ACC CAG AAG K  736  L  GTG GAC CAA TTG GCC AAT TGT GAG GAC ATT CTC ATG AAC TTC CTG  TGA  735  Appendix 8.4 8.4.1  EXT 2  EXT 2 - cDNA showing sequence and primer positions (Ref: GenBank Accession: NM_000401 Version NM_000401.1)  EXT 2 exon 1  1 tcgaggttgc tgcccggaag cctctgtagg tatctagtct gagaatcatc actttgaata 61 tttaagctat cagtgacaac ttccaccaga tggcgccaaa gtacatctgg gaccagaagg 121 gatttggatc ctgtagccag acccacaact ttaccaaacc aacatcgcag gcccaggggt 181 catttcatta acctctcaat aacatcgctc tgaattttaa tttaattttt tagtttccac 241 ttactgcttt atgacagcgg ttttagtgtg catggatagg gctaaatcat gtaaataata 301 gagaaagata caaaacaaaa atgcgttttt ttltttUU tttttggaga cagggtcttg 361 ctctatcacc caggctggag tgcagtggca cgaccacggc ccactgcagc cttgacctcc 421 tgggctcaag caatcttcct gcctctgcct cctaagtagt tgggactaca agcgtgtgct 481 acgatgccta gttaactttt tattttttgt agagatgggt cttgctctgc tgcccaggct 541 ggtctcaaac tcttgggctc aagcgatcct ctcgtttagg cctccccaaa tgctggaatt 601 acaggcgtga gccaccttgc ctcgccataa atgcttccat ttccgcctcg acaactactc 661 cacctgaagc tgttcatttc ttcttgcatt ccttccagaa aaaagttata cacatgcctg 721 aatataagca cctactttat atatttctcc ctcttgtttt tgcatatgca tagtttacct 781 aaaagtgact tgcccgctgt tttggactac gctttgatct taactaatat cttggagata 841 tttccttacc caaatatatt gcactatctc acattactca aatcaatcaa attccataat 901 ttatttcgat tgtgtctagc atttcgctat gattagaaag aatgctgtca tggaactttt 961 tgacaaacat tgttgagaat atccataggg caaactccgt acagagagct tgttggaatg 1021 aagggtacca gcattttccg tttgatggat agtaccaaat tgccctccag gaatgttata 1081 cgctcaccag aactgattat aataaaacgt ctacatattt gttagtttta taagcaacgc 1141 gtggtgtctc gtttgggttt aaggattctt taattatgaa tgagg i ctgtc tgagcatttc exon 1  147  1201 actgcggagc ctgagcgcgc ctgcctggga aaacactgca gcggtgctcg gactcctcct 1261 gtccagcagg aggcgcggcc cggcagctcc cgcatgcgca gtgcgctcgg tgtcagacgg 1321 cccggatccc ggttaccggc ccctcgctcg ctgctcgcca gcccagactc ggccctggca 1381 gtggcggctg gcgattcgga ccgatccgac ctgggcggag gtggcccgcg ccccgcggca 1441 tgagccggtg accaagctcg gggccgagcg ggaggcagcc gtggccgagjg taagcgcggc intron 1  1501 tctccagggc agcggccggg cgggcgctga ggcgagggct ctggcctccg ggggccgctg 1561 ctgggtcggg acaagggccg agggagcgcg gccgcgcgga ggctccctgg aggcccgtgg 1621 gctgcga E X T 2 exon l a  1 tcctccggcg gcggccgcgc tttcagcatc ttggtaccca cctgttctcc tagccaacct 61 tcgcccccag tccgctcctt cctttcctcc tgcgacccgc cctccgccct ccgcggcgac 121 ccctcccttc ctgctgccac cttcccgcca g l^cacaggga tctgattcct cccaggggga exon la  181 tgtcctgcgc ctcagggtcc ggtggtggcc tgcggcatcc cttgcggtgc cagaagccgt 241 gggacgag gt acggaagggg ccaggggcat gtaaggccgg ggactgggtg gtcgggggcg y  intron la  301 tgtcaggccg gggactgggt gaccgggaac tagatggccg ggggcgtgtt a E X T 2 exon l b  1 acattcagtc tgttgcagtg tcatatgtca tgtagcctct ggaaaatgga agtgaataaa 61 gcaaacgtca gtattaaact agtataagcc ctttgaaagg gcctgggatg cctgaagcat 121 acttcaagaa ccagtgttct aagattttgg tatgaagcat ttgctagcct cctaaactga 181 gctctgaagc gtttcctctt ttttttctga actctggaat aatttgtgga aaattggaat 241 tattatttct tgaatatttg gtagaacttg gaaaattttc tgggcctgga attttcttta 301 taggaagatt tttaaacttt tgattcagtg tctttaajtgt tatagagcta ctcagagttg exon lb  361 ctgtttctcc ttgagatgct tttglgtaagt atattttaaa ataatttttc catgttatct intron lb  All gagttttcaa atgtactggc ataaattcat tgataccatc ttatctttta aatatatgca 481 gcatttagag ttatgttccc cttttcaggt atttatttgc acctttttcc ttgaattctt 148  541 gattaatctt accagaattt tattagtctg tttttaaaca aacaactttt agccttgttg 601 accatttcta ttttgttttt taattaattt ctgtgctttt atttattatt ttctcctgtt 661 atctttggtt ttactttgtt gtgatatgtc ttttttatag ttaatctgca actcataaag 721 atttgtgaag ctcatgtgtg aatacagttt ttgttccctt aacctcaatt ttgtcataca 781 tagaagctat ttttcaagct atggaggacc cattattggg tggtataatt gatctagtag 841 gt  E X T 2 exon 2  1 tgcactccag cctgagtgac agagtgaaac cctgtctcaa aacaaaacaa aacaaaaaaa 61 aaggttgaat agtcttttca agtgtcattt gccatcctaa atacttggtt tttcttattt 121  I  •  ctctcccctg gtgacce g gagtgtgaggaagaggctgtct gtgtcattat gtgtgcgtcg ex 2a exon 2 r  r  181 gtcaagtata atatccgggg tcctgccctc atcccaagaa tgaagaccaa gcaccgaatc 241 tactatatca ccctcttctc cattgtcctc ctgggcctca ttgccactgg catgtttcag 301 ttttggcccc attctatcga gtcctcaaat gactggaatg tagagaagcg cagcatccgt 361 gatgtgccgg ttgttaggct gccagcc gacagtcccatcc cagagcge gg ggatctcagt *  ex 2A26  ^  421 tgcagaatgc aca cgtgttt tgatgtctat cgctgtg get tcaacccaaa gaacaaaatc *  ex 2AH  ^  481 aaggtgtata tetatgetet gaaaaagtac gtggatgact ttggcgtctc tgtcagcaac 541 accatctccc gggagtataa tgaactgetc atggccatct cagacagtga ct actacact  U——  601 gatgacatca accg gg cctg tctgtttgtt ccctcc atcg atgtgcttaa ccagaacaca ex2A4  >  *<—-—ex 2A25  661 ctgcgcatca aggagacagc acaagegatg gcccagctct. ctag gtatct cacactcata <  intron 2  721 cagcccagcc cccaggagat acttgagt gg ccctcaggga actaaaggg a agggaaggat <  ex 2b  >  781 gggaatgett ctgctcttga gttggtttcc cgatgctgtc ttcttgeagg acggggtgtg 841 ttggagggac tgac  E X T 2 exon 3  1 tatatttcca aattatgaca taattttatg ttcttttact atataacttt aagggttgca 149  61 tagtattcca ttttgcagat gttctaccat atatttaacc aggcttctct aatgtatttt 121 gtgtttcttt aaccaaatgg tgaacatttg ggtagttttc aacttttcat tattagaagc 181 aggtctgtat gggacaagct tgaagtacac gtgcgttcat ttttcccctg tcatggagcc 241 agacttgtgt ctgatgtgct gttgggattt ccaggagttt gctttgcata cctgagaagc 301 ggccctattt gggcttgggg atccttgata gttgttgtct agtaactgac tcttgtcttt 361 tcatalgttga cacattaatt ctccca catt ttaaattttt tgacad gtgg gatcgaggta ^  ex 3a  ~* exon 3  ^  421 cgaatcacct gttgttcaac atgttgcctg gaggtccccc agattataac acagccctgg 481 atgtccccag agacagj gtag gaggcatatg tggggctgtc cttatgat ggl gttcaagatc <—  intron 3  ^<— ex3b  541 attttgttc la tgtgaaatta tattcctaaa tctaccacat actttgtaat cagaattgtt 601 tattaaacta gaaaattgtc ataagtattt tcctcctgaa gatttagaag tgcttaaatc 661 ttttatggaa aaccagttag ggcttatgtc ctggcatacc ctctaaaact gttttcccac 721 tctggattgt gcacttctga gtgtaacaca tccagccccc aaaagtgtga caggcttgtg 781 ctacctctct ctgaattcgg gagcatttgc cacaagtaga tgcacagctt actgagagaa 841 ggt EXT 2 exon 4 1 gtaaatgtgt ttatttataa agtatgacta gggagaggtg aatgggatct gagggaggta 61 gcagagaggc tgtccgtaag gtgtcttctg gactatgatg tgtttcaaaa actgggaagt 121 aaggaaaggg tatttaggac cccgggggaa ggctggtgat tcaaggatag aacgcagctg 181 atggccccga gatgcgtgta taaggcattg tctttataga aaactgactc tgtaaacgtt 241 agctggtttt gataataaag actcagtaat tcctgttcct ctccacagtg tgtatca) gaa 301 taaagtcctt tctttctcat cgl tttaacaa aatactttgc tttcag g igcc ctgttggctg ex 4a  ->exon 4  361 gtggcggctt ttctacgtgg acttaccggc aaggctacga tgtcagcatt cctgtctata 421 gtccactgtc agctgaggtg gatcttccag agaaaggacc^agg j gtaaggt acattcatcc intron 4  481 ca [gccaggtg tgcctttactgkatctgtga gatgttgatg aggtttagtg tggtgggcat ^  ex 4b  * 150  541 caaagcaacc aatacatcag ttacagggta gggtccttga ggcactgagg cacccatctt 601 tcccacctcc atgcagtctc attcatcttg cagttttctc tgtctcctta aattcacagt 661 gctgtctacc aagttttcta agccaggaat ccatgtggta tccttaactc cgttctctcc 721 tttgtttcct atatcaaagt aagaagtcgt attgattctg catcctaaat acttcctatg 781 tctgtctgct ccccgaa E X T 2 exon 5  1 aaaatcagtg gagtgaagac tggtaaggaa cacttactgt cgtaagttta atatcaaagt 61 ttgtcttacc tggactaaca taccagct gc aattttccaa tcacctg ttt ttttcccttg ^  ex 5a  ^  121 tag tccacgg caatacttcc tcctgtcatc tcaggtgggt ctccatcctg agtacagaga 1  >  exon 5  181 ggacctagaa gccctccagg tcaaacatgg agagtcagtg ttagtactcg ataaatgcac 241 caacctctca gagggtgtcc tttctgtccg taagcgctgc cacaagcacc aggtcttcga 301 ttacccacag gtgctacag I g tgagtgtcat tcatta cctc tcgcaaaggc tcaggj agagt U  <——' intron 5  ex 5b  >  361 ttgcttacat gggttaaaat tgagcccagc gaacctgagt tgtttttcag catgcaacta 421 gaattaccca gggggaagaa aacatagcat tgctctttac tggacatgta gaccttcagg 481 tacttggatg tctggtgtct tgtgttcgtg caaagctgct tggcctatga gagtctatac 541 tcctttcaga tattcattat acttcaaaaa ga  E X T 2 exon 6  1 tgaggtaagt actgtaagag atgtcagaca gtgtgccgtg gtgtgtttac atagtacata 61 gggcttaaag agacccattt gcaggaagtc acgttgttag ctgtctaagg gaagactttg 121 acattgacct tgaacatttt cagaaggcca acagtggtgg cattgaagca atactgaaga 181 gtagaaatat taatacaaaa cattgcagcc atttaaactt ttcaagtttt acaggtgtga 241 gctgttgtct tttggcattt ttgtgtcaag atgcctcagt attgcttggc gtcaaccctt 151  I  301 gtagaaactttgtggtctgtagggatcaaagttagtggatcagcaaaa  ctagtttgtaat ex 6a  361 ctcttgcctcitttgtgttcctgcag gaggctactttctgtgtggttcttc gtggagctcg \  exon 6  All gctgggccag gcagtattga gcgatgtgtt acaagctggc tgtgtcccgg ttgtcattgc 481 agactcctat attttgcctt tctctgaagt tcttgactgg aagag gtggg tagtacctcc intron 6  541 tagtaas etc tacattagtg gttctgcgte tattacaaat aaaatctcct caggtcattg <  L  ex 6b  —  601 taatgtatac cctgttcaag aactactaca gatagttttt ctctattttc cattaggaga 661 gttagtacac tggtctagag cagttcacaa accaaggcca gtttgeagge tggctgtttt 721 tgtaagtcaa gttttattga aacacagccg tgtcccttcc tttaegtata gtctggctgt 781 tattgtgtca cattggtaga gttaagtaat tgcaacagaa attggatgac atacagggct 841 taaatatcac tatctggect ttcatcacag gggtccccaa ctcccgggct gtggcctgtt 901tggagccggg E X T 2 exon 7  1 tatgecagat aaatgaatag atttgeatag atagctaaag gagaaaagta tttgttaact 61 tagaatggaa taaaggaaga gtgtactagg tgggtgggat ttcacatgea aaggccctct 121 ggtagggcag agcatggtgt gttcaagtga ctgaaataat accagtgtgg ctagagcaca 181 ctagtggagt ggaggcaggg tgaaagatta atggagtagg gagtgggagg taaaaaaatg 241 gagctgtaag agaactcctt tgagaagttc agccagtgaa gaagggaggg gaaagagaca 301 atacttaccg gaa gggatgt ggggctgaag gagg tttggg atgttgtttc tgcttgtgaa ^  ex 7a  ^  361 atgaaacaag actgtgtgta gaaatgcttt ctgtgaaggg ctgtgtgtat gtaaactgtt 421 ttgctgttgt ctccagagca tctgtggttg taccagaaga aaagatgtca gatgtgtaca *• exon 7  481 gtattttgea gagcatcccc caaagacaga ttgaagaaat gcagagaSag Igtaagaggcc intron 7  541 aagtcttggg gaggtgacat gggtggtacc gaaatggtgg cctt gactgg atacagaggg <  ex 7b  601 acaggag ctg aatgectgag tggggtttac ttcctccact agatcaacta gecaaactga > 661 aacgaaagga aattaatgtt aggtgagttg catcaaataa ggtttgaaat aataactctc 721 agagaactgt gcagaggtaa gcctactgca attttagggt cttaccatag cagatgeaaa 152  781 gctgaagctc tttggagggt ttgtagtcac agcaggtgat agtcgtagtg actaagacag 841 ccatggaagc tggaccattt cagggcaata cttctgtgta gctattgacc atgatacatt 901 gcggcacaaa ctagcccagc tt E X T 2 exon 8  1 tttatctgga tactaattgt aagagtatgt acatatgtat aaattcattg agctgtacac 61 aagatttgtg cactttatgt tatataagac aaaatactat aaactctgcc ataacacatg 121 gatattctca tcatcacata atttatcttc tatcttaatt gaatccaatg tgcatttcac 181 ttgctaacat tttattttga ctgcatttga taaatgccaa cttctgatgg cagctggctt 241 gaacagcagg gagcatatgc cctaggcacc cccatcccta caactttggg aataaaggaa 301 ttagcctaac ctggagttga ctatgataga gtatctagtt ttcccactct gtctclgcttg L<—ex 8a  361 ctcacttaaa ad agcattat tttttttata g gcccggtgg ttctgggaag cgtacttcca y  I  ^.  exon 8  421 gtcaattaaa gccattgccc tggccaccct gcagattatc aatgaccgga tctatccata 481 tgctgccatc tcctatgaag aatggaatga ccctcctgct gtg gtaagtg aattcca| gtg intron 8  541 ctagccac at gaggcatggt ccagctgtca gggtgggtgg aaggaaaaat gtactaccat ex 8b  ^  601 tgtaaaggtt atttaaattc tagctttcta agatgagagt gtgcttttta tacttg gggc *~ex8c  661 ctgataaggg cagcataal tt ttgaaacact gacaaaagta aaaaatacgg aagcagcagc 721 ttccagtgtg ttttaagtgc ttacaaagac tgtctattta ttgcagagat aagtaaggag 781 gcatgggtct tgttggaaat caaagacatc ccggtgactt ttgcaattgt aatgcttaga 841 gcttttgaaa aacttctgta age E X T 2 exon 9  1 gtctcttctc ccatctcttt gtccttgtag atttatattc ttttatattc atcaactgcc 61 tttttattgg gtttggggag agaatggaga taaacgcatg ctttaatctg tcatgtttaa 121 ctagaattct tttctcagct gcaaaagttc tcagctcctt ttccagtgat atcagaacca 181 aacttaatta gtccatgcaa attttgagga ggggaagact ttgagcagtt gcttagctct 153  241 gggatctgtc ctggtaaaag ccatcaagcc tgccatgttt gggtttgctg acgatattgg 301 gtcagccata ttgtta cage tgcttttctg acccg tgtta atctgtcctc ttgtagj aagt <—  ex 9a  >  361 ggggcagcgt gagcaatcca ctcttcctcc cgctgatccc accacagtct caagggttca exon 9  421 ccgccatagt cctcacctac gaccgagtag agagectett ccgggtcatc actgaagtgt 481 ccaaggtgcc cagtctatcc aaactacttg tcgtctggaa taatcagaat aaaaaccctc 541 caga^ag gtaa gaagectta ^ tgcctctctc agctggatc a attttggatg gccaaattat intron 9  ex 9b  601 tcacatcctt tgttttaaat aaattttcct gctttgtcaa tagcaatacc atttctgaga 661 cagcatgcct ccatttttct cagtcatctc attcttgttc tagggtggcc catctaactc 721 caagccctgg catactctgt agecacaagt g  E X T 2 exon 10  1 gaagecaatt tgttcattct agttaggaca gtattgagaa ttagtagtgt tacaaggatt 61 tagagaggat aaatatgtat gtatatagta tgtgtgtata tatgtagtat atatgtgtat 121 gtgeagtata tatatttttt attataacaa agatgeatet gtgagaatct cccctgacac 181 agttctacct atggatttga tgagagccgt ggatacaagc tgattctccc atctcatttg 241 tgatgtcatg cttttactac tttatct |cct cacaaaagtt aggag |aatag taaatacctt ex 10a  301 ttctcttttt ccag I attdtc tctggcccaa aatccgggtt ccattaaaag ttgtgaggac -> exon 10  361 tgctgaaaac aagttaagta acegtttett cccttatgat gaaatcgaga cagaagctgt 421 tetggecatt gatgatgata teattatget gacctctgac gagctgeaat ttggttatga 481 \  Igtaagga intron 10  Igg ttttacacag tgtgttt lata tgtttaatat tacttcctat gaetgettgt ex 10b  >  541 cttttctaaa aaagagtatt atatttcctt cttaaaagtc agagttctaa aatcttccag 601 tagagtccaa aaggtgtgcg taagagtgtg ggttatgaag ctgttctttg aagcactgga 661 gaaaccctat tccaaaatgg caactgtgcc ctccactggt tttgggaact cccaagggag 721 agtcccaggg gacaatttca aaagagcatc tatagcattt aacaagcact taattgatgt 781 ctccttgaat accacttccc ttgactcaag cagct  154  E X T 2 exon 11  1 taatacaaat cagggcagtt gagttgcagg gttccattta tccttcattt tgtgaattca 61 ttaagaatgg aacctatttc attaatcata tgttaagaga ttgcgtacct tggcccaact 121 cagtaagcta ttacccccac attaattgaa attcccaagc atatacaaga agattgggag 181 gaagtcagaa tcagcatctg tctttgagtt ttggcagaat aactaacacc tgtttgatgg 241 aacatctcca gaatcccatt atgaccttct taggttatga tggtttgaac ctaggaagtc 301 tgttgatacc tgtttggata actcagcact gaatggttgc tgtctgaatt ggg hcttgat  •  U—  ex 11a  V  361 tgttattatg tgtctgtcct tag Igtct^gc gggaatttcc tgaccggttg gtgggttacc ->•  exon 11  421 cgggtcgtct gcatctctgg gaccatgaga tgaataagtg gaagtatgag tctgagtgga 481 cgaatgaagt gtccatggtg ctcactgggg cagcttttta tcacatiglgta agggggcgca ^—I  intron 11  541 gtcctlggcaa ggtgacaaaa ctgaglagaat gatacacatt ttatttgacc caatttaatt ex lib  *  601 tttcatacct gccaagaggg cttagaaaag ccatattgtg tgacagtatt ttacaaataa 661 agctatcctt tttctaatta taaaagtaat gcacgctcat agtagaaaat atgaaaatag 721 aatgaagaaa agttacttgt aatcctgtca cttcgagata accattttat cattcaggtg 781 ctatttccag cttgccgttt atttatttac ttacttgtat gtatacacag acagttgtaa 841 atattctcat tagcctgctt tttcatggat tgtattgtga gccttttctc atgtcattga 901 catttcttca taaacagtta cttgttagca taattaagat accattactt aatgttttag 961 aagtcatgta taactatttt gctatcgtgg atattacttt ctaaattttg ctattt E X T 2 exon 12  1 tttagctgta ttcatatcga ttgtttgttg ttagctcagc actactgcct catatttttc 61 aggtctctag atccagaaat gggttttttt attttgtaat aagcaaacaa aaaaacccaa 121 aaactaaggt ttacaattca tgggatttac agtagtagac tatgtatgct ttattttttt 181 tgacccaata atttctacac tatttcatat atatggtagt tttagaattg cctcattttt 241 cttctacttt aaaaagcaca cactttggta gaaaatgacc atattgaaca tgcttggtca 301 cttgaccaaa agcattctaa tgcctccttt tacccttcct attaatacag ccttgtgatt 155  361 aatcttatga gagaaagctt gtccccatgc cttggctatg ctgl cccctta tttatcagct \ —  ex 12a  All aaaggd aact gctatttttg aatatttctt ctttctgtct cacttgacag I tatttWtt r  I  exon 12  481 acctgtatac ctacaaaatg cctggggata tcaagaactg ggtagatgct catatg^acj t 541 gtgaagatat tgccatgaac ttcctggtgg ccaacgtcac gggaaaagca gttatcaagg intron 12  601 tagga ggctc tgccactcac ttg ctttgtg atcttgggca aatatctatt atctgagcct ^—  ex 12b  *  661 aggaagttct tgtaactata aattaaatat aggactagat aaactttaag ctctatttta 721 gtttaaggtt ctatgattga tgcggtcaca ttgggaaatt gaagctaggc tttgacaatt 781 taaacatatt ttcttttttt atacagtttc tttagttgca gtttttaacc ttcagtatca 841 gaataaaggc tatgatgatc agtctataaa tcaaaaaatt atattctcaa agctg  EXT 2 exon 13 1 gggaagctgt atttcatcgc ccttatgggt acaagaacaa atggtgttta tacaaggacc 61 ttggcagtga gaaaacagtc attaaacagg aattaaggag cttgtcatca ccacttcttt 121 ccagttacag aaggcaaaag ccctccaagc cttttttatt gggcccttgt gagttctgcc 181 gttggctgag ccagacagag ttgaatggag gaatggcgag gtgtgtgtgt gtgtgtgtgc 241 acgcgcatgc aacatctcag cttacaacac aaaagaatgc agtgtggtgt cacaagcatg 301 attttattl^t ccttgacact gacagccagg^ tatgtttttg tcctcctctg gcag gtaacc ex 13a  exon 13  361 ccacgaaaga aattcaagtg tcctgagtgc acagccatag atgggctttc actagaccaa 421 acacacatgg tggagag gta agtgagcctc caaccaaaag I gcgccttag cctctgatct if ^  intron 13  ex 13b  481 etaJtttcctg ccttag gect gtttatgggg ctttgttgga gatataagga cagcagctgg 541 tagecatagt cacctccatg tgcactgtgg gaattgggtt agttcaagcc caggtcaccc 601 aaagaattaa tttggaatgc tactcactca atttgtaatg gctggaaggg tcttaaaaat 661 atagtgggcc ttaagctcca gaagccaaat tctccatgtg gactaagcag ttaaccatct 156  721 acagtcattg agtggaagct agttaattcc aaggaaatac tggattattt ttc  EXT 2 exon 14 1 cctttttaag aacctgggag cagactgtgg ctactgagct ttttttgttg atgttgaaca 61 ttatgtattt tgctgttatc tctcaacctc ttgaacatac tatcttttct ccctgccccc 121 atccttctca ttctgct caa acccctcctc cccacctcct d tccaaatcc cacag Igtce^g ex 14a  >  181 agtgcatcaa caagtttgct tcagtcttcg ggaccatgcc tctcaaggtg gtggaacacc exon 14  241 gagctgaccc tgtcctgtac aaagatgact ttcctgagaa gctgaagagc ttccccaaca 301 ttggcagctt atgaaacgtg tcattggtgg aggtctgaat gtgaggctgg gacagaggga 361 gagaacaagg cctcccagca ctctgatgtc agagtagtag gttaagggtg gaaggttgac 421 ctacttggat ctt  ggcatgc acccacctaa cccacj ac tttct caagaacaag aacctagaat ^  ex 14c  481 gaatatccaa gcacctcgag ctatgcaacc tctgttcttg tatttcttat gatctctgat 541 gggttcttct cgaaaatgcc aagtggaaga ctttgtggca tgctccagat ttaaatccag 601 ctgaggctcc ctttgttttc agttccatgt aacaatctgg aaggaaactt cacggacagg 661 aagactgctg gagaagagaa gcgtgttagc ccatttgagg tctggggaat catgtaaagg 721 gtacccagac ctcactttta gttatttaca tcaatgagtt ctttcaggga accaaaccca 781 gaattcggtg caaaagccaa acatcttggt gggatttgat aaatgccttg ggacctggag 841 tgctgggctt gtgcacagga agagcaccag ccgctgagtc aggatcctgt cagttccatg 901 agctattcct ctttggtttg gctttttgat atgattaaaa ttatttttta ttcctttttc 961 tactgtgtct taaacaccaa ttcctgatag tccaaggaac cacctttctc ccttgatata 1021 tttaactccg tctttggcct gacaacagtc ttctgcccat gtctgggaac acacgccagg 1081 aggaatgtct gataccctct gcatcaagcg taagaaggtc ccaaatcata accattttaa 1141 gaacagatga ctcagaaacc tccagaggaa tctgtttgct tcctgattag atccagtcaa 1201 tgttttaaag gtattgtcag agaaaaacag agggtctgta ctagccatgc aaggagtcgc 157  1261 tctagctggt acccgtaaaa gttgtgggaattgtgacccccatcccaagg ggatgccaaa 1321 atttctctca ttcttttggt ataaacttaa cattagccag ggaggttctg gctaacgtta 1381 aatgctgcta tacaactgct ttgcaacagt tgctggtata tttaaatcat taaatttcag  1561 agattagcca cagtttgggc tttagccaca acatatgtcc ccaaaacaca aaatacataa  8.4.2  1  EXT 2Translation  M C A S V K Y N I R G P A L I A T G TGT GCG TCG GTC A A G TAT A A T ATC CGG GGT CCT GCC CTC ATC  16  P R M K T K H R I Y Y I T L F C C A A G A A T G A A G A C C A A G C A C C G A A T C T A C TAT A T C A C C CTC TTC  31  S I V L L G L I A T G M F Q F TCC ATT GTC CTC C T G G G C CTC A T T GCC A C T GGC A T G TTT C A G TTT  46  15  30  45  W P H S I E S S N D W N V E K TGG CCC CAT TCT ATC G A G TCC TCA A A T G A C TGG A A T GTA G A G A A G R  60  61  S I R D V P V V R L P A D S CGC A G C A T C CGT G A T GTG CCG GTT GTT A G G CTG C C A GCC G A C A G T  75  76  P I P E R G D L S C R M H T C CCC ATC CCA G A G CGG GGG GAT CTC AGT TGC A G A A T G CAC A C G TGT  90  91  F D V Y R C G F N P K N K I K TTT G A T GTC T A T C G C TGT G G C TTC A A C C C A A A G A A C A A A A T C A A G  105  106  V  Y I Y A L K K Y V D D F G V GTG TAT A T C TAT GCT CTG A A A A A G TAC GTG GAT G A C TTT GGC GTC  120  121  S V S N T I S R E Y N E L L M TCT GTC A G C A A C A C C ATC TCC CGG G A G TAT A A T G A A CTG CTC A T G  135  136  A I S D S D Y Y T D D I N R A GCC ATC TCA GAC AGT GAC TAC TAC ACT GAT GAC ATC A A C CGG GCC  150  151  C  L F V P S I D V L N Q N T L TGT CTG TTT GTT CCC TCC A T C G A T G T G CTT A A C C A G A A C A C A C T G  166  R l K E T A Q A M A Q L S R W CGC ATC A A G G A G A C A GCA C A A GCG A T G GCC C A G CTC TCT A G G TGG D  181  R G T N H L L F N M L P G G GAT CGA GGT A C G A A T CAC CTG TTG TTC A A C A T G TTG CCT G G A GGT  158  165  180  195  196  P P D Y N T A L D V P R D R A CCC CCA GAT TAT A A C A C A GCC CTG GAT GTC CCC A G A GAC A G G GCC  211  L L A G G G F S T W T Y R Q G CTG TTG GCT GGT GGC G G C TTT TCT A C G T G G A C T T A C C G G C A A G G C  225  226  Y D V S I P V Y S P L S A E V TAC GAT GTC AGC ATT CCT GTC TAT AGT CCA CTG TCA GCT G A G GTG  240  241  D L P E K G P G P R Q Y F L L GAT CTT C C A G A G A A A G G A C C A GGT C C A CGG C A A T A C TTC CTC CTG  255  256  S S Q V G L H P E Y R E D L E TCA TCT C A G GTG GGT CTC CAT CCT G A G TAC A G A G A G G A C CTA G A A  270  A 271  L Q V K H G E S V L V L D K GCC CTC C A G GTC A A A CAT GGA G A G TCA GTG TTA GTA CTC GAT A A A  210  2850  286  C T N L S E G V L S V R K R C TGC A C C A A C CTC T C A G A G GGT GTC CTT TCT GTC CGT A A G CGC TGC  301  H K H Q V F D Y P Q V L Q E A C A C A A G C A C C A G GTC TTC G A T T A C C C A C A G GTG CTA C A G G A G GCT  316  T F C V V L R G A R L G Q A V A C T TTC TGT GTG GTT CTT CGT G G A GCT CGG CTG GGC C A G G C A G T A  330  331  L S D V L Q A G C V P V V I A TTG A G C GAT GTG TTA C A A GCT GGC TGT GTC CCG GTT GTC ATT GCA  345  346  D S Y I L P F S E V L D W K R G A C TCC TAT A T T TTG CCT TTC TCT G A A GTT CTT G A C TGG A A G A G A  360  361  A S V V V P E E K M S D V Y S GCA TCT GTG GTT GTA C C A G A A G A A A A G A T G TCA GAT GTG TAC AGT I  376  391  L Q S I P Q R Q I E E M Q R ATT TTG C A G AGC ATC CCC C A A A G A C A G ATT G A A G A A ATG C A G A G A  Q A R W F W E A Y F Q S I K A C A G GCC CGG TGG TTC TGG G A A GCG T A C TTC C A G TCA ATT A A A GCC I  300  315  375  390  405  406  A L A T L Q I I N D R I Y P ATT GCC CTG GCC ACC CTG CAG ATT ATC A A T GAC CGG ATC TAT CCA  420  421  Y A A I S Y E E W N D P P A V TAT GCT GCC ATC TCC TAT G A A G A A TGG A A T GAC CCT CCT GCT GTG  435  436  K W G S V S N P L F L P L I P A A G TGG GGC A G C GTG A G C A A T C C A CTC TTC CTC CCG CTG A T C C C A  450  451  P Q S Q G F T A I V L T Y D R C C A C A G TCT C A A GGG TTC A C C GCC A T A GTC CTC A C C TAC G A C C G A  465  466  V  E S L F R V I T E V S K V P GTA G A G A G C CTC TTC C G G GTC A T C ACT G A A GTG TCC A A G GTG CCC  480  481  S L S K L L V V W N N Q N K N AGT CTA TCC A A A CTA CTT GTC GTC TGG A A T A A T C A G A A T A A A A A C  495  159  496  P P E D S L W P K I R V P L K CCT C C A G A A GAT TCT CTC TGG CCC A A A A T C CGG GTT C C A TTA A A A  510  511  V V R T A E N K L S N R F F P GTT GTG A G G A C T GCT G A A A A C A A G TTA A G T A A C CGT TTC TTC CCT  525  526  Y D E I E T E A V L A I D D D TAT GAT G A A ATC G A G A C A G A A GCT GTT CTG GCC ATT GAT GAT GAT  541  I I M L T S D E L Q F G Y E V A T C A T T A T G CTG A C C TCT G A C G A G C T G C A A TTT GGT TAT G A G GTC  555  556  W R E F P D R L V G Y P G R L TGG C G G G A A TTT CCT G A C C G G TTG G T G GGT T A C C C G GGT CGT CTG  570  571  H L W D H E M N K W K Y E S E CAT CTC TGG G A C CAT G A G A T G A A T A A G TGG A A G TAT G A G TCT G A G  540  585  586  W T N E V S M V L T G A A F Y T G G A C G A A T G A A G T G TCC A T G G T G CTC A C T G G G G C A GCT TTT TAT  600  601  H K Y F N Y L Y T Y K M P G D C A C A A G T A T TTT A A T T A C C T G T A T A C C T A C A A A A T G CCT G G G G A T  615  616  I  K N W V D A H M N C E D I A ATC A A G A A C TGG GTA GAT GCT CAT A T G A A C TGT G A A GAT ATT GCC  630  631  M N F L V A N V T G K A V I K A T G A A C TTC CTG GTG GCC A A C GTC A C G G G A A A A G C A GTT A T C A A G  645  646  V T P R K K F K C P E C T A I GTA A C C C C A C G A A A G A A A TTC A A G TGT CCT G A G TGC A C A GCC A T A  660  661  D G L S L D Q T H M V E R S E GAT G G G CTT T C A CTA G A C C A A A C A C A C A T G GTG G A G A G G T C A G A G  675  676  C I N K F A S V F G T M P L K TGC A T C A A C A A G TTT GCT T C A GTC TTC G G G A C C A T G CCT CTC A A G  691  V V E H R A D P V L Y K D D F GTG GTG G A A C A C C G A GCT G A C CCT GTC CTG TAC A A A G A T G A C TTT P  706  E K L K S F P N I G S L CCT GAG AAG CTG AAG AGC TTC CCC AAC ATT GGC AGC TTA TGA  160  690  705  Appendix 8.5 Genotyping Appendix 8.5.1  Short Tandem Repeats (STR) markers  Marker name Chromosome Gene D-number  A03/04  85  547  A01/02  905  13  216  221  8 EXT 1 D8S555  8 EXT 1 D8S85  85 EXT 1 D8S547  11 EXT 2 D11S905  11 EXT 2 D11S1313  19 EXT 3 D19S216  19 EXT 3 D19S221  Gene symbol Heterozygote Frequency # o f alleles Allele frequencies  Z24446 75.0 %  N/A  Z24154 71.4%  11 EXT 2 D11S9 03 Z16529 82.1%  Z16575 71.4%  Z23608 89.3%  Z16743 81.5%  Z17017 89.29%  5 1 - .012 2 - .332 3-.188 4 - .250 5-.219  6 1 - .054 2-.107 3 - .321 4 - .464 5-.018 6 - .036  6 1 - .125 2-.411 3 - .036 4-.161 5 - .196 6 - .071  8 1 - .143 2 - .214 3 - .411 4 - .107 5 - .054 6-.018 7 - .036 8-.018  10 1 - .018 2-.125 3 - .232 4-.071 5 - .089 6-.143 7 - .054 8-.196 9 - .054 10-7018  5 1 - .259 2-.315 3 - .241 4-.130 5 - .056  Size o f fragments  1 - .177 2-.173 3 - .167 4-.169 5 - .165 6 - .175 7 - .171  1 - .083 2-.081 3 - .079 4 - .075 5 - .073  1 - .193 2-.191 3 - .189 4 - .187 5 - .195 6 - .185  1 - .101 2 - .099 3 - .105 4 - .107 5 - .103 6 - .109  1 - .222 2 - .224 3-.210 4 - .226 5 - .208 6 - .228 7-.212 8 - .220  1 - .202 2 - .198 3 - .196 4 - .200 5 - .192 6 - .190 7 - .204 8 - .194 9 - .188 1 0 - .184  1 - .191 2 - .185 3 - .179 4 - .187 5 - .189  10 1 - .232 2 - .089 3 - .089 4 - .071 5 - .071 6 - .179 7 - .107 8 - .125 9 - .018 1 0 - .018 1 - .207 2 - .209 3 - .201 4 - .195 5 - .197 6 - .199 7 - .205 8 - .203 9 - .211 1 0 - .191  P C R Temp  58°C  58°C  60°C  59°C  60°C  58°C  60°C  1 - .464 2-.214 3 - .107 4 - .036 5 - .089 6 - .018 7 - .071  78.9 %  161  60°C  8.5.2  Short Tandem Repeats (STR) Primer Sequences  Marker Name A03/04  Sequence  caagatggattcaaagccaaa  cattcctaaggagggttcca 85  agctatcatcaccctataaaat  547  tttaaaatgcatgtggccttc  ccttgcccatcacttacac tacacacagcctcatggctc  A01/02  caacacttcgatgttccttcc  905  tctcctgtccctcacacaca  13  taacgatttncaacgtctaagc  agctgagagcgcatgtataa acaggggccaaataggtttc  gggaattttgacttcatatgca  216  ggagacctctggctaggta  221  gagcaagactctgactcaac  aggtacttagttactgactttg acccagtctccagtagcag  162  Appendix 8 . 6 Data 8.6.1  C y r i l l i c F a m i l y Pedigrees  0-  -o  1:1  l:2 GH 1-4  Blood 1:3  11:4  11:5  11:6  -o  11:1 GH 1-3  ll:2 GH 1-2  Blood Cells 111:1 GH 1-1  Family 1  163  m-  "0 l:2  1:1  ll:3 NI2-7  If IV:4  IV:5  IV:6  -O 111:10  IV:7  L>  IV:8  ll:4 NI 2-6  ll:7  5  D-  lll:6  111:11  •A" •  111:12  IV:9  lll:8  IV:10  IV:11  •  lll:7  D-  6±  IV:12  IV: 1 NI2-2  164  ll:2  III2 NI2-5  111:1 NI 2-4  ^  Family 2  -o  11:1  Blood C«IIs ^  IV:2 NI2-1  B  IV:3 NI2-3  o-  11:1 H O 3-1  111:12 H O 3-5  1  Blood  IV:3 H O 3-2  IV:4 H O 3-3  lll:3 HO 3-13  Blood  IV:5 H O 3-6  111:14 H O 3-14  ^ | ^ Blood  IV:6 H O 3-7  ^Bbod  IV:7 HO 3-16  111:15 H O 3-9  Blood  IV:8 HO 3-15  ^ [^B  IV:9 HO 3-17  IV:10 H O 3-11  —o  112  11:4  AT  IV: 1 HO 3-19  Family 3  165  IV:2 HO 3-20  111: 5 H 0 3-  lll:4 H O 3-8  T^Btoo.  IV:11 HO 3-12  IV:12 Hoi 3-10  JZr 1:1  11:1  Blood  o  lll:2  111:1 HE  4-2  4  HE  4-1  Blood  IV: 1 HE  Blood  4-4  Blood  IV:2 HE  4-3  Family 4  166  0 - r - 0 1:1  l:2  o  Blood  ll:2 TA5-6  I  Blood  Q  11:3 :3 TA5-5  ll:4  Blood  :4  Blood  111:1 TA5-1  lll:2 TA5-2  Blood  4  IV: 1 TA5-3  Blood  IV:2 TA5-4  Family 5  167  111 3  Q 1:1  4=  Blood  #  •  ll:4  ll:2 B l 6-3  Blood  O  l:2  LT l:5  Blood Cells  ti lll:3 B l 6-2  lll:2 B l 6-1  Family 6  168  Q  l:2  111:1  Blood  Blood  O  U IV:2  IV: 1 FR  8-4  FR  8-2  4  1  FR8-1  FR8-3  Blood Cells  V:1  IV:3  V:2  Family 8  169  IV:6  IV:4  IV: 5  D-  l:2  1:1  ll:2 BO 16-5  •o  11 :B  ll:9  V— 111:1 BO 16-2  lll:2 BO 16-3  Blood  6" IV: 1 BO 16-1  IV:2 BO 16-4  F a m i l y 16  170  11:3  ll:6  ll:5  CHr-6 ll:7  o  D-  Blood  -o  1:4  1:3  11:1 KE 17-7  1:1  l:2  KE 17-5  KE 17-6  1  ll:2  o  11:3  KE 17-2  Blood  111:1 KE 17-1  Blood  11:4  Blood  lll:2  111 3  KE 17-3  KE 17-4  F a m i l y 17  171  111:4  111:5  111:6  •  1:1  I  Blood Cells  IV: 1  WH  18-1  l:2  1  Blood  IV:2 WH  IV: 3  18-4  WH  Family  172  18  18-5  8.6.2  Short tandem repeat (STR) Gels  Family 1 EXT 1  Family Marker Member  Family Marker Member  1_1: c, a 1_2: a, b 1_3: c, c  1, 4  1_2: 2, 4 1 3: 1, 3  Family 2 EXT 1  EXT 2 Family Marker Member  Family Marker Member  2_1: b, c 2_2: b, c 2_3: a, d 2_4:a, b 2_5:c d  2_1: 3, 1 2_2: 1, 2 2_3: 3, 2 2_4:1, 3 2 5:l 2  r  r  173  Family 3  EXT 1  Family Member  Marker  3-1  2, 3  3-2  1, 1  3-3  1, 1  3-4  (1)  3-5 3-6  1, 1 2, 3  3-7  3, 3  3-8  1, 1  3-9  1, 1  3-10  1, 1  3-11  1, 1  3-12  1, 1  3-13  1, 1  3-14 3-15  1, 1 1, 3  3-16  1, 3  3-17  1, 1  3-18  2, 3  3-19  2, 3  3-20  2, 3  3-21  1, 4  174  Family 3  EXT 2  Family Member  Marker  3-1  g,e  3-2  a, e  3-3  c, e  3-4  c, c  3-5  c, c  3-6  g> c  3-7 3-8  d, g  3-9  e,  3-10  e, d  3-11  e, d  3-12 3-13  d, e  3-14  c, d  3-15  d,  3-16  d, d  3-17 3-18  b, d  3-19 3-20  f, d  3-21  d, d  175  Family 5  EXT 1  EXT 2 Family Marker Member  Family Marker Member  5 : 2, 2 5_2 1, 2 5_3 2, 1 54 2, 1  5_1: 5_2: 5_3: 5 4:  Family 8  EXT 1  EXT 2  Family Marker Member  Family Marker Member  8_1: 1, 1 8_2: 1, 1 83: 2, 1 8 4: 1,2  8_l:b, a 8_2:a a 8_3: b a 8 4: b b  176  a, d a, a d, a a, a  F a m i l y 16  F a m i l y 17  EXT  1  EXT 2  Family Marker Member  Family Marker Member  17_1: a, d 17_2: d, b 17_3: c, b 17_4:c, b 17_5:d, d 17_6:b, d  17_1: 3, 1 17_2: 4, 1 17_3: 1, 4 17_4:1, 1 17_5:1, 4 17_6:1, 2  177  Family 18  EXT 2  EXT 1  Family Marker Member  Family Marker Member  mk . mk  am  18_1: a, c 18_2: a, d 18_3: c, c 18_4:a, c  18 1: 2, 3 18 2: 1, 2 18_3: 2, 3 18 4:1, 2  178  8.6.3 Phenotype Data 8.6.3.1. Core Data 8.6.3.1.1 Lesion Quality Core Data  EXT  Stage o f  Total #  %  Mutation  lesions  small  %  SUBJECT  GENDER  Big  6-01  male  42  35.7  Big  6-02  male  27  22.2  33.3  Big  6-03  female  28  35.7  46.4  Sense  Severity  medium  26.2  Boe  16-01  Female  1  SS  severe  Late  32  15.0  33.3  Boe  16-02  Male  1  SS  severe  Late  36  13.8  33.3  16-05  Female  1  SS  severe  Late  20  25.0  35.0  Fri  8-01  Male  2  SS  severe  Late  33  13.8  39.0  Fri  8-02  Boe  Female  2  SS  severe  Late  18  27.7  16.6  Ghu  1-01  Female  1  MS  mild  Late  28  29.6  33.3  Ghu  1-03  Male  1  MS  mild  Late  26  36.0  34.3  H e g 4-01  female  25  20.0  44.0  H e g 4-03  female  33  48.5  33.3  H e g 4-04  female  42  38.1  28.5  Hoi  3-01  Female  2  NS  severe  Early  11  18.0  45.0  Hoi  3-02  Female  2  NS  severe  Early  13  7.7  23.1  Hoi  3-04  Male  2  NS  severe  Early  13  7.7  23.1  Hoi  3-08  Female  2  NS  severe  Early  18  38.8  16.0  Hoi  3-10  Female  2  NS  severe  Early  9  22.2  22.2  Hoi  3-15  Female  2  NS  severe  Early  14  14.3  64.3  Hoi  3-19  Male  2  NS  severe  Early  39  43.5  30.7  Hoi  3-22  Female  2  NS  severe  Early  12  50.0  47.6  Hoi  3-23  Male  2  NS  severe  Early  28  18.0  21.4  Ker  17-01  Male  2  FS  severe  Early  36  33.3  25.0  Ker  17-02  Female  2  FS  severe  Early  24  27.7  47.0  17-05  Male  2  FS  severe  Early  11  18.2  45.5  Nic  2-01  Male  2  NS  severe  Early  27  37.0  25.9  Nic  2-02  Male  2  NS  severe  Early  29  24.1  31.0  Nic  2-04  Male  2  NS  35.0  Ker  severe  Early  20  55.0  Tab  5-01  Male  2  MS  mild  Early  16  71.4  14.2  Tab  5-03  Female  2  MS  mild  Early  14  57.1  14.2  Whi  18-01  Male  1  NS  severe  Early  53  47.0  33.3  Whi  18-02  Male  1  NS  severe  Early  34  32.3  11.7  179  8.6.3.1.1 Lesion Quality Core Data (continued) % SUBJECT  % large  pelvic  %pelvic  flatbone  Lesion  flatbone  flare  %flare  Rank 1  B i g 6-01  33.3  4  9.5  1  2.4  26  61.9  15  B i g 6-02  44.4  2  7.4  1  3.7  13  48.1  6  B i g 6-03  21.4  2  7.1  0  0  17  60.7  6  Boe  16-01  51.5  2  6.3  4  12.5  10  31.3  5  Boe  16-02  50.0  3  8.3  3  8.3  29  80.6  5  16-05  40.0  1  5.0  1  5.0  2  10.0  5  Fri  8-01  45.4  4  12.1  4  12.1  3  9.1  5  Fri  8-02  55.5  0  0.0  0  • 0.0  1  5.6  5  1-01  37.0  3  10.7  3  10.7  3  10.7  7  1-03  16.0  0  0.0  2  7.7  4  15.4  9  H e g 4-01  32.0  2  8.0  1  4.0  13  52.0  5  H e g 4-03  18.2  1  3.0  0  0.0  14  42.4  16  9.5  3  7.0  0  0  17  16.7  16  H o i 3-01  36.0  0  0.0  0  0.0  6  54.5  2  H o i 3-02  69.2  0  0.0  1  7.7  3  23.1  1  Hoi  3-04  69.2  0  0.0  0  0.0  7  53.8  1  H o i 3-08  44.4  0  0.0  0  0.0  3  16.7  7  H o i 3-10  55.5  0  0.0  0  0.0  0  0.0  2  H o i 3-15  21.4  0  0.0  0  0.0  0  0.0  2  H o i 3-19  25.6  4  10.3  4  10.3  14  35.9  17  6  Boe  Ghu Ghu  Heg  4-04  3-22  8.3  2  16.7  2  16.7  1  8.3  H o i 3-23  25.0  0  0.0  0  0.0  21  75.0  5  Ker  17-01  41.7  1  2.8  1  2.8  17  47.2  6  Ker  17-02  25.0  3  12.5  3  12.5  6  25.0  7  Ker  17-05  Hoi  36.3  0  0.0  0  0.0  1  9.1  2  N i c 2-01  37.0  0  0.0  0  0.0  7  25.9  10  N i c 2-02  44.8  0  0.0  0  0.0  10  34.5  7  N i c 2-04  10.0  0  0.0  1  5.0  12  60.0  13  Tab  5-01  14.2  0  0.0  0  0.0  10  62.5  11  Tab  5-03  28.5  0  0.0  0  0.0  6  42.9  9  Whi  18-01  19.6  7  13.2  8  15.1  26  49.1  22  Whi  18-02  55.9  8  23.5  8  23.5  25  73.5  11  180  8.6.3.1.2 Limb Alignment Core Data  Subject  Gender  B i g 6-01  male  EXT  Sense  Severity  Stage o f  #of  Carpal  Carpal  Mutation  lesions  SlipR  SlipL  42.0  8.0  27.0  too immature to see  10.0 to immature to see  28.0  2.0  3.0  32.0  3.0  4.0  B i g 6-02  male  B i g 6-03  female  B o e 16-01  female  1  SS  severe  Late  B o e 16-02  male  1  SS  severe  Late  36.0  8.0  5.0  B o e 16-05  female  1  SS  severe  Late  20.0  4.0  0.0  F r i 8-01  male  2  SS  severe  Late  33.0  2.0  3.0  F r i 8-02  female  2  SS  severe  Late  18.0  2.0  6.0 2.0  Ghu  1-01  female  1  MS  mild  Late  28.0  2.0  Ghu  1-03  male  1  MS  mild  Late  26.0  9.0  8.0  H e g 4-01  female  25.0  2.0  12.0  H e g 4-03  female  33.0  1.0  0.0  H e g 4-04  female  42.0  4.0  6.0  H o i 3-01  female  2  NS  severe  Early  11.0  2.0  6.0  H o i 3-02  female  2  NS  severe  Early  13.0  4.0  5.0  H o i 3-04  male  2  NS  severe  Early  13.0  -8.0  -5.0  H o i 3-08  female  2  NS  severe  Early  18.0  2.5  3.0  H o i 3-10  female  2  NS  severe  Early  9.0  7.0  5.0  H o i 3-15  female  2  NS  severe  Early  14.0  -5.0  -5.0  H o i 3-19  male  2  NS  severe  Early  39.0  1.0  1.0  H o i 3-22  female  2  NS  severe  Early  12.0  3.0  6.0  H o i 3-23  male  2  NS  severe  Early  28.0  missing r wrist f i l m  9.0  02  female  2  FS  severe  Early  24.0  3.0  6.0  K e r 17-01  male  2  FS  severe  Early  36.0  3.0  2.0  K e r 17-05  male  2  FS  severe  Early  11.0  5.0  3.0  N i c 2-01  male  2  NS  severe  Early  27.0  6.0  3.0  N i c 2-02  male  2  NS  severe  Early  29.0  2.0  3.0  N i c 2-04  male  2  NS  severe  Early  20.0  5.0  5.0  T a b 5-01  male  2  MS  mild  Early  16.0  3.0  2.0  T a b 5-03  female  2  MS  mild  Early  14.0  2.0  1.0  Whi  18-01  male  1  NS  severe  Early  53.0  6.0  Whi  18-02  male  1  NS  severe  Early  34.0  5.0 r arm and forearm not filmed  Ker  17-  181  8.0  8.6.3.1.2 Limb Alignment Core Data (continued)  Subject  Rad  Rad  Uln  Inclin  Inclin  Short  Uln  R  L  R  Short L  Rad Bow  Rad  R  Bow L  Rad Head  Rad Head  Disclocation  Disclocation  R  L  N  Y  7.0  N  N  9.0  N  N  9.0  20.0  N  Y  8.0  7.0  11.0  Y  N  11.0  9.0  9.5  N  N  4.0  1.0  9.0  8.0  N  N  2.0  4.0  8.0  8.0  N  N  22.0  1.0  1.0  6.0  10.5  N  N  37.0  34.0  -3.0  -6.0  12.0  8.5  N  N  H e g 4-01  17.0  0.0  -2.0  12.0  7.0  4.0  N  N  H e g 4-03  24.0  23.0  6.0  3.0  7.0  7.0  N  N  H e g 4-04  23.0  31.0  3.0  8.0  11.0  17.0  N  Y  H o i 3-01  28.0  24.0  -4.0  3.0  8.0  5.0  N  N  H o i 3-02  34.0  30.0  -2.0  -1.0  11.0  6.0  N  N  H o i 3-04  23.0  29.0  0.0  -1.0  4.0  8.0  N  N  H o i 3-08  22.0  28.0  1.0  -9.0  5.5  7.0  N  N  H o i 3-10  26.0  22.0  -5.0  -3.0  6.0  7.0  N  N  H o i 3-15  21.0  26.0  7.0  2.0  7.0  8.0  N  N  H o i 3-19  25.0  27.0  -1.0  -3.0  6.0  12.0  N  N  H o i 3-22  21.0  23.0  0.0  1.0  4.0  4.0  N  N  H o i 3-23  missing r wrist film  30.0  missing r wrist film  11.0  9.0  9.0  missing r wrist film  N  11.0  24.0  -11.0  -7.0  9.0  11.0  N  N  30.0  34.0  -2.0  -9.0  7.0  12.0  N  N  B i g 6-01  39.0  31.0  -7.0  -10.0  9.0  12.0  B i g 6-02  22.0  25.0  0.0  6.0  5.0  B i g 6-03  26.0  28.0  1.5  1.0  8.0  27.0  36.0  3.0  2.0  28.0  32.0  0.0  05  27.0  33.0  0.0  F r i 8-01  27.0  28.0  F r i 8-02  28.0  21.0  Ghu  1-01  20.0  Ghu  1-03  B o e 1601 B o e 1602 B o e 16-  Ker  17-  02 K e r 1701 K e r 1705  21.0  19.0  1.0  2.0  6.0  5.0  N  N  N i c 2-01  27.0  27.0  1.5  2.0  4.0  7.0  N  N  N i c 2-02  22.0  38.0  -10.0  -5.0  8.0  6.0  N  N  N i c 2-04  21.0  20.0  -10.0  2.0  10.0  5.0  N  N  T a b 5-01  28.0  28.0  -3.0  -5.0  12.0  9.0  N  N  T a b 5-03  21.0  24.0  1.0  0.0  10.0  10.0  Y  Y  29.0  35.0  -8.0  -2.0  11.0  9.0  N  N  22.0  r arm and forearm not filmed  5.0  r arm and forearm not filmed  r arm and forearm not filmed  Y  W h i 1801  W h i 1802  r arm and forearm not filmed  182  31.0  8.6.3.1.2 Limb Alignment Core Data (continued)  Subject  E l b Jt  E l b Jt  Fem  Fem A A  Fem NS  Fem NS  Fem M A  Fem M A  R  L  A A R  L  AngR  AngL  R  L  B i g 6-01  -7.0  12.0  -19.0  -9.0  150.0  150.0  -11.0  -4.0  B i g 6-02  -17.0  -11.0  -6.0  -12.0  135.0  135.0  0.0  -3.0  B i g 6-03  -4.0  -3.0  12.0  11.0  145.0  142.0  B o e 16-01  17.0  3.0  0.0  -5.0  176.0  170.0  B o e 16-02  19.0  12.0  4.0  11.5  129.0  140.0  8.0  -10.0  0.0 n/a  -4.0 n/a  -3.0  -14.0  2.5  -6.5  122.0  137.0  13.5  2.0  F r i 8-01  -12.0  -19.0  -5.0  -4.0  147.0  149.0  8.0  0.0  F r i 8-02  -16.0  -16.0  -6.0  -5.0  122.0  125.0  3.0  0.0  G h u 1-01  -24.0  -18.0  -17.0  6.0  148.0  145.0  6.0  3.0 9.0  B o e 16-05  G h u 1-03  -22.0  -3.0  0.0  2.0  135.0  148.0  8.0  H e g 4-01  -19.0  8.0  -5.0  -13.0  137.0  133.0  3.0  0.0  H e g 4-03  -5.0  -9.0  -12.0  14.0  139.0  135.0  -30.0  -6.5  H e g 4-04  -13.0  14.0  0.0  -7.0  139.0  149.0  5.0  0.0  H o i 3-01  -4.0  -15.0  -15.0  7.0  147.0  145.0  -9.0  6.0  H o i 3-02  13.0  9.0  10.0  10.0  133.0  134.0  2.5  3.0  H o i 3-04  29.0  15.0  10.0  9.0  139.0  137.0  8.0  5.0  H o i 3-08  -19.0  2.5  -15.0  -9.0  135.0  129.0  -5.0  6.0  H o i 3-10  -13.0  -16.0  -10.0  -11.0  139.0  132.0  -3.0  -6.0  H o i 3-15  9.0  12.0  6.0  15.0  142.0  150.0  H o i 3-19  2.0  -11.0  -12.0  -14.0  146.0  139.0  H o i 3-22  -15.0  -11.0  -11.0  -15.0  140.0  135.0  -3.0  -8.0  H o i 3-23  -12.0  -12.5  -15.0  -16.0  148.0  147.0  -10.0  -6.0  Ker  4.0 n/a  4.0 n/a  17-  2.0  -3.0  -9.0  0.0  148.0  140.0  -4.0  6.0  K e r 17-01  -13.0  -12.0  -22.0  -11.0  127.0  130.0  -12.0  -1.0  K e r 17-05  -17.0  -7.0  5.0  -3.0  132.0  127.0  4.0  4.0  N i c 2-01  -8.0  -30.0  -10.0  -5.0  149.0  142.0  0.0  4.0  N i c 2-02  -14.0  -18.0  0.0  -11.0  150.0  135.0  6.0  -6.0  9.0  -11.0  -6.0  -3.0  133.0  130.0  4.0  8.0 -2.0  02  N i c 2-04 T a b 5-01  -13.0  -8.0  3.0  3.0  145.0  123.0  3.0  T a b 5-03  -14.0  -11.0  -4.0  -2.0  140.0  155.0  1.0  3.0  2.0  0.0  0.0  -7.0  151.0  142.0  5.0  -7.0  -7.0  -11.0  -12.0  141.0  143.0  -3.0  -3.0  W h i 18-01  W h i 18-02  r arm and forearm not filmed  183  8.6.3.1.2 Limb Alignment Core Data (continued)  Subject  Sharps  Sharps  Fib  R  L  HtR  Ankle Jt Fib Ht L  Ankle Jt R  L  B i g 6-01  48.0  44.0  70.0  69.0  -41.0  -16.0  B i g 6-02  48.0  44.0  68.0  62.5  0.0  6.0  B i g 6-03  39.0  41.0  51.0  57.0  14.0  12.0  B o e 16-01  43.0  40.0  50.0  58.0  3.0  3.0  B o e 16-02  36.5  40.0  53.0  56.0  32.0  21.0  B o e 16-05  37.5  37.5  39.0  40.0  3.0  2.0  F r i 8-01  42.0  44.0  57.0  63.0  0.0  -4.0  33.0  28.0  49.0  -3.0  -5.0  52.0  52.0  -6.0  1.0 2.0  36.0  F r i 8-02 Ghu  1-01  Ghu  n/a  n/a  1-03  37.0  33.0  53.0  67.0  -7.0  H e g 4-01  41.0  39.0  75.0  62.0  19.0  3.0  H e g 4-03  37.0  42.0  63.0  66.0  -9.0  -9.0  H e g 4-04  39.5  45.0  69.0  68.0  -5.0  -10.0  H o i 3-01  32.0  35.0  32.5  H o i 3-02  51.0  50.0  62.0  H o i 3-04  40.0  41.0  H o i 3-08  40.0  39.0  H o i 3-10  39.0  H o i 3-15  -12.0  -2.0  78.0  2.0  14.5  45.0  52.0  25.0  11.0  53.0  58.0  6.0  -11.0  39.0  37.5  23.0  -7.0  -5.0  50.0  45.0  62.0  48.0  0.0  H o i 3-19  36.0  42.0  28.5  37.0  H o i 3-22  43.0  39.0  55.0  47.0  -10.0  -9.0  H o i 3-23  42.0  38.0  60.0  57.0  6.0  21.0  02  51.0  47.0  61.0  77.0  -20.0  -18.0  K e r 17-01  42.0  48.0  63.0  63.0  -10.0  -7.0  K e r 17-05  35.0  38.0  62.5  64.8  -9.0  -9.0  N i c 2-01  46.0  46.5  58.0  48.0  0.0  3.0  N i c 2-02  39.0  41.0  51.0  50.0  -7.0  -11.0  N i c 2-04  35.0  34.0  55.0  46.0  8.0  58.0  30.0  long films didn't include distal ankle  13.0 long films didn't include distal ankle  52.0  41.0  0.0  0.0  Ker  n/a  2.0 n/a  17-  46.0  T a b 5-01 T a b 5-03  n/a  41.0 n/a  W h i 1801  40.0  47.0  64.0  64.0  -31.0  -34.0  35.0  35.0  54.0  32.0  -21.0  -7.0  W h i 1802  184  8.6.3.1.2 Limb Alignment Core Data (continued)  EXT  % Wt  % Wt  Bear R  Bear L  Subject  Gender  B i g 6-01  male  15.0  40.0  B i g 6-02  male  58.0  39.0  B i g 6-03  female  34.0  40.0  B o e 16-01  female  1  SS  severe  68.0  60.0  B o e 16-02  male  1  SS  severe  76.0  40.0  Boe16-05  female  1  SS  severe  70.0  74.0  F r i 8-01  male  2  SS  severe  51.0  51.0  F r i 8-02  female  2  SS  severe  58.0  50.0  Ghu  1-01  female  1  MS  mild  20.0  70.0  Ghu  1-03  male  1  MS  mild  Sense  Severity  85.0  81.0  H e g 4-01  female  56.0  46.0  H e g 4-03  female  37.0  33.0  H e g 4-04  female  50.0  50.0  H o i 3-01  female  2  NS  severe  19.0  79.0  female  2  NS  severe  61.5  56.7  H o i 3-04  male  2  NS  severe  67.0  63.0  H o i 3-08  female  2  NS  severe  19.0  59.0  H o i 3-10  female  2  NS  severe  45.0  24.0  H o i 3-15  female  severe  male  NS  severe  H o i 3-22  female  2 2 2  NS  H o i 3-19  NS  severe  32.0  15.0  H o i 3-23  male  2  NS  severe  2.0  23.5  Hoi  Ker  3-02  58.0  n/a  38.0  n/a  17-  female  2  FS  severe  30.0  75.0  Ker  17-01  male  2  FS  severe  11.0  37.0  Ker  17-05  male  2  FS  severe  68.0  68.0  N i c 2-01  male  2  NS  severe  54.0  62.0  N i c 2-02  male  2  NS  severe  77.0  29.0  N i c 2-04  male  2  NS  severe  68.0  78.0  02  T a b 5-01  male  2  MS  mild  56.0  52.0  T a b 5-03  female  2  MS  mild  61.0  65.0  Whi  18-01  male  1  NS  severe  69.0  57.0  Whi  18-02  male  1  NS  severe  54.0  49.0  185  8.6.3.1.3 Limb segments and percentile height core data  SUBJECT  GENDER  B i g 6-01  male  B i g 6-02 B i g 6-03  male female  B o e 16-01  Female  B o e 16-02  Male  Boe16-05  Female  Fri  8-01  Male  Fri  8-02  Female  Heg  4-01  female  Heg  4-03  female  Heg  4-04  female  Ghu  1-01  Female  Ghu  1-03  Male  EXT  Sense  MS  Mild  Early  MS  Mild  Early  1  NS  Severe  Early  3  43.0  43.0  79.0  1  NS  Severe  Early  3  41.0  43.0  81.0  Late Late  Severe  Late  MS  Mild  Late  MS  Mild  Late  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  FS  Severe  Early  FS  Severe  Early  Whi  18-01  Male  Whi  18-02  Male  3-10  Female  Hoi  3-15  Female  H o i 3-19  Male  Hoi  3-22  Female  Hoi  3-23  Male  Ker  17-01  Male  Ker  17-02  Female  Ker  17-05  Male  Side  Severe  Female  Hoi  Side  Late  Male  Female  Side  Severe  T a b 5-03  3-08  (%ile)  ss ss ss  T a b 5-01  Hoi  Mutation  Late  Male  Male  -Left  Severe  N i c 2-04  Female  3-04  Right  Severe  Male  3-02  Hoi  Length  Tot Leg  -Left  SS  N i c 2-02  Hoi  Length -  Height  SS  Male  Female  Arm  Length Stage o f  1 1 1 2 2  N i c 2-01  3-01  Tot  Arm  50 38 30 3 39 9 25 25 8 38 60 3 5 25 24 18 25 51 95 50 90 60 18 8 3 51 77 85 15 63  1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  Hoi  Severity  Tot  FS  Severe  Early  NS  Severe  Early  NS  Severe  Early  NS  Severe  Early  186  40 31 44 43.0 47.5 50.0 47.0 55.5 42.5 48.0 43.0 38.0 52.5 53.5 49.5 56.0 52.0 53.0 40.0 55.0 56.0 53.5 50.5 45.5 57.0 44.0 52.5 59.0 54.0 36.5  41 30 49.5 42.0 47.0 51.0 44.0 55.5 48.0 47.0 44.0 37.0 51.0 50.0 46.5 55.5 54.5 53.0 40.5 55.0 57.0 53.5 52.5 46.0 53.5 44.5 50.5 58.5 54.5 38.5  74 46 83 75.0 86.5 86.0 77.5 89.0 86.0 86.0 87.0 62.0 82.5 90.0 77.0 89.5 81.0 85.5 67.5 88.5 97.0 92.0 86.5 81.5 86.5 74.5 94.0 95.0 88.0 61.5  8.6.3.1.3 Limb segments and percentile height core data (continued) Tot Leg Length SUBJECT  Right  Upper  Lower  Upper  Lower  Upper  Lower  Upper  Lower  Side  ArmR  ArmR  Arm L  Arm L  LegR  LegR  LegL  LegL  B i g 6-01  75.5  25  20  24  18  38.5  28.5  35.5  31.5  B i g 6-02  47  16.5  15  18  14.5  24  19  26  17.5  B i g 6-03  85  28.5  23  25  20.5  47.5  31.5  44  35  Boe16-01  75.5  26.0  21.5  26.0  18.5  39.5  30.0  38.5  30.5  B o e 16-02  89.0  27.0  21.5  28.5  19.0  43.5  37.5  42.0  39.5  Boe16-05  85.0  30.0  24.0  30.0  23.0  42.0  35.0  43.0  37.0  F r i 8-01  77.5  28.5  21.0  30.0  21.0  37.5  32.0  38.5  31.0  F r i 8-02  89.5  35.0  26.0  35.5  25.0  47.0  37.0  47.0  37.0  G h u 1-01  83.0  23.0  17.5  22.0  19.5  30.0  25.5  29.0  25.5  24.5  42.5  34.5  43.0  32.5  G h u 1-03  90.0  30.0  23.0  33.0  H e g 4-01  84.0  27.5  21.0  24.5  19.0  43.5  33.5  47.0  34.0  H e g 4-03  63.5  27.0  21.0  28.0  23.0  49.0  33.5  43.0  33.0  H e g 4-04  83.5  25.0  21.0  26.0  18.0  45.0  35.0  49.0  35.0  H o i 3-01  89.0  30.0  22.0  32.5  25.0  49.5  33.0  48.5  34.0  H o i 3-02  77.5  27.5  23.5  29.5  24.5  45.0  33.5  44.5  31.0  H o i 3-04  89.0  30.5  29.5  30.5  26.5  52.0  39.5  51.0  38.5  H o i 3-08  90.0  31.0  24.0  34.5  25.0  46.0  37.0  46.0  37.0  H o i 3-10  85.5  31.0  24.0  31.0  22.5  44.0  35.0  43.5  37.0  H o i 3-15  67.5  23.0  18.0  21.5  19.5  34.0  29.5  33.0  29.0  H o i 3-19  89.0  35.5  25.5  37.0  25.0  45.0  38.0  44.5  43.0  H o i 3-22  97.0  33.0  27.5  33.5  27.5  53.0  39.0  50.5  43.5  H o i 3-23  90.5  31.5  25.0  33.5  23.5  46.0  38.5  46.0  40.0  K e r 17-01  89.5  31.5  25.0  31.0  24.0  44.5  36.5  41.5  38.0  K e r 17-02  81.0  28.0  18.5  30.0  18.0  41.5  32.5  40.0  36.0  K e r 17-05  86.5  32.0  27.0  33.5  27.0  41.0  35.0  41.5  36.0  N i c 2-01  78.0  29.0  19.5  27.5  21.0  40.5  31.0  38.0  32.0  N i c 2-02  92.0  33.0  24.0  33.0  24.5  46.5  34.5  45.5  37.0  N i c 2-04  95.5  38.0  24.0  37.5  29.5  46.5  37.0  44.5  44.0  T a b 5-01  88.5  31.0  26.0  29.5  28.0  45.5  39.5  44.5  38.0  T a b 5-03  61.5  22.5  17.5  21.0  17.5  31.0  26.5  32.5  26.0  W h i 18-01  79.0  28.0  18.0  27.0  20.0  39.0  31.0  39.0  32.0  W h i 18-02  82.0  28.0  21.0  26.0  15.0  38.0  33.0  37.5  30.0  187  8.6.4.2 Pearson Correlation Matrix  1  2  3  4  5  6  7  8  Correlation  #  carpal  carpal  rad  rad  ulnar  ulnar  rad  Matrix  lesions  slip r  slip 1  inclin r  inclin 1  short r  short 1  bow r  0.738  0.361  0.665  -0.362  0.456  0.268  0.27  0.543  0.755  -0.495  -0.194  0.022 -0.104  1  # lesions  2  c a r p a l slip r  0.433  1  3  c a r p a l slip 1  0.738  0.27  0.346  -0.18  -0.297  0.192  4  rad inclin r  0.361  0.543  0.346  1  0.594  -0.14  0.022  0.34  5  rad inclin 1  0.03  0.755  -0.18  0.594  1  -0.437  -0.323  0.089  6  u l n a r short r  -0.362  -0.495  -0.297  -0.14  -0.437  0.475  -0.268  7  u l n a r short 1  0.456  -0.494  0.192  0.022  -0.323  0.475  8  rad bow r  0.268  0.22  -0.104  0.34  0.089  -0.268  0.01  9  rad bow 1  0.749  0.427  0.349  0.463  0.312  -0.582  0.247  10  elb jt r  0.473  0.104  0.319  -0.55  0.159  -0.656  0.093  0.08  11  elb jt 1  0.465  0.78  0.507  -0.47  -0.397  -0.133  0.324  0.128  12  fern a a r  -0.041  0.42  -0.475  0.226  0.297  0.275  0.479  0.355  13  fem a a 1  -0.397  0.37  0.004  0.32  -0.161  0.301  -0.402  -0.028  14  fem ns r  0.256  0.599  0.17  0.418  0.556  -0.532  -0.03  0.271  15  fern ns 1  0.364  0.614  0.144  0.709  0.444  0.079  0.352  0.312  16  fem m a r  0.217  0.143  0.379  0.213  0.24  0.388  0.593  0.355  17  fem m a 1  -0.495  -0.08  -0.382  -0.285  -0.021  0.124  -0.638  -0.12  18  sharps r  -0.557  -0.073  -0.462  -0.082  0.064  0.012  -0.537  0.212  19  sharps 1  -0.229  -0.116  -0.261  -0.377  -0.17  -0.278  -0.432  0.297  20  fib ht r  0.37  1  0.352  -0.355  -0.394  -0.275  -0.02  0.085  0.433 1  1  1  1  0.01 1 0.64  21  fib ht 1  0.326  0.1  0.452  -0.159  -0.332  0.148  0.039  -0.434  22  a n k l e jt r  -0.505  0.137  -0.564  -0.202  -0.141  861  0.384  -0.281  23  a n k l e jt 1  -0.552  -0.257  -0.496  -0.2  0.131  0.689  0.213  -0.398  24  % wt b e a r r  0.304  -0.027  -0.326  0.253  0.348  0.078  0.35  0.559  25  % wt b e a r 1  -0.087  0.244  -0.42  0.114  0.307  -0.113  -0.405  0.376  26  % ped  -0.352  0.404  -0.58  -0.09  0.218  -0.557  -0.576  -0.08  27  % sess  0.33  0.132  0.173  0.042  -0.317  0.267  0.146  0.278  28  % distal  29  %prox  30  % pelvic  31  %diaph  -0.244  0.288  32  %flat bones  0.002  -0.201  33  %complex  0.109  0.282  34  %simple  -0.446  35  %flared  0.164  36  % not  -0.084  37  %ofl  38  %of4  39  avg #  40  % left  41  0.224  0.142  0.504  0.278  -0.114  0.249  0.158  -0.232  -0.518  0.229  -0.79  -0.407  0.083  -0.07  -0.106  0.295  0.82  -0.277  0.724  0.51  0.083  -0.413  0.279  0.265  -0.04  0.06  -0.115  0.217  -0.17  -0.219  0.027  -0.053  0.278  -0.021  -0.229  -0.487  -0.05  0.435  0.346  -0.607  -0.149  0.656  0.087  -0.337  -0.535  -0.389  0.514  -0.211  -0.387  -0.327  0.118  0.104  0.29  -0.528  0.1  0.146  0.28  -0.058  -0.045  -0.304  0.552  0.043  -0.096  -0.228  -0.249  -0.155  -0.195  -0.238  -0.244  -0.286  0.529  0.098  -0.233  -0.16  0.336  0.345  0.243  0.373  -0.324  1  0.028  0.738  0.361  0.03  -0.362  0.456  0.268  -0.087  0.433  -0.27  0.479  0.434  -0.325  -0.215  0.787  % right  0.087  0.104  0.27  -0.479  -0.434  0.325  0.042  -0.787  42  %ht  -0.47  -0.104  -0.186  -0.353  -0.145  0.253  -0.075  -0.475  43  1 a r m upper  -0.28  -0.336  -0.28  -0.376  -0.01  0.126  -0.335  -0.432  44  1 a r m lower  -0.744  -0.305  -0.607  -0.519  -0.078  0.274  -0.099  -0.37  45  total a r m 1  -0.492  -0.42  -0.466  -0.514  -0.085  0.225  0.383  -0.439  flared  188  8.6.4.2 Pearson Correlation Matrix (continued) 1  #  2  3  4  5  6  7  8  carpal  carpal  rad  rad  ulnar  ulnar  rad  slip r  slip 1  bow r  inclin r  inclin 1  short r  short 1  0.72  -0.426  0.447  0.218  0.131  -0.288  -0.073  -0.027  r a r m upper  -0.23  0.181  -0.296  -0.393  0.016  0.015  -0.027  -0.308  48  r a r m lower  -0.607  -0.321  -0.523  -0.526  -0.139  0.312  -0.048  -0.485  49  total a r m r  -0.514  0.555  -0.464  -0.639  -0.194  0.209  -0.74  -0.455  50  r a r m ratio  0.384  -0.527  0.167  0.017  0.196  -0.383  0.085  0.128  51  ALD  0.242  0.162  0.415  0.268  -0.423  0.265  0.522  0.302  52  I leg u p p e r  -0.177  -0.143  -0.082  -0.655  -0.609  0.405  0.272  -0.404  53  1 leg l o w e r  -0.264  -0.662  -0.35  -0.726  -0.262  0.199  0.299  -0.49  54  total leg 1  -0.056  -0.446  -0.178  -0.592  -0.233  0.072  0.347  -0.369  55  1 leg r a t i o  0.347  -0.402  0.601  0.354  -0.525  0.299  0.376  0.273  56  r leg u p p e r  -0.188  0.227  -0.121  -0.661  -0.414  0.279  0.262  -0.57  57  r leg l o w e r  -0.262  -0.528  -0.423  -0.607  -0.294  0.19  0.149  -0.157  58  total leg r  -0.12  -0.574  -0.229  -0.484  -0.022  -0.03  0.158  -0.403  59  r leg r a t i o  0.128  -280  0.525  -0.024  -0.157  0.134  0.44  -0.679  60  LLD  0.297  0.118  0.26  -0.439  -0.485  0.308  0.047  -0.339  lesions  46  ratio I a r m  47  189  8.6.4.2 Pearson Correlation Matrix (continued)  9  1  10  Correlation  rad  elbjt  Matrix  bow 1  r  # lesions  11 elb j t 1  12  13  14  15  16  17  fem a a  fem a a  fem ns  fem ns  fem  fem  r  1  r  1  ma r  ma 1  0.749  0.473  0.465  -0.041  -0.397  0.256  0.364  0.217  -0.495  0.427  0.104  0.078  0.42  -0.037  0.599  0.614  0.143  -0.008  -0.004  0.17  0.144  -0.379  -0.382  c a r p a l slip  2  r c a r p a l slip  3  1  0.349  0.319  0.507  -0.475  4  rad inclin r  0.463  -0.055  -0.047  0.058  0.132  0.418  0.709  0.213  -0.285  5  rad inclin 1  0.312  0.159  -0.397  0.226  -0.161  0.556  0.444  0.24  -0.021  -0.582  -0.656  -0.133  0.297  0.301  -0.532  0.079  0.388  0.124  0.247  0.093  0.324  0.275  -0.402  -0.03  0.352  0.593  0.345  0.64  0.08  0.128  0.479  -0.028  0.271  0.312  0.355  0.342  1  0.452  0.415  0.355  -0.441  0.411  0.484  0.27  0.986  0.263  -0.704  0.421  -0.307  -0.127  0.67  0.01  0.194  0.098  0.256  -0.186  0.875  ulnar short  6  r ulnar short  7  1  8  r a d bow r  9  r a d bow I  10  elb jt r  0.452  11  elb j t 1  0.415  12  fem a a r  0.263  0.01  0.058  1  -0.158  0.281  0.487  0.741  0.423  13  fem a a 1  -0.441  -0.704  0.194  0 158  I  -0.084  0.17  -0.311  0.34  14  fem ns r  0.484  0.421  0.098  0.281  -0.084  1  0.467  0.077  0.023  15  fem ns 1  0.27  -0.307  0.256  0.487  0.17  0 467  0.53  0.234  16  fem m a r  -0.55  -0.127  -0.186  0.741  -0.311  0.077  0.53  17  fem m a 1  -0.268  -0.563  -0.498  -0.489  0.44  -0.357  -0.28  -0.156  18  sharps r  0.003  -0.254  0.085  0.253  0.636  0.011  -0.121  -0.184  19  sharps 1  0.153  0.095  0.386  0.507  0.342  -0.011  -0.321  -0.278  0.108  20  fib ht r  -0.114  0.269  0.72  0.615  0.208  0.063  -0.17  -0.249  -0.098  21  fib ht 1  -0.575  -0.204  0.407  -0.023  0.235  -0.545  -0.09  -0.211  0.168  22  a n k l e jt r  -0.587  -0.626  -0.251  -0.165  0.333  -0.21  0.213  0.517  0.194  23  a n k l e jt 1  0.38  -0.498  -0.238  -0.105  -0.348  0.049  0.276  0.363  0.075  0.106  0.101  -0.279  -0.103  0.158  0.253  0.402  0.902  -0.062  1 0.09  0.09 1  1  1  0.456 1' 0.246  % wt b e a r  24  r % wt b e a r  25  1  0.052  -0.423  -0.375  0.508  0.197  -0.018  0.266  0.36  0.7  26  % ped  -0.064  0.12  0.237  -0.145  0.088  0.31  -0.121  -0.666  0.034  27  % sess  -0.281  -0.028  -0.121  -0.422  0.459  -0.292  -0.136  0.3  0.534  28  % distal  0.044  -0.069  0.158  -0.118  -0.163  0.283  0.215  -0.055  -0.345  29  %prox  0.789  0.011  -0.055  0.296  -0.368  0.074  -0.122  0.178  0.543  30  % pelvic  -0.147  0.363  0.412  -0.431  0.136  0.068  0.316  -0.03  0.23  31  %diaph  -0.375  -0.556  -0.162  0.406  -0.028  -0.599  -0.016  -0.251  0.245  32  bones  0.559  0.254  -0.35  -0.372  -0.234  -0.036  -0.311  -0.024  0.234  33  %complex  -0.726  0.53  0.038  0.094  0.289  0.677  0.12  -0.05  0.1  34  %simple  0.409  -0.58  -0.401  0.221  -0.134  -0.684  -0.416  -0.03  0.89  35  %flared  0.379  0.605  0.47  -0.041  0.164  0.681  0.133  -0.148  0.678  36  flared  0.015  -0.593  -0.431  0.425  0.35  -0.645  -0.08  0.202  0.345  37  % of 1  0.075  0.115  0.254  -0.425  -0.526  0.375  -0.165  -0.244  0.093  38  % of 4  0.749  0.071  -0.397  0.043  -0.397  -0.223  0.151  0.339  0.78  39  avg #  0.4  0.473  0.465  -0.359  0.017  0.256  0.364  0.217  0.7  %flat  % not  190  L6.4.2 Pearson Correlation Matrix (continued) 9 rad bow 1  40  % left  41 42  10  11  elbjt elb jt 1  r  12  13  14  15  fem a a  fem a a  fem ns  fem ns  fem  fem  r  1  r  1  ma r  ma 1  16  17  -0.4  0.133  -0.203  -0.266  -0.017  0.575  0.297  0.21  0.9  % right  -0.656  -0.133  0.203  -0.27  0.008  -0.575  -0.297  -0.21  0.65  % ht  -0.389  0.188  -0.305  -0.137  -0.502  0.213  -0.401  -0.175  0.456  1 arm 43  upper  -0.725  -0.05  -0.735  -0.282  -0.037  -0.415  -0.521  -0.007  0.23  44  I a r m lower  -0.511  -0.255  -0.759  -0.112  -0.394  -0.314  -0.547  -0.107  0.134  45  total a r m 1  0.567  -0.07  -0.709  -0.237  -0.765  -0.432  -0.549  -0.02  0.65  46  ratio 1 a r m  -321  0.512  0.01  -0.308  -0.603  -0.101  0.038  0.169  0.897  -0.517  0.254  -0.76  -0.052  -0.351  -0.274  -0.593  0.029  0.568  r arm  47  upper r arm  48  lower  -0.522  -0.104  -0.596  -0.113  -0.422  -0.498  -0.536  -0.075  0.123  49  total a r m r  0.163  0.023  -0.508  -0.053  -0.557  -0.402  -0.632  -0.086  0.343  50  r a r m ratio  -0.032  0.537  0.376  -0.165  0.574  0.181  -0.272  0.134  0.34  51  ALD  -0.334  -0.195  -0.084  -0.11  -0.465  -0.13  0.088  -0.065  0.56  52  1 leg u p p e r  -0.348  0.108  -0.309  -0.159  -0.596  -0.374  -0.448  0.033  0.67  53  1 leg l o w e r  -0.174  0.206  -0.359  -0.027  -0.742  -0.281  -0.513  0.068  0.76  54  total leg 1  0.118  0.33  0.488  -0.176  0.422  -0.253  -0.498  0.079  0.435  55  1 leg r a t i o  -0.401  -0.229  -0.219  -0.185  -0.529  -0.075  0.266  -0.1  0.346  56  r leg u p p e r  -0.172  0.215  -0.366  -0.06  -0.619  -0.287  -0.519  -0.05  0.876  57  r leg l o w e r  -0.171  0.102  -0.5  -0.567  -0.757  -0.352  -0.442  0.151  0.345  58  total leg r  -0.363  0.349  0.286  -0.74  0.207  -0.187  -0.48  0.044  0.234  59  r leg r a t i o  -0.241  0.188  0.289  -0.009  0.123  0.124  -0.097  -0.356  0.113  60  LLD  -0.214  -0.041  0.456  -0.234  0.145  -0.064  0.009  0.132  0.135  191  8.6.4.2 Pearson Correlation Matrix (continued) 18  19  Correlation  sharps  sharps  Matrix  r  1  1  # lesions  2  20 fib ht r  21  22  fib ht  ankle  1  jtr  23 ankle jt 1  24  25  26  % wt  % wt  %  bear r  bear 1  ped  -0.557  -0.229  0.37  0.326  -0.505  -0.552  0.304  -0.087  0.352  c a r p a l slip r  0.073  -0.116  0.1  0.137  -0.257  -0.027  0.244  0.404  0.132  3  c a r p a l slip 1  0.462  -0.261  0.352  0.452  -0.564  -0.496  -0.326  -0.42  0.058  4  r a d inclin r  0.082  -0.377  -0.355  -0.159  -0.202  -0.02  0.253  0.114  -0.09  5  rad inclin 1  0.064  -0.17  -0.394  -0.332  -0.141  0.131  0.348  0.397  0.218  0.012  -0.278  -0.275  0.148  0.861  0.689  0.078  -0.113  0.557 0.576  u l n a r short  6  r u l n a r short  7  1  -0.537  -0.432  -0.02  0.039  0.384  0.213  0.35  -0.405  8  r a d bow r  0.212  0.297  0.085  -0.434  -0.281  -0.398  0.559  0.376  -0.08  9  rad bow 1  0.268  0.003  0.153  -0.114  -0.575  -0.587  0.38  0.106  0.053  10  elb jt r  0.254  0.095  0.269  -0.204  -0.626  -0.498  0.101  -0.423  0.12  11  elb jt 1  0.0865  0.342  0.72  0.407  -0.251  -0.238  -0.279  -0.375  0.237  12  fem a a r  0.253  -0.011  -0.148  -0.438  0.453  0.507  0.615  -0.023  0.165  13  fem a a 1  0.636  -0.321  0.208  0.235  0.235  0.333  -0.348  0.158  0.197  14  fem ns r  0.011  -0.278  0.063  -0.545  -0.21  0.049  0.253  -0.018  0.31  15  fem ns 1  -0.121  0.108  -0.17  -0.09  0.213  0.276  0.402  0.266  0.121  16  fem m a r  -0.184  0.156  -0.249  -0.211  0.517  0.363  0.902  0.36  0.666  17  fem m a 1  0.246  0.108  -0.098  0.168  0.194  0.075  -0.062  0.7  0.034  18  sharps r  0.821  0.3  -0.102  0.079  0.254  -0.098  0.138  0.408  19  sharps 1  0.685  0.062  -0.242  -0.165  -0.12  0.055  0.44  20  fibhtr  0.489  -0.343  -0.17  -0.123  -0.15  0.101  21  fib ht 1  -0.113  0.896  -0.323  -0.027  0.214  22  0.895  0.226  0.082  0.368  23  0.109  -0.048  -0.13  24  0.483  0.618  1 0.821  1  0.3  0.685  1  -0.102  0.062  0.489  a n k l e jt r  0.079  -0.242  -0.343  -0.113  a n k l e jt 1  0.254  -0.165  -0.32  -0.17  % wt b e a r r  -0.098  -0.12  -0.123  -0.323  0.226  0.109  25  % wt b e a r 1  0.138  0.055  -0.15  -0.027  0.082  -0.048  0.483  26  % ped  0.408  0.44  0.101  -0.214  -0.368  -0.13  -0.618  27  % sess  -0.131  0.02  0.272  0.303  -0.006  -0.185  0.456  0.189  28  % distal  0.012  -0.148  0.291  0.244  0.153  0.308  -0.027  -0.277  -0.33  29  %prox  0.436  0.479  -0.087  -0.562  0.22  0.18  0.153  0.16  0.431  30  % pelvic  -0.529  -0.293  0.067  0.265  -0.654  -0.68  -0.005  -0.16  0.068  31  %diaph  -0.244  -0.377  -0.511  0.254  0.044  -0.111  -0.45  0.184  0.119  32  %flat bones  0.117  0.043  0.171  0.345  -0.013  0.185  0.093  -0.022  0.258  33  %complex  0.175  0.229  -0.057  -0.76  -0.477  -0.332  0.191  0.096  0.418  34  %simple  0.023  -0.052  -0.066  0.392  0.456  0.224  -0.102  0.39  0.342  1  192  1 .896  1  1  1 -0.128  0.128 1 0.797  8.6.4.2 Pearson Correlation Matrix (continued) 18  35  19  Correlation  sharps  sharps  Matrix  r  1  %flared %  20 fib ht r  21  22  fib ht  ankle  1  jtr  23  24 %  ankle jt 1  wt  bear r  25  26  % wt  %  bear 1  ped  0.315  0.45  0.389  -0.306  -0.34  -0.037  -0.058  -0.433  0.562  not  36  flared  -0.31  -0.447  -0.33  0.331  0.34  0.04  0.128  0.424  -0.65  37  % of 1  0.605  0.693  0.434  -0.155  -0.113  -0.024  -0.038  0.379  38  % of 4  -0.528  -0.71  -0.694  -0.455 7.95E05  0.167  0.178  0.137  -0.168  -313  39  avg #  -0.557  -0.229  0.37  0.326  -0.505  -0.552  0.304  -0.087  0.352  40  % left  0.331  0.175  -0.257  -0.816  -0.174  -0.085  0.428  0.252  0.201  41  % right  -0.331  -0.175  0.257  0.816  0.174  0.085  -0.428  -0.252  0.201  42  % ht  0.088  -0.057  -0.096  -0.37  0.387  0.528  -0.182  -0.505  0.046  43  1 a r m upper  -0.498  -0.558  -0.071  0.149  -0.016  -0.031  0.118  -0.22  44  1 a r m lower  -0.496 4.69E04  -0.161  -0.484  -0.284  0.421  0.31  -0.115  0.224  0.005  45  total a r m 1  -0.337  -0.373  -0.557  -0.165  0.306  0.135  -0.088  0.112  0.105  46  ratio 1 a r m  -0.779  -0.513  -0.132  0.246  -0.452  -0.536  0.172  -0.126  0.326  r arm  47  upper  -0.449  -0.401  -0.478  -0.215  0.063  -0.078  0.092  0.073  0.238  48  r a r m lower  -0.205  -0.276  -0.57  -0.18  0.365  0.236  -0.241  -0.094  0.055  49  total a r m r  -0.294  -0.268  -0.449  -0.202  0.297  0.134  -0.161  -0.038  0.022  50  r a r m ratio  -0.283  -0.306  -0.079  -0.147  -0.364  -0.441  0.431  0.234  0.413  51  ALD  -0.483  0.26  0.45  0.325  -0.082  -0.093  0.005  -0.162  0.349  52  1 leg u p p e r  0.314  -0.345  -0.148  -0.031  0.355  0.107  -0.152  -0.428  0.258  53  1 leg l o w e r  -0.531  -0.247  -0.221  -0.113  0.329  0.163  -0.064  -0.207  0.087  54  total leg 1  -0.419  -0.394  -0.277  -0.127  0.138  -0.068  0.013  -0.173  0.194  55  1 leg ratio  -0.619  -0.104  0.205  0.201  -0.063  -0.147  -0.151  -0.335  0.276  56  r leg u p p e r  -0.074  -0.363  -0.204  -0.053  0.308  0.152  -0.206  -0.427  -0.14  57  r leg l o w e r  -0.52  -0.282  -0.391  -0.284  0.265  -0.011  0.045  -0.042  0.118  58  total leg r  -0.445  -0.432  -0.406  -0.191  0.084  -0.055  0.004  -0.099  0.097  59  r leg r a t i o  -0.044  -0.079  0.346  0.414  0.042  0.284  -0.435  -0.666  0.003  60  LLD  -0.14  0.08  0.646  0.546  0.305  0.214  0.036  -0.163  0.377  193  8.6.4.2 Pearson Correlation Matrix (continued) 27  28  29  30  31  32  33  34  35  %  %  %  %  %  %  %distal  %prox  pelvic  diaph  flatbones  complex  simple  flared  Correlation Matrix  % sess  1  # lesions  -0.495  -0.194  0.022  0.427  0.104  0.078  0.665  0.362  0.456  2  c a r p a l slip r  -0.297  0.192  -0.104  0.349  0.319  0.507  0.755  -0.495  -0.194  3  c a r p a l slip 1  4  rad inclin r  -0.14  0.022  0.34  0.463  0.055  -0.047  -0.18  -0.297  0.192  -0.437  -0.323  0.089  0.312  0.159  -0.397  0.594  -0.14  0.022  0.475  -0.268  -0.582  0.656  -0.133  -0.89  -0.437  -0.323  0.01  0.247  0.093  0.324  -0.437  0.765  0.475  0.08  0.128  -0.323  0.475  0.346  0.452  0.415  0.089  -0.268  0.01  0.09  0.312  -0.582  0.247  0.159  -0.656  0.093  -0.397  -0.133  0.324  5  rad inclin I  6  u l n a r short r  0.475  1  7  u l n a r short 1  -0.268  0.01  1  8  rad bow r  -0.582  0.247  0.64  9  rad bow  1  -0.656  0.093  0.08  0.452  1  0.64 1  1  10  elb jt r  -0.133  0.324  0.128  0.415  0.09  11  elb jt  1  0.275  0.479  0.355  0.263  0.01  0.058  12  fem a a r  0.301  -0.402  -0.028  -0.441  0.704  0.194  0.297  0.275  0.479  13  fem a a  1  -0.532  -0.03  0.271  0.484  0.421  0.098  -0.161  0.301  -0.402  14  fem ns r  0.079  0.352  0.312  0.27  0.307  0.256  0.556  -0.532  -0.03  15  fem ns  1  0.388  0.593  0.355  -0.55  0.127  -0.186  0.444  0.079  0.352  16  fem m a r  0.124  -0.638  -0.12  -0.268  0.563  -0.498  0.24  0.388  0.593  I  1  17  fem m a  0.012  -0.537  0.212  0.003  0.254  0.085  -0.021  0.124  -0.638  18  sharps r  -0.278  -0.432  0.297  0.153  0.095  0.386  0.064  0.012  -0.537 -0.432  19  sharps 1  -0.275  -0.02  0.085  -0.114  0.269  0.72  -0.17  -0.278  20  fib ht r  -0.576  -0.08  -0.352  0.404  -0.58  -0.09  0.218  -0.275  -0.02  21  fib ht  1  0.146  0.278  0.33  0.132  0.173  0.042  -0.317  0.148  0.039  22  a n k l e jt r  0.158  -0.232  0.224  0.142  0.504  0.278  -0.114  861  0.384  23  a n k l e jt 1  -0.106  0.295  -0.518  0.229  -0.79  -0.407  0.083  0.689  0.213  24  % wt b e a r r  0.279  0.265  0.82  -0.277  0.724  0.51  0.083  0.078  0.35  25  % wt b e a r 1  -0.17  -0.219  -0.244  0.288  -0.04  0.06  -0.115  -0.113  -0.405  26  % ped  -0.229  -0.487  0.002  -0.201  0.027  -0.053  0.278  -0.557  -0.576  27  % sess  0.656  0.109  0.282  -0.05  0.435  0.346  0.267  0.146  28  % distal  0.087  0.337  -0.535  -0.389  0.249  0.158  29  %prox  1 -0.327  0.118  0.104  0.29  -0.07  -0.106  30  % pelvic  0.058  -0.045  -0.304  -0.413  0.279  31  %diaph  -0.195  -0.238  0.217  -0.17  32  %flat bones  0.345  -0.021  -0.229  33  %complex  -0.607  -0.149  1 -0.211 0.1 0.043  K  1 1 1«  -0 446  -0.096  -0.084  -0.286  0.529  -0.228  -0.249  0.373  -0.324  0.098  -0.233  -0.16  0.456  0.268  0.028  0.738  1  1  1  1 0.361  1  34  %simple  -0.215  0.787  -0.087  0.433  -0.27  0.479  0.434  35  %flared  0.042  -0.787  0.087  0.104  0.27  -0.479  -0.434  36  % not f l a r e d  -0.075  -0.475  -0.47  -0.104  0.186  -0.353  -0.145  0.552  0.043  37  % of 1  -0.335  -0.432  -0.28  -0.336  -0.28  -0.376  -0.01  -0.244  -0.286  38  % of 4  -0.099  -0.37  -0.744  -0.305  0.607  -0.519  -0.078  0.243  0.373  194  1 -0 211 -0.528  1  8.6.4.2 Pearson Correlation Matrix (continued) 27  28  29  Correlation Matrix  41  % sess  30  31  32  34  33  35  %  %  %  %  %  %  %distal  %prox  pelvic  diaph  flatbones  complex  simple  flared  -0.308  -0.23  0.181  0.296  -0.393  0.016  0.325  0.042  % right  -0.027  42  % ht  -0.093  0.005  -0.162  -0.349  0.113  -0.024  -0.038  0.253  -0.075  43  1 a r m upper  0.107  -0.152  -0.428  -0.258  0.178  0.137  -0.168  0.126  -0.335  44  1 a r m lower  0.163  -0.064  -0.207  -0.087  0.552  0.304  -0.087  0.274  -0.099  45  total a r m 1  -0.068  0.013  -0.173  -0.194  0.085  0.428  0.252  0.225  0.383  46  ratio 1 a r m  -0.147  -0.151  -0.335  -0.276  0.085  -0.428  -0.252  -0.288  -0.073  47  r a r m upper  0.152  -0.206  -0.427  -0.14  0.528  -0.182  -0.505  0.015  -0.027  48  r a r m lower  -0.011  0.045  -0.042  -0.118  0.016  -0.031  0.118  0.312  -0.048  49  total a r m r  -0.055  0.004  -0.099  -0.097  0.31  -0.115  0.224  0.209  -0.74  50  r a r m ratio  0.284  -0.435  -0.666  -0.003  0.135  -0.088  0.112  -0.383  0.085  51  ALD  0.214  0.036  -0.163  -0.377  0.536  0.172  -0.126  0.265  0.522  52  1 leg u p p e r  -0.466  -0.514  -0.085  -0.362  0.456  0.268  0.073  0.405  0.272  53  1 leg l o w e r  0.447  0.218  0.131  -0.325  0.215  0.787  -0.262  0.199  0.299  54  total leg I  -0.296  -0.393  0.016  0.325  0.042  -0.787  -0.233  0.072  0.347  55  1 leg ratio  -0.113  -0.024  -0.038  0.253  0.075  -0.475  -0.525  0.299  0.376  56  r leg u p p e r  0.178  0.137  -0.168  0.126  0.335  -0.432  -0.414  0.279  0.262  57  r leg l o w e r  -0.552  0.304  -0.087  0.274  0.099  -0.37  -0.294  0.19  0.149  58  total leg r  -0.085  0.428  0.252  0.225  0.383  -0.439  -0.022  -0.03  0.158  59  r leg ratio  0.085  -0.428  -0.252  -0.288  0.073  -0.027  -0.157  0.134  0.44  60  LLD  0.528  -0.182  -0.505  0.015  0.027  -0.308  -0.485  0.308  0.047  195  8.6.4.2 Pearson Correlation Matrix (continued) 36  37  38  39  40  41  42  43  44  % Correlation  not  %of  %of  Matrix  flared  1  4  1  % avg #  % left  right  % ht  1  arm  upper  arm  lower  1  # lesions  0.268  1  0.738  0.361  0.665  -0.362  0.456  0.268  2  c a r p a l slip r  0.022  0.433  1  0.27  0.543  0.755  -0.495  -0.194  0.022  3  c a r p a l slip 1  0.104  0.738  0.27  1  0.346  -0.18  -0.297  0.192  -0.104  0.433  4  rad inclin r  0.34  0.361  0.543  0.346  1  0.594  -0.14  0.022  0.34  5  rad inclin I  0.089  0.03  0.755  -0.18  0.594  1  -0.437  -0.323  0.089  0.268  0.362  -0.495  -0.297  -0.14  -0.437  1  0.475  -0.268  0.01  0.456  -0.494  0.192  0.022  -0.323  0.475  1  0.01  u l n a r short  6  r u l n a r short  7  1  8  rad bow r  0.634  0.268  0.22  -0.104  0.34  0.089  -0.268  0.01  1  9  rad bow  1  0.64  0.749  0.427  0.349  0.463  0.312  -0.582  0.247  0.64  10  elb jt r  11  elb jt  12  0.08  0.473  0.104  0.319  -0.55  0.159  -0.656  0.093  0.08  0.128  0.465  0.78  0.507  -0.47  -0.397  -0.133  0.324  0.128  fem a a r  0.355  0.041  0.42  -0.475  0.226  0.297  0.275  0.479  0.355  1  1  13  fem a a  0.028  0.397  0.37  0.004  0.32  -0.161  0.301  -0.402  -0.028  14  fem ns r  0.271  0.256  0.599  0.17  0.418  0.556  -0.532  -0.03  0.271  15  fem ns  1  0.312  0.364  0.614  0.144  0.709  0.444  0.079  0.352  0.312  16  fem m a r  0.355  0.217  0.143  0.379  0.213  0.24  0.388  0.593  0.355  17  fem m a  1  -0.12  0.495  -0.08  -0.382  -0.285  -0.021  0.124  -0.638  -0.12  18  sharps r  0.212  0.557  -0.073  -0.462  -0.082  0.064  0.012  -0.537  0.212  19  sharps 1  0.297  0.229  -0.116  -0.261  -0.377  -0.17  -0.278  -0.432  0.297  20  fib ht r  0.085  0.37  1  0.352  -0.355  -0.394  -0.275  -0.02  0.085  21  fib ht  1  0.434  0.326  0.1  0.452  -0.159  -0.332  0.148  0.039  -0.434  22  a n k l e jt r  0.281  0.505  0.137  -0.564  -0.202  -0.141  861  0.384  -0.281  23  a n k l e jt 1  0.398  0.552  -0.257  -0.496  -0.2  0.131  0.689  0.213  -0.398  24  % wt b e a r r  0.559  0.304  -0.027  -0.326  0.253  0.348  0.078  0.35  0.559  25  % wt b e a r 1  0.376  0.087  0.244  -0.42  0.114  0.307  -0.113  -0.405  0.376  26  % ped  -0.08  0.352  0.404  -0.58  -0.09  0.218  -0.557  -0.576  -0.08  27  % sess  0.278  0.33  0.132  0.173  0.042  -0.317  0.267  0.146  0.278  28  % distal  0.232  0.224  0.142  0.504  0.278  -0.114  0.249  0.158  -0.232  29  %prox  0.295  0.518  0.229  -0.79  -0.407  0.083  -0.07  -0.106  0.295  30  % pelvic  0.265  0.82  -0.277  0.724  0.51  0.083  -0.413  0.279  0.265  31  %diaph  0.219  0.244  0.288  -0.04  0.06  -0.115  0.217  -0.17  -0.219  32  %flat bones  0.487  0.002  -0.201  0.027  -0.053  0.278  -0.021  -0.229  -0.487  196  8.6.4.2 Pearson Correlation Matrix (continued) 36  37  38  39  40  41  42  43  44  %  33  Correlation  not  %of  %of  Matrix  flared  1  4  %complex  0.656  0.109  % avg #  % left  right  % ht  0.282  -0.05  0.435  0.346  1 arm  1 arm  upper  lower  -0.607  -0.149  0.656  34  %simple  0.387  0.446  0.087  -0.337  -0.535  -0.389  0.514  -0.211  -0.387  35  %flared  0.146  0.164  -0.327  0.118  0.104  0.29  -0.528  0.1  0.146  0.084  0.28  -0.058  -0.045  -0.304  0.552  0.043  -0.096  1  -0.249  -0.155  -0.195  -0.238  -0.244  -0.286  0.529  -0.16  0.336  0.345  0.243  0.373  -0.324  0.361  0.03  -0.362  0.456  0.268  0.434  -0.325  -0.215  0.787  0.325  0.042  -0.787  -0.075  -0.475  % not  36  flared  37  % of 1  0.529  1  38  %of4  0.324  39  avg #  0.268  1  0 028  40  % left  0.787  0.087  0.433  -0.27  41  % right  0.787  0.087  0.104  0.27  -0.479  i  42  %ht  0.475  -0.47  -0.104  -0.186  -0.353  -0.145  43  I a r m upper  0.432  -0.28  -0.336  -0.28  -0.376  -0.01  0.126  44  1 a r m lower  -0.37  0.744  -0.305  -0.607  -0.519  -0.078  0.274  -0.099  45  total a r m 1  0.439  0.492  -0.42  -0.466  -0.514  -0.085  0.225  0.383  -0.439  46  ratio 1 a r m  0.027  0.72  -0.426  0.447  0.218  0.131  -0.288  -0.073  -0.027  0.098  1  1  1  1  1  -0.432 1  r arm  47  upper  0.308  -0.23  0.181  -0.296  -0.393  0.016  0.015  -0.027  -0.308  48  r a r m lower  0.485  0.607  -0.321  -0.523  -0.526  -0.139  0.312  -0.048  -0.485  49  total a r m r  0.455  0.514  0.555  -0.464  -0.639  -0.194  0.209  -0.74  -0.455  50  r a r m ratio  0.128  0.384  -0.527  0.167  0.017  0.196  -0.383  0.085  0.128  51  ALD  0.302  0.242  0.162  0.415  0.268  -0.423  0.265  0.522  0.302  52  1 leg u p p e r  0.404  0.177  -0.143  -0.082  -0.655  -0.609  0.405  0.272  -0.404  53  1 leg l o w e r  -0.49  0.264  -0.662  -0.35  -0.726  -0.262  0.199  0.299  -0.49  54  total leg 1  0.369  0.056  -0.446  -0.178  -0.592  -0.233  0.072  0.347  -0.369  55  1 leg r a t i o  0.273  0.347  -0.402  0.601  0.354  -0.525  0.299  0.376  0.273  56  r leg u p p e r  -0.57  0.188  0.227  -0.121  -0.661  -0.414  0.279  0.262  -0.57  57  r leg l o w e r  0.157  0.262  -0.528  -0.423  -0.607  -0.294  0.19  0.149  -0.157  58  total leg r  0.403  -0.12  -0.574  -0.229  -0.484  -0.022  -0.03  0.158  -0.403  59  r leg r a t i o  0.679  0.128  -280  0.525  -0.024  -0.157  0.134  0.44  -0.679  60  LLD  0.339  0.297  0.118  0.26  -0.439  -0.485  0.308  0.047  -0.339  197  8.6.4.2 Pearson Correlation Matrix (continued) 45 Correlation  total  Matrix  arm  1  46  47  48  50  49  ratio  r arm  r arm  total  r arm  1  uppder  lower  arm r  ratio  arm  52  51 ALD  53  lleg  lleg  upper  lower  1  # lesions  0.749  0.473  0.465  -0.041  -0.397  0.256  0.364  0.217  0.665  2  c a r p a l slip r  0.427  0.104  0.078  0.42  -0.037  0.599  0.614  0.143  0.755  3  c a r p a l slip 1  0.349  0.319  0.507  -0.475  -0.004  0.17  0.144  -0.379  -0.18  4  rad inclin r  0.463  0.055  -0.047  0.058  0.132  0.418  0.709  0.213  0.594  5  rad inclin 1  0.312  0.159  -0.397  0.226  -0.161  0.556  0.444  0.24  -0.89  0.582  0.656  -0.133  0.297  0.301  -0.532  0.079  0.388  -0.437  ulnar short  6  r ulnar short  7  1  0.247  0.093  0.324  0.275  -0.402  -0.03  0.352  0.593  -0.323  8  rad bow r  0.64  0.08  0.128  0.479  -0.028  0.271  0.312  0.355  0.089  9  r a d bow  1  1  0.452  0.415  0.355  -0.441  0.411  0.484  0.27  0.312  0.09  0.263  -0.704  0.421  -0.307  -0.127  0.159  0.01  0.194  0.098  0.256  -0.186  -0.397  1  -0.158  0.281  0.487  0.741  0.297  10  elb jt r  0.452  1  11  elb jt  1  0.415  0.09  12  fem a a r  0.263  0.01  0.058  13  fem a a  1  0.441  0.704  0.194  0.158  1  -0.084  0.17  -0.311  -0.161  14  fem ns r  0.484  0.421  0.098  0.281  -0.084  1  0.467  0.077  0.556  15  fem ns  1  0.27  0.307  0.256  0.487  0.17  0.467  1  0.53  0.444  16  fem m a r  -0.55  0.127  -0.186  0.741  -0.311  0.077  0.53  1  0.24  17  fem m a  1  0.268  0.563  -0.498  -0.489  0.44  -0.357  -0.28  -0.156  -0.021  18  sharps r  0.003  0.254  0.085  0.253  0.636  0.011  -0.121  -0.184  0.064  19  sharps 1  0.153  0.095  0.386  0.507  0.342  -0.011  -0.321  -0.278  -0.17  20  fib ht r  0.114  0.269  0.72  0.615  0.208  0.063  -0.17  -0.249  -0.394  21  fib ht 1  0.575  0.204  0.407  -0.023  0.235  -0.545  -0.09  -0.211  -0.332  22  a n k l e jt r  0.587  0.626  -0.251  -0.165  0.333  -0.21  0.213  0.517  -0.141  23  a n k l e jt 1  0.38  0.498  -0.238  -0.105  -0.348  0.049  0.276  0.363  0.131  24  % wt b e a r r  0.106  0.101  -0.279  -0.103  0.158  0.253  0.402  0.902  0.348  25  % wt b e a r 1  0.052  0.423  -0.375  0.508  0.197  -0.018  0.266  0.36  0.307  26  % ped  0.064  0.12  0.237  -0.145  0.088  0.31  -0.121  -0.666  0.218  27  % sess  0.281  0.028  -0.121  -0.422  0.459  -0.292  -0.136  0.3  -0.317  28  % distal  0.044  0.069  0.158  -0.118  -0.163  0.283  0.215  -0.055  -0.114  29  %prox  0.789  0.011  -0.055  0.296  -0.368  0.074  -0.122  0.178  0.083  30  % pelvic  0.147  0.363  0.412  -0.431  0.136  0.068  0.316  -0.03  0.083  31  %diaph  0.375  0.556  -0.162  0.406  -0.028  -0.599  -0.016  -0.251  -0.115  32  %flat bones  0.559  0.254  -0.35  -0.372  -0.234  -0.036  -0.311  -0.024  0.278  1  198  8.6.4.2 Pearson Correlation Matrix (continued) 45  35  Correlation  total  Matrix  arm  %flared %  1  46  47  48  49  50  ratio  r arm  r arm  total  r arm  1  uppder  lower  arm r  ratio  arm  0.379  0.605  0.47  -0.041  0.164  0.681  52  51 ALD  0.133  53  Heg  lleg  upper  lower  -0.148  0.29  not  36  flared  0.015  0.593  -0.431  0.425  0.35  -0.645  -0.08  0.202  -0.304  37  % of 1  0.075  0.115  0.254  -0.425  -0.526  0.375  -0.165  -0.244  -0.238  38  %of4  0.749  0.071  -0.397  0.043  -0.397  -0.223  0.151  0.339  0.345  39  avg #  0.4  0.473  0.465  -0.359  0.017  0.256  0.364  0.217  0.39  40  % left  -0.4  0.133  -0.203  -0.266  -0.017  0.575  0.297  0.21  -0.433  41  % right  0.656  0.133  0.203  -0.27  0.008  -0.575  -0.297  -0.21  0.424  42  % ht  0.389  0.188  -0.305  -0.137  -0.502  0.213  -0.401  -0.175  -0.038  43  1 a r m upper  0.725  -0.05  -0.735  -0.282  -0.037  -0.415  -0.521  -0.007  -0.168  44  1 a r m lower  0.511  0.255  -0.759  -0.112  -0.394  -0.314  -0.547  -0.107  -0.087  45  total a r m 1  -0.07  -0.709  -0.237  -0.765  -0.432  -0.549  -0.02  0.252  46  ratio 1 a r m  -321  1  0.01  -0.308  -0.603  -0.101  0.038  0.169  -0.252  47  upper  0.517  0.254  1  -0.052  -0.351  -0.274  -0.593  0.029  -0.505  48  r a r m lower  0.522  0.104  -0.596  -0.422  -0.498  -0.536  -0.075  0.118  49  total a r m r  0.163  0.023  -0.508  -0.053  -0.402  -0.632  -0.086  0.224  50  r a r m ratio  0.032  0.537  0.376  -0.165  0.574  -0 272  0 134  0.112  51  ALD  0.334  0.195  -0.084  -0.11  -0.465  -0 13  •  -0 065  -0.126  52  1 leg u p p e r  0.348  0.108  -0.309  -0.159  -0.596  -0.374  -0.448  53  1 leg l o w e r  0.174  0.206  -0.359  -0.027  -0.742  -0.281  -0.513  0.068  54  total leg I  0.118  0.33  0.488  -0.176  0.422  -0.253  -0.498  0.079  -0.233  55  1 leg ratio  0.401  0.229  -0.219  -0.185  -0.529  -0.075  0.266  -0.1  -0.525  56  r leg u p p e r  0.172  0.215  -0.366  -0.06  -0.619  -0.287  -0.519  -0.05  -0.414  57  r leg l o w e r  0.171  0.102  -0.5  -0.567  -0.757  -0.352  -0.442  0.151  -0.294  58  total leg r  0.363  0.349  0.286  -0.74  0.207  -0.187  -0.48  0.044  -0.022  59  r leg ratio  0.241  0.188  0.289  -0.009  0.123  0.124  -0.097  -0.356  -0.157  60  LLD  0.214  0.041  0.456  -0.234  0.145  -0.064  0.009  0.132  -0.485  1  r arm  1  199  0.073 1  8.6.4.2 Pearson Correlation Matrix (continued) 54  55  56  57  58  59  Correlation  total  Heg  r leg  r leg  total  r leg  Matrix  legl  ratio  uppder  lower  legr  ratio  60 LLD  1  # lesions  0.362  0.456  0.34  -0.754  0.445  0.125  0.297  2  c a r p a l slip r  0.495  0.194  0.647  -0.576  0.233  0.324  0.044  3  c a r p a l slip I  0.297  0.192  0.99  -0.75  0.34  0.859  0.26  4  rad inclin r  -0.14  0.022  0.322  -0.756  0.78  -0.94  -0.439  5  rad inclin 1  0.437  0.323  0.538  0.34  0.98  0.23  -0.485  0.765  0.475  0.283  0.76  0.5  0.35  0.308  u l n a r short  6  r u l n a r short  7  1  0.475  0.346  0.73  0.23  0.55  -0.433  0.44  8  rad bow r  0.268  0.01  0.93  0.123  0.456  -0.354  -0.339  9  rad bow  1  0.582  0.247  0.833  0.345  0.76  -0.433  -214  10  elb jt r  0.656  0.093  0.763  0.34  0.002  -0.43  -0.041  11  elb jt  1  0.133  0.324  0.3  0.56  0.213  -0.45  0.456  12  fem a a r  0.275  0.479  883  0.83  0.04  0.94  -0.06  13  fem a a  1  0.301  0.402  0.393  0.34  -0.5  0.49  0.123  14  fem ns r  0.532  -0.03  0.482  0.09  -0.3  0.43  -0.064  15  fem ns  1  0.079  0.352  0.119  0.67  -0.3  0.87  0.009  16  fem m a r  0.388  0.593  0.299  0.69  -0.44  0.003  -0.14  17  fem m a  1  0.124  0.638  0.33  0.005  -0.564  0.9  0.08  18  sharps r  0.012  0.537  0.21  -0.56  0.04  0.54  0.636  19  sharps 1  0.278  0.432  0.222  -0.564  0.868  0.94  0.546  20  fib ht r  0.275  -0.02  0.33  0.234  0.345  0.113  0.305  21  fib ht  1  0.148  0.039  -0.38  -0.44  0.965  0.124  0.214  22  a n k l e jt r  861  0.384  -0.734  -0.2  0.674  0.13  0.036  23  a n k l e jt 1  0.689  0.213  0.823  0.609  747  0.89  -0.163  24  % wt b e a r r  0.078  0.35  0.932  0.443  0.82  0.006  -0.377  25  % wt b e a r 1  0.113  0.876  0.229  0.553  0.679  0.042  0.335  26  % ped  0.557  0.576  0.922  0.26  0.23  0.456  0.537  27  % sess  0.267  0.146  0.199  0.765  0.45  0.756  -0.289  28  % distal  0.249  0.158  0.029  0.334  0.97  0.345  -0.17  29  %prox  -0.07  0.106  0.392  0.67  0.22  0.923  -0.385  30  % pelvic  0.413  0.279  0.675  0.87  0.229  0.454  0.268  31  %diaph  0.217  -0.17  0.445  0.98  0.674  0.293  -0.562  8.6.4.2 Pearson Correlation Matrix (continued) 54  55  56  57  58  59  Correlation  total  lleg  r leg  r leg  total  r leg  Matrix  legl  ratio  uppder  lower  leg r  ratio  60 LLD  34  %simple  0.514  0.211  0.142  0.43  0.843  0.32  0.095  35  %flared  0.528  0.1  0.234  0.567  0.273  0.234  -0.019  0.552  0.043  0.566  0.54  0.009  0.345  -331  % not  36  flared  37  % of  1  0.244  0.286  0.122  0.454  0.987  0.322  0.933  38  % of 4  0.243  0.373  0.677  0.465  0.09  0.233  0.84  39  avg #  0.362  0.456  0.564  0.476  0.65  0.944  0.483  40  % left  0.325  0.215  0.678  0.2  0.43  0.758  0.93  41  % right  0.325  0.042  0.435  0.65  0.23  0.483  -0.333  42  % ht  0.253  0.075  0.789  0.67  0.19  0.493  -0.843  43  1 a r m upper  0.126  0.335  0.345  0.58  0.87  0.842  0.934  44  1 a r m lower  0.274  0.099  0.876  0.45  0.908  0:745  0.23  45  total a r m 1  0.225  0.383  567  0.678  0.654  0.39  0.383  46  ratio 1 a r m  0.288  0.073  0.998  0.345  0.876  0.398  0.203  r arm  47  upper  0.015  0.027  0.887  0.45  0.213  0.834  0.23  48  r a r m lower  0.312  0.048  -0.987  0.48  0.8  -0.842  0.432  49  total a r m r  0.209  -0.74  -0.76  0.578  0.56  0.321  0.233  50  r a r m ratio  0.383  0.085  -0.787  0.567  0.49  0.123  0.11  51  ALD  0.265  0.522  -0.765  0.45  0.65  0.432  0.002  0.35  0.922 0.74  52  1 leg u p p e r  0.405  0.272  0.789  0.576  0.7  53  1 leg l o w e r  0.199  0.299  0.098  0.333  0.567  0.23  54  total leg 1  1  0.347  0.087  0.006  0.678  0.655  0.34  55  1 leg r a t i o  0 299  1  0.554  0.433  0.098  0.544  0.299  56  r leg u p p e r  0.279  0 262  1  0.44  0.456  0.005  0.008  57  r leg l o w e r  0.19  0.149  0.667  0.87  0.35  0.009  58  total leg r  -0.03  0.158  0.453  0.234  0.343  59  r leg r a t i o  0.134  0.44  0.698  0.54  0.99  1  0.493  60  LLD  0.308  0.047  0.184  0.254  0.666  1 0.333  201  1  0.54  1  Appendix 8.7 Genotype - Phenotype Correlation Tables 8.7.1 Gene Table 8.7.1.1 Lesion Quality by Gene EXT 1 (n=7)  EXT 2 (n=19)  P-value  Power  Lesion R a n k 1  9.1+6.1  6.2 ± 4 . 6 ( n = 17)  0.21  0.25  % Rank 1  27.0 ± 10.1  31.6 ± 2 0 . 3  0.58  0.073  3.9 ± 2 . 8 ( n = 17)  0.059  0.32  22.6 ± 12.7 ( n = 17)  0.55  0.11  Variable  Lesion R a n k 2  6.412.9  (n=17)  % Rank 2  19.4 ± 7 . 5  Lesion R a n k 3  5.0 ± 1.9  1.9+1.8 ( n = 17)  <0.01  % Rank 3  16.0 ± 6 . 0  9.5 ± 6 . 9 ( n = 17)  0.042  0.57  Lesion R a n k 4  12.1 ± 4 . 1  7.1 ± 4 . 6  0.019  0.50  % Rank 4  37.7 ± 10.1  36.1 ± 1 8 . 9  (n=17)  0.83  0.053  S m a l l (%)  28.4 ± 11.7  30.8 ± 1 7 . 9  (n=19)  0.74  0.061 0.050  (n=17)  (0.0013)  0.94  M e d i u m (%)  30.6 ± 8.4  30.9 ± 13.9 ( n = 19)  0.96  L a r g e (%)  38.6 ± 15.7  36.5 ± 1 7 . 9  0.79  0.058  Average N u m b e r of Lesions  32.7 ± 10.4  19.1 ± 8 . 8 ( n = 1 7 )  < 0.01 (0.0036)  0.82  No. Pedunculated  8.7 ± 2 . 9  6.1 ± 4 . 3 ( n = 1 9 )  0.15  0.28  % Pedunculated  26.9 ± 5.3  31.2 ± 13.8 ( n = 17)  0.43  0.12  N o . Sessile  21.1+8.9  13.4 ± 6.9 ( n = 19)  0.028  0.61  % Sessile  64.3 ± 11.1  65.1 ± 14.8 ( n = 17)  0.89  0.054 0.67  (n-19)  No. Distal  13.1 ± 5 . 0  8.1 ± 4 . 4 ( n = 19)  0.020  %  Distal  40.2 ± 8.4  39.9 ± 14.5 ( n = 17)  0.97  0.051  No. Proximal  14.4 ± 5 . 2  9.4 ± 4 . 6 ( n = 19)  0.026  0.62  % Proximal  43.9 ± 8 . 9  46.4 ± 16.9 (n = 17)  0.72  0.069  No. Pelvic  3.4 ± 2 . 9  0.74 ± 1.4 ( n = 19)  < 0.01 (0.0043)  0.87  % Pelvic  9.617.5  2.3 ± 5.2 (n = 17)  0.012  0.68  1.3 ± 1.3 ( n = 19)  0.39  0.13  No Diaphyseal  1.9 ± 1.7  % Diaphyseal  6.5 ± 6 . 9  8.7 ± 12.0 (n = 17)  0.66  0.063  No. Flat Bone  4.1 ± 2 . 8  0.84 ± 1.4 ( n = 19)  < 0.01 (0.0005)  0.98  % Flat Bone  11.8 ± 6.1  3.0 ± 15.3 ( n = 17)  < 0.01(0.0019)  0.91  No. Complex  4.9 ± 5 . 9  2.7 ± 2 . 1 ( n = 19)  0.17  0.26  14.3 ± 9.3 (n = 17)  0.67  0.061 0.58  %  Complex  12.4 ± 10.3  No. Simple  25.3 ± 5.4  17.3 ± 8.6 (n = 19)  0.32  % Simple  79.5 ± 10.6  84.1 ± 9 . 5 ( n = 17)  0.31  0.20  No. Flared  14.1 ± 12.0  6 . 8 ± 5 . 9 ( n = 19)  0.047  0.52  % Flared  38.6 ± 2 9 . 7  30.4 ± 2 4 . 2 ( n = 17)  0.48  0.099 0.34  No. Not Flared  18.6 ± 7 . 8  12.9 ± 7.2 ( n = 17)  0.10  % Not Flared  61.4 ± 2 9 . 7  69.6 ± 24.2 ( n = 17)  0.48  No. Left  18.6 ± 6 . 7  10.1 ± 5 . 2 ( n = 19)  <0.01  %  56.6 ± 7 . 2  0.091 (0.0022)  0.92  49.4 ± 10.09 ( n = 17)  0.13  0.39  No. Right  14.3 ± 4 . 9  10.2 ± 4.9 ( n = 19)  0.076  0.42  %  43.8 ± 7 . 8  50.6 ± 10.9 ( n = 17)  0.15  0.34  Left  Right  202  Table 8.7.1.2. Limb Alignment by Gene Variable Normal EXT1 Values (n = 7) 1. Carpal Slip Right  5±2mm  2. Carpal Slip Left 3. R a d i a l Inclination Right  21° ± 2 °  4. R a d i a l Inclination Left 5. Ulnar Shortening Right  0 ± 1 mm  6. Ulnar Shortening Left 7. R a d i a l B o w Right  10° ± 5 °  8. R a d i a l B o w Left 9. R a d i a l Head Dislocation R 10. R a d i a l Head Dislocation L 11. E l b o w Joint Right  9° ± 3 °  12. E l b o w Joint Left 13. Femoral A . A . Right  7° ± 2 ° valgus  14. Femoral A . A . Left 15. Femoral N . S . A n g l e Right  135° ± 5 °  16. Femoral N . S . A n g l e Left 17. Femoral M . A . Right  0 ° ± 5 ° varus  18. Femoral M . A . Left 19. Sharp's Right  35° ± 4 °  20. Sharp's Left 21. Fibular Height Right  26. % Weightbear Left N u m b e r o f p a r a m e t e r s that fall beyond the n o r m a l range  Power  5.2 ± 2 . 8  2.2 ± 3 . 6  0.08  0.41  4.7 + 3.0  3.1 ± 3 . 5  0.29  0.17  28.0±5.4  24.2 ± 5 . 0  0.13  0.31  30.6 ± 6.0  26.4 ± 4.8  0.08  0.41  -1.7 ± 4 . 9  0.81  0.056  2.7 ± 5 . 8  -0.8 ± 4.9  0.14  0.30  9.0 ± 2 . 3  7.6 ± 2 . 4  0.20  0.23  14.2 + 8.4 1 dislocation  <0.01  0.87  2 dislocations  7.7 + 2.4 1 dislocation 1 dislocation  -1.8 ± 18.5  -5.6 ± 12.9  0.58  0.082  -3.9 ± 10.2  -8.5 ± 11.3  0.35  0.14  -3.1 ± 7 . 8  -5.6 ± 9 . 1  0.53  0.093  -1.6 ± 8 . 3  -3.4 ± 9 . 0  0.64  0.073  143.1 ± 17.7  140.1 ± 8 . 0  0.55  0.088  146.4 ± 11.0  137.1 ± 9 . 1  0.04  0.56  6.3 ± 5 . 4  -0.1 ± 6 . 0  0.03  0.59  -1.0 ± 7 . 0  1.1+5.0  0.42  0.12  38.5 ± 3 . 1  41.4 ± 5 . 7  0.29  0.23  38.5 ± 5 . 4  41.4 ± 4 . 9  0.31  0.16  51.6 ± 11.7  0.94  0.051  -1.2 ± 3 . 9  52.0 ± 8.0 5 2 . 2 + 13.8  51.8 ± 14.4  0.95  0.052  0 ° ± 5°  -9.8 ± 13.6  -1.8 ± 10.1  0.14  0.062  -5.5 ± 14.4  -1.0 ± 10.6  0.42  0.052  61.0 ± 2 2 . 4  46.5 ± 22.2  0.18  0.36  65.2 ± 11.9 15/24  51.4 ± 19.7 4/24  0.12  0.21  24. A n k l e Joint A n g l e Left 25. % Weightbear Right  P-value  50 ± 10  22. Fibular Height Left 23. A n k l e Joint A n g l e Right  EXT 2 (n = 19)  50 ± 10  203  Table 8.7.1.3. Segment Lengths and Percentile Height by Gene Variable  EXTl (n = 7)  EXT 2 (n = 19)  P-value  Power  Total L e g Length-Right  79.6 ± 8.3  84.9 ± 9 . 1  0.19  0.24 0.49  U p p e r L e g - Right  39.2 ± 4 . 5  44.0 ± 5.5  0.052  L o w e r L e g - Right  32.4 ± 3 . 9  34.9 ± 3 . 6  0.12  0.32  Total L e g Length - Left  78.9 ± 8.4  84.3 ± 9.3  0.19  0.24  Upper L e g - Left  38.9 ± 4 . 9  43.2 ± 5 . 1  0.063  0.45  L o w e r L e g - Left  32.4 ± 4.6  36.2 + 4.8  0.087  0.39  Total A r m Length - Right  44. 9 ± 5 . 1  50.7 ± 5 . 7  0.026  0.62 0.49  Upper A r m - Right  27.4 + 2.4  30.6 ± 3 . 8  0.052  L o w e r A r m - Right  20.9 ± 2.4  23.6 ± 3 . 4  0.071  0.43  Total A r m Length - Left  45.0 ± 5.2  51.1 ± 5 . 9  0.027  0.62  U p p e r A r m - Left  27.5 ± 3 . 5  31.2 ± 4 . 4  0.059  0.47  L o w e r A r m - Left  19.9 ± 3 . 1  23.9 ± 3 . 3  0.011  0.77  Percentile Height  9.33 ± 13.3  42.5 ± 29.0  <0.01 (0.0081)  0.80  204  8.7.2 Gender Table 8.7.2.1. Lesion Quality by Gender Variable  Males  Females  (n = 14)  (n = 12)  P-value  Lesion R a n k 1  9.1 ± 5 . 9  4.612.6  0.03  % Rank 1  27.8 ± 16.8  32.3119.1  0.55  Lesion R a n k 2  5.313.4  3.812.4  0.24 0.19  % Rank 2  18.8 ± 9 . 6  2 5 . 0 1 12.8  Lesion R a n k 3  3.712.5  1.811.7  0.04  % Rank 3  12.417.0  10.317.6  0.49  Lesion R a n k 4  10.015.0  6.9+4.5  0.13  % Rank 4  36.5116.3  36.7117.9  0.97  S m a l l (%)  32.2117.9  2 7 . 8 1 14.7  0.50  M e d i u m (%)  28.8 + 9.3  3 3 . 1 1 15.6  0.39  L a r g e (%)  35.1117.4  3 9 . 4 1 17.1  0.53  Average N u m b e r of Lesions  28.1111.5  1 7 . 2 1 7.2  0.01  No. Pedunculated  7.915.0  5.612.3  0.16  % Pedunculated  27.4112.8  36.0110.4  0.12  N o . Sessile  19.118.5  11.315.4  0.01  % Sessile  68.0 + 14.6  58.216.7  0.08  No. Distal  11.215.4  7.413.9  0.05  %  Distal  40.5 1 1 4 . 5  39.5111.1  0.86  No. Proximal  13.415.1  7.713.6  < 0.01 (0.0035)  % Proximal  49.3112.1  41.4119.5  0.30  No. Pelvic  1.912.8  0.911.2  0.26  % Pelvic  5.217.6  3.515.7  0.55  No Diaphyseal  1.1 1 1.1  1.911.6  0.13  % Diaphyseal  5.418.3  12.9113.7  0.16  No. Flat Bone  2.212.9  1.211.5  0.26  % Flat Bone  6.317.5  4.816.2  0.59  No. Complex  4.414.5  1.911.4  0.07  % Complex  14.517.1  12.218.0  0.51  No. Simple  23.118.7  15.216.3  0.01  % Simple  83.918.7  83.318.0  0.88  No. Flared  12.619.1  4.314.9  0.01  % Flared  45.0 1 2 5 . 2  18.5117.8  0.01  No. Not Flared  15.118.9  14.016.1  0.74  % Not Flared  55.0125.2  81.5117.8  <0.01 (0.0079)  No. Left  15.217.2  9.014.3  0.02  % Left  5 2 . 1 1 10.6  51.0110.0  0.83  No. Right  13.415.6  8.813.5  0.02  % Right  47.9110.6  49.3 1 1 0 . 1  0.77  205  Table 8.7.2.2. Limb Alignment by Gender Variable  Normal  Males  Females  (n = 14)  (n = 12)  1. Carpal Slip Right  Values 5 ± 2mm  3.4 ± 4 . 3 (n=12)  2.5 ± 2 . 7  0.52  3.8 ± 3 . 6  3.3 ± 3 . 3  0.70  26.5 ± 4 . 6 (n=12)  23.8 ± 5 . 8  0.22  28.8 ± 5 . 7  26.1 ± 4 . 7  0.20  -2.5 ± 4 . 6 (n=12)  -0.58 ± 4 . 5  0.30  0.0 ± 5 . 6 8.1 ± 2 . 7 (n=13)  0.33 ± 5 . 1 7.7 + 2.1  0.88  10.0 ± 6 . 4 1 dislocation  8.8 ± 4 . 1 1 dislocation  0.58  1 dislocation  2 dislocations  -3.8 ± 15.2 (n=13)  -5.6 ± 13.4  0.77  -7.9 ± 11.7  -6.5 ± 10.7  0.74  3.4 ± 4 . 3 (n=12)  2.5 ± 2 . 7  0.52  3.8 ± 3 . 6  3.3 ± 3 . 3  0.70  140.9 ± 8.3  141.0 ± 14.1  0.97  138.0 + 8.1  141.4 ± 12.5  0.41  2.2 + 6.7 (n=13)  0.55 ± 6 . 2 (n=ll)  0.53  -0.39 + 6.0 (n=13)  1.7 ± 4 . 7 (n=ll)  0.36  39.7 ± 3 . 9 (n=13)  42.3 ± 6.6 (n=10)  0.25  40.7 + 4.9 (n=13)  40.5 ± 5.3 (n=10)  0.93  54.5 ± 9.4 (n=13)  48.7 ± 11.7  0.18  2. Carpal Slip Left 3. R a d i a l Inclination Right  21° ± 2 °  4. R a d i a l Inclination Left 5. Ulnar Shortening Right  0 ± 1 mm  6. Ulnar Shortening Left 7. R a d i a l B o w Right  10° ± 5 °  8. R a d i a l B o w Left 9. R a d i a l H e a d Dislocation R 10. R a d i a l Head Dislocation L 11. E l b o w Joint Right  9 ° ± 3°  12. E l b o w Joint Left 13. Femoral A . A . Right  7° ± 2 ° valgus  14. Femoral A . A . Left 15. Femoral N . S . A n g l e Right 16. Femoral N . S . A n g l e Left 17. Femoral M . A . Right  135°±5°  0° ± 5° varus  18. Femoral M . A . Left 19. Sharp's Right  35° ± 4 °  20. Sharp's Left 21. Fibular Height Right  50 ± 10  22. Fibular Height Left  P-value  0.71  51.9 ± 15.9  0.99  (n=H) -3.7 ± 7 . 6  0.92  -2.0 ± 14.7 (n=ll)  -2.3 ± 8.2  0.95  55.2 ± 24.9 (n=12)  45.1 ± 19.9  0.29  26. % Weightbear Left  54.2 ± 17.9 (n=12)  55.5 ± 2 0 . 4 (n=12)  0.87  Number of parameters that fall beyond the normal range  9  12  23. A n k l e Joint A n g l e Right  51.8 ± 12.8 (n=13) 0 ° ± 5°  24. A n k l e Joint A n g l e Left 25. % Weightbear Right  50 ± 10  -4.2 ± 14.9 (n=ll)  206  Table 8.7.2.3. Segment Lengths and Percentile Height by Gender Variable  Males (n = 14)  Females (n = 12)  P-value  Total L e g Length-Right  86.4 ± 5 . 5  80.2 ± 11.3  0.08  Upper L e g - Right  43.4 + 3.9  41.9 ± 7 . 2  0.49  L o w e r L e g - Right  35.5 ± 2 . 9  32.8 ± 4.2  0.065  Total L e g Length - Left  85.8 ± 6 . 2  79.4 ± 11.2  0.081  Upper L e g - Left  42.6 ± 3 . 7  41.3 ± 6 . 9  0.54  L o w e r L e g - Left  36.5 ± 4 . 5  33.6 ± 5 . 3  0.14  Total A r m Length Right U p p e r A r m - Right  50.4 ± 5.2  47.6 ± 6.9  0.25  31.0 ± 3 . 0  28.3 ± 4 . 1  0.072  L o w e r A r m - Right  23.6 ± 3 . 1  22.0 ± 3 . 4  0.23  Total A r m Length - Left  50.8 + 5.6  47.7 ± 6.9  0.20  Upper A r m - Left  31.3 ± 3 . 5  28.9 ± 5 . 1  0.18  L o w e r A r m - Left  23.5 ± 3 . 9  22.1 ± 3 . 4  0.36  Percentile Height  32.3 ± 28.2  35.1 ± 3 2 . 4  0.82  207  8.7.3 Mutation Type Table 8.7.3.1. Lesion Quality by Mutation Type Variable Lesion R a n k 1 % Rank 1 Lesion R a n k 2 % Rank 2 Lesion R a n k 3 % Rank 3 Lesion R a n k 4 % Rank 4 S m a l l (%) M e d i u m (%) L a r g e (%) Average Number of L e s i o n s No. Pedunculated % Pedunculated N o . Sessile % Sessile No. Distal % Distal No. Proximal % Proximal No. Pelvic % Pelvic No Diaphyseal % Diaphyseal No. Flat Bone % Flat Bone No. Complex  Missense  Nonsense  Splice Site  Frameshift  p-value  Power  (n=4)  (n=14) 7.6 ± 6 . 4  (n=5) 5.0 ± 0.0 19.0 ± 5 . 5 6.0 ± 4 . 1  (n=3) 5.0 ± 2 . 6 21.3 ± 6 . 9 6.7 ± 3 . 1 31.5 ± 13.1 2.3 ± 2 . 3 9.0 ± 4 . 9 9.7 ± 8 . 1 37.9 ± 14.0 26.4 ± 7 . 6 39.2 ± 12.3 34.3 ± 8 . 5 23.7 ± 12.5  0.57 0.054 0.53 0.38 0.55 0.42 0.22 0.47 0.045 0.49 0.19 0.81  0.17 0.62 0.18 0.24  8.3 ± 6.7 35.5 ± 16.3 15.3 ± 8 . 1 64.5 ± 16.3 7.3 ± 5.0 18.2 ± 0 . 0 11.7 ± 5.5 50.6 ± 3 . 7 1.3 ± 1.5 0.0 ± 0 . 0 2.0 ± 1.0 11.4 ± 13.8 1.3 ± 1.5 0.0 ± 0 . 0 2.3 ± 1.5  0.93 0.69 0.81 0.90 0.84 0.39 0.76 0.92 0.89 0.79 0.61 0.92 0.89 0.78 0.92  0.074 0.13 0.10 0.080 0.095 0.23 0.11 0.075 0.083 0.10 0.15 0.075 0.081 0.11 0.075  10.3 ± 3 . 5  12.6 ± 8 . 5  22.8 ± 6.5 82.6 ± 7 . 9  21.3 ± 13.1 87.4 ± 8 . 5 8.0 ± 8 . 2  0.86 0.76 0.85 0.89  0.089 0.12 0.091 0.081 0.084  9.0 ± 1.6 48.3 ± 2 1 . 6 4.0 ± 3 . 8 16.0 ± 12.2 2.5 ± 1.9 10.8 ± 5 . 9 5.5 ± 3 . 0 24.8 ± 5 . 9 48.5 ± 19.3 24.0 ± 1 1 . 3 23.9 ± 10.8 21.0 ± 7 . 0  30.0 ± 16.9 4.4 ± 2 . 6 21.5 ± 11.8 2.6 ± 2 . 3 10.1 ± 7 . 6 8.4 ± 5 . 1 38.3 ± 20.2 29.7 ± 15.8 30.7 ± 13.9 37.3 ± 2 0 . 1 22.9 ± 12.8  23.8 ± 9 . 6 4.2 ± 2 . 6 16.0 ± 7 . 1 12.6 ± 3 . 6 41.2 ± 7 . 6 19.1 ± 6 . 7 3 1 . 4 ± 8.6 48.5 ± 5 . 9 27.8 ± 8 . 2  % Complex  6.3 ± 2 . 1 30.8 ± 8.9 14.8 ± 5 . 7 69.2 ± 8.9 9.0 ± 4 . 7 42.3 ± 17.5 9.5 ± 3 . 0 48.1 ± 19.5 0.75 ± 1.5 2.7 ± 5 . 4 1.5 ± 2 . 4 6.0 ± 8 . 4 1.3 ± 1.5 4.6 ± 5 . 5 2.8 ± 0.96 14.9 ± 9 . 4  14.6 ± 9 . 3 63.6 ± 15.7 9.6 ± 5 . 9 41.1 ± 12.6 10.1 ± 5 . 7 44.9 ± 15.9 1.5 ± 2.8 4.5 ± 7 . 9 1.1 ± 1.3 7.2 ± 12.1 1.7 ± 2 . 9 5.6 ± 7 . 9 3.7 ± 4 . 8 14.3 ± 11.2  No. Simple % Simple No. Flared  18.3 ± 7 . 5 85.1 ± 9 . 4 5.8 ± 3.1  18.2 ± 9 . 0 82.2 ± 11.3 9.6 ± 8 . 9  % Flared No. Not Flared  32.9 ± 24.3 15.3 ± 9 . 6  36.5 ± 2 5 . 3 13.2 ± 7 . 1  % Not Flared No. Left % Left  67.1 ± 2 4 . 3  72.7 ± 3 1 . 4  12.5 ± 2 . 6 61.7 ± 10.2  63.5 ± 2 5 . 3 12.1 ± 8 . 7 49.1 ± 9 . 8  No. Right % Right  8.5 ± 4 . 7 38.3 ± 10.2  10.7 ± 4 . 5 50.9 ± 9 . 8  13.6 ± 5 . 6 47.3 ± 7.4  6.7 ± 4 . 6 30.4 ± 13.5  6.6 ± 2.7 24.9 ± 9.4 18.8 ± 7 . 2 66.9 ± 11.9 10.8 ± 3 . 0 39.8 ± 8 . 3 12.8 ± 6 . 1 44.1 ± 10.9 2.0 ± 1.6 6.3 ± 4 . 5 2.0 ± 1.0 8.2 ± 5 . 3 2.4 ± 1.8 7.6 ± 5 . 2 3.0 ± 1.6  9.0 ± 11.7 27.3 ± 3 1 . 4 18.8 ± 8 . 3 14.4 ± 2 . 9 53.3 ± 6 . 9  208  27.1 ± 19.2 14.7 ± 4 . 5 68.7 ± 19.8 9.7 ± 4 . 9 40.8 ± 7.9 14.0 ± 8 . 2 59.2 ± 7 . 9  0.88 0.57 0.92 0.83 0.039 0.39 0.044  0.17 0.22 0.36 0.20 0.65 0.19 0.37 0.10  0.18 0.075 0.096 0.67 0.24 0.65  Table 8.7.3.2. Variable  imb Alignment by Mutation Type Normal  Missense  Nonsense  Splice  Frameshift  (n=4)  (n=14)  Site  (n=3)  P" value  Power  Values  5±2mm  4.0 ± 3 . 4 3.3 ± 3 . 2 26.5 ± 7.9  2.0 ± 4 . 4 3.6 ± 4 . 2 24.9 ± 4 . 1  3.8 ± 2 . 5 3.6 ± 2 . 3 27.4 ± 0 . 5 5  3.7 ± 1.2 3.7±2.1 20.7 ± 9 . 5  0.71 0.99 0.36  0.13 0.052 0.25  27.0 ± 5 . 3  27.2 ± 5 . 1  30.0 ± 5 . 8  25.7 ± 7 . 6  0.70  0.13  0 ± 1 mm -1.0 ± 2 . 3  -2.5 ± 5 . 1  1.8 ± 1.8  -4.0 ± 6.2  0.25  0.32  -4.7 ± 5 . 9  0.14 ± 4 . 8  5.2 ± 4.2  -4.7 ± 5.9  0.033  0.69  10.0 ± 2 . 8  7.2 ± 2 . 5  8.4 ± 0.89  7.3 ± 1.5  0.20  0.37  9.5 ±0.91 1 dislocation  8.9 ± 6 . 7 0  9.3 ± 3 . 8 0  0.88  0.086  1 dislocation -18.3 ± 5 . 6  1 dislocation -1.6 ± 13.9  11.3 ± 5 . 0 1 dislocation 1 dislocation 1.0 ± 16.2  -9.3 ± 1 0 . 0  0.13  0.45  -10.0 ± 6 . 3 -4.5 ± 8.8  -6.6 ± 12.7 -5.6 ± 9 . 1  -6.8 ± 13.6 -0.9 ± 4.5  -7.3 ± 4.5 -8.7 ± 13.5  0.97 0.66  0.063 0.14  2.3 ± 3 . 3  -4.4 ± 10.4  -1.8 ± 7 . 5  -4.7 ± 5.7  0.59  0.16  142.0 ± 5 . 7  142.4 ± 6 . 2  135.7 ± 11.0  0.81  0.10  142.8 ± 13.8  138.6 ± 6 . 5  132.3 ± 6 . 8  0.41  0.23  4.5 ± 3 . 1  -0.27 ± 5.7  139.2 ± 22.9 144.2 ± 16.8 8.1 ± 4 . 3  -4.0 ± 8.0  0.027  0.73  3.3 ± 4 . 5  0.0 ± 6 . 0  -2.0 ± 5.4  3.0 ± 3 . 6  0.48  0.19  41.5 ± 6 . 4 37.0 ± 5 . 7 53.8 ± 2 . 9  40.6 ± 5 . 5 40.8 ± 4 . 9 51.3 ± 11.3  39.6 ± 3 . 4 38.6 ± 4 . 6 43.5 ± 12.7  42.7 ± 8.0 44.3 ± 5.5 62.2 ± 1.0  0.90 0.35 0.15  0.078 0.25 0.43  47.5 ± 15.8  49.2 ± 13.9  52.5 ± 10.1  68.3 ± 7.6  0.17  0.39  -4.3 ± 3 . 8  -3.2 ± 14.1  0.75 ± 2.9  -13.0 ± 6.1  0.48  0.19  1.0 ± 1.0  -1.1 ± 14.4  -1.0 ± 4.1  -11.3 ± 5.9  0.55  0.17  55.5 ± 2 6 . 8  48.1 ± 2 3 . 2  61.8 ± 8 . 9  36.3 ± 2 9 . 0  0.50  0.19  67.0 ± 12.0  48.7 ± 20.9  58.8  60.0 ± 20.2  0.34  0.26  13  11  12  (n=5)  1. Carpal Slip Right 2. Carpal Slip Left 3. Radial Inclination Right 4. Radial Inclination Left 5. Ulnar Shortening Right 6. Ulnar Shortening Left 7. Radial Bow Right 8. Radial Bow Left 9. Radial Head Dislocation R lO.Radial Head Dislocation L 11. Elbow Joint Right 12. Elbow Joint Left 13. Femoral A.A. Right 14. Femoral A.A. Left 15. Femoral N.S. Angle Right 16. Femoral N.S. Angle Left 17. Femoral M.A. Right 18. Femoral M.A. Left 19. Sharp's Right 20. Sharp's Left 21. Fibular Height Right 22. Fibular Height Left 23. Ankle Joint Angle Right 24. Ankle Joint Angle Left 25. % Weightbear Right 26. % Weightbear Left Number of parameters that fall beyond the normal range  21° ± 2 °  10° ± 5 °  9° ± 3 °  7° ± 2 ° valgus  135° ± 5 °  0°±5° varus  35° ± 4 ° 50 ± 10  0°± 5°  50 ± 10  209  =b  11.1  0  12  Table 8.7.3.3. Segment Lengths and Percentile Height by Mutation Type Variable Missense Nonsense Splice Site Frameshift p-value (n=4) (n=14) (n=5) (n=3)  Power  Total L e g Length-Right  74.3 13.8  86.3 ± 8.4  83.3 ± 6.5  85.7 ± 4 . 3  0.16  0.42  Upper L e g - Right  37.3 ± 7 . 9  44.0 ± 5 . 3  41.9±3.7  42.3 ± 1.9  0.21  0.36  L o w e r L e g - Right  31.5 ± 6 . 7  34.7 ± 3 . 2  34.3 ± 3.2  34.7 ± 2.0  0.55  0.17  Total L e g Length - Left  73.5 ± 13.8  85.4 ± 9 . 0  82.8 ± 6.2  84.8 ± 2.9  0.18  0.39  Upper L e g - Left  37.2 ± 7.7  43.0 ± 5 . 1  41.8 ± 3 . 5  41.0 ± 0 . 8 7  0.29  0.29  L o w e r L e g - Left  30.5 ± 5 . 9  36.5 ± 5 . 3  35.0 ± 4 . 0  36.7 ± 1.2  0.22  0.35  Total A r m Length Right Upper A r m - Right  45.3 ± 5.4  50.3 ± 6 . 1  47.9 ± 5.4  50.7 ± 4 . 1  0.53  0.18  26.6 ± 4 . 5  30.9 ± 3 . 9  29.3 ± 3.5  30.5 ± 2 . 2  0.28  0.29  L o w e r A r m - Right  21.0 ± 4 . 2  22.7 ± 3.0  22.8 ± 2 . 1  23.5 ± 4.4  0.75  0.12  Total A r m Length - Left  45.3 ± 9 . 3  50.2 ± 6.4  48.6 ± 4 . 6  51.0±5.8  0.58  0.16  Upper A r m - Left  26.4 ± 5 . 8  31.2 ± 4 . 8  30.0 ± 3 . 5  31.5 ± 1.8  0.32  0.27  L o w e r A r m - Left  22.4 ± 4.8  23.2 ± 3 . 9  21.3 ± 2 . 7  23.0 ± 4 . 6  Percentile Height  21.5 ± 2 8 . 2  51.3 ± 3 2 . 1  20.2 ± 14.3  9.7 ± 7 . 6  0.048  0.64  210  8.7.4 Mutation Severity Table 8.7.4.1. Lesion Quality by Multation Severity Variable Severe Mild (n=22) (n=4) Lesion Rank 1 6.7 ± 5.5 (n=20) 9.0 ± 1.6 % Rank 1 26.7 ± 15.2 48.3 ±21.6 Lesion Rank 2 4.8 ± 2.9 4.0 ±3.8 % Rank 2 22.8 ± 11.2 16.0 ± 12.2 Lesion Rank 3 2.9 ± 2.4 2.5 ± 1.9 % Rank 3 11.5 ±7.6 10.8 ±5.9 Lesion Rank 4 9.2 ±5.1 5.5 ±3.0 % Rank 4 38.9 ± 17.2 24.8 ±5.9 Small (%) 26.8 ± 13.7 48.5 ± 19.3 Medium (%) 32.0 ± 12.5 24.0 ± 11.3 Large (%) 39.4 ± 17.0 23.9 ± 10.8 Average Number of 23.5 ± 11.8 21.0 ±7.0 Lesions No. Pedunculated 6.9 ± 4.4 6.3 ±2.1 % Pedunculated 29.8 ± 12.7 30.8 ±8.9 No. Sessile 15.6 ±8.6 14.8 ±5.7 % Sessile 63.9 ± 14.3 69.2 ± 8.9 No. Distal 9.5 ±5.2 9.0 ± 4.7 % Distal 39.6 ± 12.2 42.3 ± 17.5 No. Proximal 10.9 ±5.6 9.5 ±3.0 % Proximal 45.2 ± 14.3 48.1 ± 19.5 No. Pelvic 1.6 ±2.4 0.75 ± 1.5 % Pelvic 4.8 ±7.0 2.7 ±5.4 No Diaphyseal 1.5 ± 1.2 1.5 ±2.4 % Diaphyseal 8.4 ± 11.2 6.0 ±8.4 No. Flat Bone 1.8 ±2.5 1.3 ± 1.5 % Flat Bone 5.8 ±7.1 4.6 ±5.5 No. Complex 3.4 ±3.9 2.8 ± 0.96 % Complex 13.5 ±9.6 14.9 ±9.4 No. Simple 19.7 ±8.9 18.3 ±7.5 % Simple 82.3 ± 10.1 85.1 ±9.4 No. Flared 9.3 ± 9.0 5.8 ± 3.1 % Flared 32.8 ±26.4 32.9 ±24.3 No. Not Flared 14.5 ±7.5 15.3 ±9.6 % Not Flared 67.2 ± 26.4 67.1 ±24.3 No. Left 12.3 ±7.3 12.5 ±2.6 % Left 49.5 ± 9.4 61.7 ± 10.2 No. Right 11.8 ± 5.2 8.5 ±4.7 % Right 50.6 ±9.5 38.3 ± 10.2  211  P-value  Power  0.42 0.024 0.66 0.29 0.76 0.85 0.18 0.12 0.011 0.24 0.095 0.69  0.12 0.64 0.070 0.17 0.060 0.054 0.25 0.32 0.76 0.19 0.37 0.067  0.77 0.89 0.85 0.49 0.85 0.71 0.62 0.73 0.50 0.58 0.95 0.69 0.66 0.75 0.76 0.79 0.77 0.62 0.45 0.99 0.85 0.99 0.96 0.029 0.25 0.028  0.059 0.052 0.054 0.10 0.054 0.064 0.076 0.063 0.098 0.082 0.050 0.067 0.070 0.060 0.060 0.058 0.060 0.076 0.11 0.050 0.054 0.050 0.050 0.60 0.20 0.61  Table 8.7.4.2. Limb Alignment by Mutation Severity Variable Normal Severe Mild Values (n=22) (n=4)  P-value  1. 2. 3. 4.  0.53 0.86 0.59 0.83  Carpal Carpal Radial Radial  Slip Right Slip Left Inclination Right Inclination Left  5 ± 2mm 21° ± 2 °  5. U l n a r Shortening Right 6. Ulnar Shortening Left  0 ± 1 mm  7. Radial B o w Right 8. Radial B o w Left 9. Radial Head Dislocation R 10.Radial H e a d Dislocation L 11. E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N . S . A n g l e Right 16. Femoral N . S . A n g l e Left 17. Femoral M . A . Right 18. Femoral M . A . Left 19. Sharp's Right 20. Sharp's Left 2 1 . Fibular Height Right 22. Fibular Height Left 23. A n k l e Joint A n g l e Right 24. A n k l e Joint A n g l e Left 25. % Weightbear Right 26. % Weightbear Left  10° ± 5 °  N u m b e r of parameters  9° ± 3 ° 7° ± 2 ° valgus 135° ± 5 °  0° ± 5° varus 35° ± 4 ° 50 ± 10 0°±5°  5 0 ± 10  2.7 ± 3 . 7 3.6 ± 3 . 5 24.9 ± 4.9 27.6 ± 5 . 5 -1.7 ± 4 . 9 0.6 ± 5 . 5 7.5 ± 2 . 1 9.5 ± 5.9 1 dislocation  4.0 ± 3 . 4 3.3 ± 3 . 2 26.5 ± 7.9 27.0 ± 5 . 3 -1.0 ± 2 . 3 -2.5 ± 3.5 10.0 ± 2 . 8 9.5 ± 0 . 9 1 dislocation  2 dislocations  1 dislocation  -2.1 ± 13.8 -6.8 ± 11.7 -5.0 ± 8 . 9 -3.9 ± 9 . 1 140.7 ± 11.9  -18.3 ± 5 . 6 -10.0 ± 6 . 3 -4.5 ± 8.8 2.3 ± 3 . 3 142.0 ± 5 . 7  0.03 0.60 0.92 0.20 0.84  139.0 ± 9 . 8  142.8 ± 13.8  0.51  0.9 ± 6 . 8 0.1 ± 5 . 6 40.7 ± 5 . 4 40.9 ± 4 . 9 51.3 ± 11.7 52.7 ± 13.9 -3.9 ± 12.2  4.5 ± 3 . 1 3.3 ± 4 . 5 41.5 ± 6 . 4 37.0 ± 5 . 7 53.8 ± 2 . 9 47.5 ± 15.8 -4.3 ± 3.8  0.31 0.29 0.84 0.30 0.69 0.51 0.95  -2.6 ± 12.3 49.1 ± 2 2 . 4 52.5 ± 19.2  1.0 ± 1.0 55.5 ± 2 6 . 8 67.0 ± 12.0  0.62 0.62  7/24  5/24  8/24  that fall b e y o n d the n o r m a l range  212  0.79 0.28 0.05 0.99  0.16  Table 8.7.4.3. Segment Lengths and Percentile Height by Mutation Severity Variable Mild Severe P-value Power (n=4) (n=22) Total Leg Length74.3 ± 13.7 85.2 ±7.2 0.022 0.65 Right Upper Leg - Right  37.3 ± 7.9  43.7 ±4.7  0.031  0.59  Lower Leg - Right  31.5 ±6.7  34.8 ±3.0  0.11  0.34  Total Leg Length Left  73.5 ± 13.8  84.5 ± 7.5  0.025  0.63  Upper Leg - Left  37.3 ±7.7  42.9 ± 4.5  0.048  0.51  Lower Leg - Left  30.5 ±5.9  36.0 ±4.4  0.038  0.55  Total Arm Length Right  45.3 ±8.8  49.8 ±5.4  0.17  0.26  Upper Arm - Right  26.6 ±4.5  30.3 ± 3.4  0.067  0.44  Lower Arm - Right  21.0 ±4.2  23.2 ±3.1  0.23  0.21  Total Arm Length Left  45.3 ± 9.3  50.2 ±5.6  0.15  0.28  Upper Arm - Left  26.4 ±5.8  30.9 ±3.9  0.059  0.46  Lower Arm - Left  22.4 ± 4.8  22.9 ±3.6  0.79  0.058  Percentile Height  21.5 ±28.2  35.8 ±29.9  0.39  0.13  213  8.7.5  Mutation Location  Table 8.7.5.1. Lesion Quality by Muttation Location Variable Early Late P-Value (n=19) (n=7) Lesion Rank 1 % Rank 1 Lesion Rank 2 % Rank 2 Lesion Rank 3 % Rank 3 Lesion Rank 4 % Rank 4 Small (%) Medium (%) Large (%) Average Number of Lesions No. Pedunculated % Pedunculated No. Sessile % Sessile No. Distal % Distal No. Proximal % Proximal No. Pelvic % Pelvic No Diaphyseal % Diaphyseal No. Flat Bone % Flat Bone No. Complex % Complex No. Simple % Simple No. Flared % Flared No. Not Flared % Not Flared No. Left % Left No. Right % Right  Power  7.5 ± 6.0 33.6 ± 19.9 3.9 ± 2 . 6 20.6 ± 12.4 2.3 ± 2 . 2 9.6 ± 6 . 9 7.5 ± 5 . 0 36.0 ± 19.2 (n=17) 32.8 ± 17.8 30.3 ± 14.1 35.1 ± 18.2 2 1 . 2 ± 12.1  5.9 ± 1.6 22.1 ± 7 . 6 6.3 ± 3 . 5 24.3 ± 8.7 4.1 ± 2 . 2 15.7 ± 6 . 2 11.3 ± 3 . 7 37.9 ± 8.6  0.48 0.16 0.085 0.48 0.072 0.058 0.084 0.81  0.10 0.28 0.39 0.10 0.43 0.47 0.39 0.056  22.9 ± 8.9 32.1 ± 7 . 1 42.2 ± 13.2 27.6 ± 6.7  0.18 0.75 0.36 0.21  0.25 0.061 0.14 0.22  6.8 ± 4 . 6 31.8 ± 13.1 14.2 ± 8 . 5 63.3 ± 14.7 8.8 ± 5 . 6 39.4 ± 14.3 10.3 ± 5 . 3 47.2 ± 16.3 1.3 ± 2 . 5 3.7 ± 7.4 1.2 ± 1.2 7.9 ± 12.1 1.5 ± 2 . 6 4.6 ± 7 . 5 3.4 ± 4 . 1  6.9 ± 2.4 25.6 ± 8.2 19.0 ± 5 . 9 68.5 ± 10.4 11.3 ± 2.8 41.7 ± 8 . 6 12.0 ± 5 . 3 42.2 ± 10.8 1.9 ± 1.6 6.1 ± 4 . 8 2.1 ± 1.7 8.4 ± 6.7 2.4 ± 1.5 8.1 ± 4 . 4 2.9 ± 1.3 10.0 ± 3 . 1 23.3 ± 5 . 4 84.9 ± 7 . 7 7.4 ± 9.9 23.2 ± 2 6 . 6 20.1 ± 7 . 2 76.8 ± 26.6 14.4 ± 2.4 53.4 ± 5 . 6 13.3 ± 4 . 6 47.0 ± 6 . 0  0.97 0.27 0.19 0.41 0.27 0.69 0.47 0.46 0.59 0.46 0.13 0.91 0.37 0.27 0.73 0.22 0.17 0.50 0.63 0.25 0.019 0.25 0.35 0.58 0.25 0.65  0.050 0.18 0.24 0.12 0.18 0.067 0.11 0.11 0.080 0.11 0.31 0.051 0.14 0.18 0.063 0.21 0.26 0.097 0.074 0.19 0.68 0.19 0.14 0.082 0.19 0.072  15.3 ± 10.7 18.1 ± 9 . 2 81.9 ± 10.6 9.3 ± 8 . 1 36.8 ± 2 4 . 8 12.3 ± 6 . 7 63.2 ± 2 4 . 8 11.6 ± 7 . 7 50.8 ± 11.9 10.6 ± 5 . 3 49.2 ± 11.9  214  Table 8.7.5.2. Limb Alignment by Mut ation Location Variable Normal Early Late Values (n=19) (n=7)  P-Value  1. 2. 3. 4. 5. 6.  0.25 0.68 0.13 0.28 0.08 0.10  Carpal Slip Right Carpal Slip Left R a d i a l Inclination Radial Inclination U l n a r Shortening Ulnar Shortening  5 ± 2mm Right Left Right Left  7. R a d i a l B o w Right 8. R a d i a l B o w Left 9. R a d i a l Head Dislocation R 10. R a d i a l H e a d Dislocation L 11. E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N . S . A n g l e Right 16. 17. 18. 19. 20. 21. 22.  Femoral N . S . A n g l e Left Femoral M . A . Right Femoral M . A . Left Sharp's Right Sharp's Left Fibular Height Right Fibular Height Left  23. 24. 25. 26.  A n k l e Joint A n g l e Right A n k l e Joint A n g l e Left % Weightbear Right % Weightbear Left  of parameters that fall beyond the n o r m a l  Number  21° ± 2 ° 0 ± 1 mm 10° ± 5 °  9° ± 3 ° 7° ± 2 ° valgus 135° ± 5 °  0°±5°varus 35° ± 4° 50 ± 10 0°±5° 50 ± 10  2.4 ± 3 . 7 3.4 ± 3 . 7 24.1 ± 5.2 26.8 ± 5 . 1 -2.6 ± 4.9 -0.9 ± 5.0 7.6 ± 2 . 5 9.0 ± 5 . 8 1 dislocation  4.3 ± 3 . 0 4.0 ± 2 . 6 27.7 ± 5 . 0 29.4 ± 5 . 9 1.0 ± 2 . 3 3.0 ± 5 . 5 8.6 ± 1.9 10.8 ± 4 . 2 1 dislocation  1 dislocation  1 dislocation  -4.2 ± 13.0 -7.1 ± 11.0 -5.6 ± 9 . 3 -3.9 ± 9 . 2 141.3 ± 7 . 0  -5.9 ± 17.7 -7.9 ± 12.0 -3.1 ± 7 . 1 -0.1 ± 6 . 8 139.9 ± 19.2  0.80 0.87 0.53 0.33 0.77  137.6 ± 8 . 3 -0.6 ± 5 . 8 0.6 ± 5.4 41.2 ± 5 . 7 41.4 ± 4 . 8 53.4 ± 10.5 50.9 ± 15.2  144.9 ± 13.8 7.8 ± 3 . 4 0.7 ± 6.2 39.1 ± 3 . 2 37.5 ± 4 . 7 46.5 ± 10.9 54.8 ± 9 . 9 -1.7 ± 4 . 4 -0.2 ± 3 . 4 58.7 ± 2 2 . 2  0.11 <0.01 0.97 0.44 0.12 0.18 0.56  -4.7 -2.9 47.3 51.7  ± ± ± ±  13.0 13.3 22.7 19.7  11  64.3 ± 12.7 11  range  215  0.39 0.47  0.59 0.63 0.30 0.15  Table 8.7.5.3. Segment Lengths and Percentile Height by Mutation Location Variable Early Late P-Value Power (n=19) (n=7) Total L e g Length-Right U p p e r L e g - Right L o w e r L e g - Right Total L e g Length - Left U p p e r L e g - Left L o w e r L e g - Left Total A r m Length - Right U p p e r A r m - Right L o w e r A r m - Right Total A r m Length - Left U p p e r A r m - Left L o w e r A r m - Left Percentile Height  84.7 43.6 34.7 83.9 42.7 35.9  ± 9.0 ±5.6 ±3.6 ±9.2 ± 5.2 ±4.9  50.0 ± 5 . 9 30.2 ± 3 . 7 23.1 ± 3 . 5 50.0 ± 6 . 5 30.5 ± 4 . 5 23.3 ± 3 . 9 40.2 ± 3 1 . 3  80.5 ± 9.2 40.3 ± 5.4 33.1 ± 4 . 3 79.8 ± 9 . 3 40.1 ± 5 . 2 33.3 ± 4 . 8 46.8 ± 6 . 3 28.5 ± 3 . 8 22.1 ± 2 . 7 47.6 ± 5.9 29.3 ± 4.4 21.5 ± 3 . 9 15.6 ± 14.1  216  0.31 0.19 0.34 0.32 0.28 0.25 0.24 0.31 0.48 0.39 0.54 0.26 0.058  0.16 0.24 0.15 0.16 0.18 0.19 0.20 0.16 0.10 0.13 0.089 0.19 0.47  8.7.6  Gene and Gender  Table 8.7.6.1 Lesion Quality by Gene and Gender Variable  EXT1  EXT 1  EXT 2  EXT 2  P-value  P-value  Males  Females  Males  Females  EXT  Gender  (n=4)  (n=3)  (n=10)  (n=9) 0.032  Lesion R a n k 1  11.8 + 7.3  5.711.2  7.915.3  4.312.9  0.18  % Rank 1  30.8+11.9  2 2 . 0 1 5.2  33.1 1 2 2 . 2  29.9119.3  0.59  0.56  Lesion R a n k 2  7.012.9  5.713.5  4.613.5  3.1 1 1.7  0.064  0.22  % Rank 2  18.815.3  20.3111.1  18.8111.3  26.8113.6  0.55  0.20  Lesion R a n k 3  5.8 + 2.2  4.011.0  2.812.0  1.0  0.0006  0.013  8.016.7  0.049  0.47  5.3 1 3 . 3  0.018  0.097  % Rank 3  15.8 + 5.9  16.317.6  10.9 + 7.2  Lesion R a n k 4  12.8 + 3.9  11.314.9  8.8 + 5.0  10.93  % Rank 4  34.8110.0  41.7110.6  37.2118.9  34.8120.3  0.84  0.97  S m a l l (%)  32.3 1 1 3 . 8  23.217.5  32.2120.0  29.3 1 1 6 . 5  0.75  0.51  M e d i u m (%)  28.2110.9  33.910.98  29.119.1  32.9118.2  0.96  0.41  L a r g e (%)  35.4120.5  42.8 1 7 . 7  34.9117.2  38.2119.5  0.79  0.55 0.0032  A v g # of  37.3 1 1 1 . 4  26.716.1  24.019.5  13.613.2  0.0011  9.013.2  8.3 1 3 . 1  8.1+5.6  4.9 1 0 . 9  0.27  0.15  24.114.1  30.514.9  28.9113.9  38.8111.6  0.24  0.097  lesions No. Pedunculated  % Pedunculated N o . Sessile  24.3 1 10.9  17.0 + 3.5  17.617.1  9.4 1 5 . 2  0.037  0.015  % Sessile  6 4 . 4 1 15.3  64.114.3  67.7 1 1 6 . 0  55.215.7  0.75  0.15  No. Distal  14.815.7  11.014.0  9.814.8  6.2 + 3.2  0.016  0.038  %  Distal  40.0110.8  40.416.0  40.7 1 1 6 . 5  39.2112.8  0.97  0.86  No. Proximal  17.315.0  10.713.8  11.814.4  6.713.1  0.0095  0.0016  % Proximal  46.818.0  40.1110.1  48.5113.8  42.1123.7  0.81  0.37  N o . Pelvic  4.513.7  1.0  0 . 9 0 1 1.7  0 . 5 6 1 1.1  0.0038  0.19  %  11.3 + 9.8  7.313.0  2.514.9  2.115.9  0.015  0.51  No Diaphyseal  2.0  3.012.0  1.211.2  1.711.5  0.50  0.11  %  2.411.6  11.917.8  6.419.1  13.4116.5  0.57  0.12  0 . 6 7 1 1.1  0.0004  0.15  3.0  0.0026  0.52  Pelvic  Diaphyseal  10.82  11.0  No. Flat Bone  5.3 1 3 . 2  2.711.5  1.0  % Flat Bone  13.717.4  9.413.9  2.0 1 4.9  11.6  217  16.1  Table 8.7.6.1 Lesion Quality by Gene and Gender (continued) Variable  EXT 1 Males (n=4)  EXT 1 Females (n=3)  EXT 2 Males (n=10)  EXT 2 Females (n=9)  P-value EXT  P-value Gender  No. Complex  6.8 ± 7 . 7  2.3 ± 1.5  3.4 ± 2 . 5  2.1 ± 1 . 3  0.24  0.15  14.1 ± 9 . 1  0.61  0.47  % Complex  15.6 ± 13.1  8.2 ± 3 . 9  14. 9 ± 7 . 9  No. Simple  26. ± 5.6  23.3 ± 5 . 5  23.1 ± 8 . 9  12.3 ± 4 . 2  0.039  0.0076  % Simple  73.6 ± 10.1  87.4 ± 4 . 7  85. 1 ± 7.9  81.2 ± 8 . 9  0.33  0.59  No. Flared  21.0± 11.5  5.0+4.4  9.2 + 5.7  4.1+5.3  0.019  0.0043  % Flared  54.6 ± 29.4  17.3 ± 12.1  40.6 + 23.7  18.9 ± 2 0 . 3  0.43  0.0097  No. Not Flared  16.3 ± 9.8  21.7 ± 3 . 5  14.6 ± 9 . 1  11.1 ± 3 . 9  0.10  0.73  % Not Flared  45.4 ± 29.4  82.7 ± 12.1  59.3 ± 2 3 . 7  81.1 ± 2 0 . 3  0.43  0.0097  N o . Left  21.8 + 7.4  14.3 ± 2 . 1  13.3 ± 5 . 4  7.7 ± 3 . 4  0.0030  0.0079  % Left  58.2 ± 8.9  54.5 ± 5 . 1  52.1 ± 1 1 . 9  49.3 ± 11.8  0.27  0.52  No. Right  15.5 ± 5.5  12.7 ± 4 . 5  13.2 ± 5 . 7  7.4 ± 2 . 4  0.11  0.022  % Right  41.8 ± 8.9  46.5 ± 6 . 6  47.9 ± 11.9  50.7 ± 11.8  0.31  0.48  218  Table 8.7.6.2. Limb Alignment by Gene and Gender Variable  1. Carpal Slip Right  Normal  E X T l  EXT 1  EXT 2  EXT 2  P-value  P-value  Values  Males  Females  Males  Females  EXT  Gender  (n=4)  (n=3)  (n=10)  (n=9)  7.3 ± 2 . 1  2.1 ± 4 . 1  3.0 ± 1.0  2.3 ± 3 . 1  0.076  0.4941 0.68  5 ± 2mm  2. Carpal Slip Left  6.8 ± 1.5  2.6 ± 3 . 5  2.0 ± 2 . 0  3.7 ± 3 . 7  0.27  31.3 ± 4 . 9  24.9 ± 3.3  24.7 ± 4.0  23.6 ± 6 . 5  0.13  0.21  30.8 ± 5 . 9  28.0 ± 5 . 7  30.3 ± 7 . 4  24.7 ± 2 . 9  0.079  0.19  -3.7 ± 4 . 0  -2.2 ± 4.9  1.3 ± 1.5  -1.2 ± 5 . 1  0.81  0.32  1.3 ± 6 . 4  -0.50 ± 5 . 5  4.7 ± 5 . 5  -1.1 ± 4 . 5  0.15  0.87  10.0 ± 2 . 6  7.5 ± 2 . 6  8.0 ± 1.7  7.6 ± 2 . 2  0.21  0.71  14.9 ± 10.8 1 dislocation 1 dislocation  8.1 ± 2 . 5 0  13.3 ± 5 . 8 0  0.0061  0.53  1 dislocation  0  7.3 ± 2 . 2 1 dislocation 1 dislocation  -0.33 ± 20.6  -4.9 ± 14.4  -3.3 ± 2 0 . 5  -6.3 ± 11.8  0.59  0.77  0.50 ± 8 . 2  -11.4 ± 11.4  -9.7+11.2  -5.4 ± 10.9  0.34  0.73  -1.8 ± 6 . 4  -5.2 ± 9 . 9  -4.8 ± 10.6  -6.0 ± 8.8  0.54  0.68  -1.4 ± 10.4  -5.5 ± 7 . 8  -1.8 ± 6 . 8  -1.1 ± 10.1  0.65  0.40  139 ± 9 . 4  141.6 ± 8 . 2  148.7 ± 2 7 . 0  138.4 ± 7 . 9  0.55  0.97  143.3 ± 3 . 4  135.9 ± 8 . 6  150.7 ± 17.2  138.3 ± 9 . 9  0.040  0.38  4.5 ± 5 . 2  1.2 ± 7 . 4  9.8 ± 5 . 3  -1.5 ± 4 . 4  0.033  0.49  -2.8 ± 8.3  0.68 ± 4 . 9  2.5 ± 0 . 7 1  1.615.2  0.43  0.37  37.3 ± 2 . 5  40.3 ± 4 . 1  40.3 ± 3 . 9  42.8 ± 7 . 3  0.30  0.26  38.3 ± 7 . 6  41.4 ± 4 . 2  38.8 ± 1.8  40.9 ± 5 . 9  0.33  0.93  57.0 ± 6 . 1  53.8 ± 10.4  47.0 ± 7.0  49.2 ± 13.2  0.94  0.19  54.3 ± 19.4  51.1 ± 11.5  50.0 ± 9.2  52.6 ± 18.3  0.96  0.99  -19.7 ± 12.1  1.6 ± 11.6  0.0± 5.2  -4.9 ± 8 . 1  0.099  0.90  -13.0 ± 18.7  2.1 ± 11.0  2.0 ± 1.0  -3.7 ± 9 . 1  0.40  0.95  69.3 ± 15.5  50.4 ± 26.3  52.7 ± 2 8 . 3  42.6 ± 17.9  0.19  0.29  26. % Weightbear Left  62.3 ± 16.7  51.5 ± 8 . 4  68.0 ± 7.2  51.3 ± 2 1 . 9  0.14  0.87  P a r a m e t e r s outside  16  13  10  8  3. R a d i a l Inclination Right 4. R a d i a l Inclination Left  21° ± 2 °  5. U l n a r Shortening Right 6. U l n a r Shortening Left 7. R a d i a l B o w Right 8. R a d i a l B o w Left  0 ± 1 mm  9. R a d i a l Head Dislocation R lO.Radial Head Dislocation L 11. E l b o w Joint Right  10° ± 5 °  9° ± 3 °  12. E l b o w Joint Left 13. Femoral Right 14. Femoral Left 15. Femoral Angle R 16. Femoral Angle L 17. Femoral Right  A.A.  7° ± 2 ° valgus  A.A. N.S.  135°±5°  N.S. M.A.  18. Femoral M . A . Left 19. Sharp's Right  0°±5° varus  35° ± 4 °  20. Sharp's Left 21. Fibular Height Right 22. Fibular Height Left 23. A n k l e Joint A n g l e Right  50 ± 10  0°±5°  24. A n k l e Joint A n g l e Left 25. % Weightbear Right  50 ± 10  of n o r m a l range  219  Table 8 . 7 . 6 . 3 . Segment Lengths and Percentile Height by Gene and Gender Variable  EXT 1 Males (n=4)  EXT1 Females (n=3)  EXT 2 Males (n=10)  EXT 2 Females (n=9)  P-value EXT  P-value Gender  Total L e g LengthRight Upper L e g - Right  83.4 ± 4.2  74.7 ± 10.8  87.6 ± 5 . 7  82.1 ± 11.5  0.082  0.18  40.8 ± 2.7  37.2 ± 6.3  44.5 ± 4.0  43.4 ± 7 . 1  0.47  0.058  L o w e r L e g - Right  34.012.7  30.2 ± 4.8  36.2 ± 2 . 9  33.7 ± 3 . 9  0.059  0.11  Total L e g Length Left Upper L e g - Left  82.3 ± 3.2  74.3 ± 12.0  87.2 ± 6.6  81.1 ± 11.2  0.080  0.18  40.4 + 2.6  36.8 ± 7 . 1  43.6 + 3.8  42.8 + 6.5  0.53  0.069  L o w e r L e g - Left  33.5 ± 4 . 1  31.0 ± 5 . 8  37.8 ± 4 . 1  34.5 ± 5 . 2  0.13  0.084 0.028  Total A r m Length - Right  46.0 ± 3 . 8  43.3 ± 7 . 1  52.2 ± 4.7  49.1 ± 6 . 6  0.22  Upper A r m Right Lower A r m Right Total A r m Length -Left Upper A r m - Left  28.3 ± 1.3  26.3 ± 3.5  32.1 ± 2 . 9  29.0 ± 4.2  0.058  0.043  20.9 ± 2 . 1  21.0 ± 3 . 3  24.7 ± 2.8  22.3 ± 3 . 6  0.21  0.069  46.0 ± 5 . 1  43.7 ± 6 . 0  52.9 ± 4 . 6  49.1 ± 6 . 9  0.17  0.026  28.6 ± 3 . 1  26.0 ± 4 . 0  32.3 ± 3 . 2  29.9 ± 5.3  0.17  0.058  19.6 ± 3 . 9 12.5 ± 17.7  20.3 ± 2.4 5.0 ± 3 . 5  25.0 ± 2 . 8 40.2 + 28.3  22.7 ± 3 . 6 45.1 + 31.4  0.30  0.011  0.79  0.011  L o w e r A r m - Left Percentile Height  220  8.7.7  Gene and Mutation Type  Table 8.7.7.1 Lesion Quality by Gene and Mutation Type Variable  EXT1  EXT 2  Missense  Missense  P-value  Power  EXT1  EXT 2  P-  Nonsense  Nonsense  value  Power  (n=2)  (n=2)  (n=2)  (n=12)  Lesion R a n k 1  8.0 ± 1.4  10.0 ± 1.4  0.29  0.14  16.5 ± 7 . 8  6.1 ± 5 . 1  0.026  0.64  % Rank 1  30.0 ± 7 . 1  66.5 ± 3 . 5  0.022  0.88  37.0 ± 7 . 1  28.9 ± 18.0  0.55  0.086  Lesion R a n k 2  7.0 ± 2 . 8  1.0 ± 0 . 0  0.096  0.39  7.0 ± 4 . 2  3.9 ± 2 . 2  0.13  0.31  % Rank 2  25.5 ± 9 . 2  6.5 ± 0 . 7 1  0.10  0.37  15.5 ± 4 . 9  22.5 ± 12.4  0.46  0.11  Lesion R a n k 3  4.0 ± 1.4  1.0 ± 0 . 0  0.096  0.39  5.0 ± 2 . 8  2.2 ± 2 . 1  0.11  0.34  % Rank 3  6.5 ± 0 . 7 1  0.17  0.24  3.0 ± 0 . 0  0.038  0.72  11.0 ± 2.8 15.0 ± 1.4  9.9 ± 8.2  Lesion R a n k 4  15.0 ± 5 . 7 8.0 ± 1.4  7.3 ± 4 . 6  0.87 0.041  0.053 0.55  % Rank 4  29.5 ± 3 . 5  20.0 ± 1.4  0.072  0.48  36.5 ± 14.8  38.6±21.5  0.89  0.052  S m a l l (%)  32.8 ± 4 . 5  64.3 ± 10.1  0.057  0.57  39.7 ± 10.4  28.0 ± 16.3  0.36  0.14  Medium  33.8± 0.71  14.2 ± 0 . 0  0.0007  1.0  22.5 ± 15.3  32.1 ± 13.9  0.39  0.13  L a r g e (%)  26.5 ± 14.8  21.4± 10.1  0.72  0.058  37.8 ± 25.7  37.2 ± 20.5  0.97  0.050  Average  27.0 ± 1.4  15.0 ± 1.4  0.013  0.97  43.5 ± 13.4  19.4 ± 9 . 3  0.007 1  0.86  7.5 ± 2 . 1  5.0 ± 1.4  0.29  0.14  11.5 ± 2.1  5.9 ± 4 . 5  0.12  0.33  27.6 ± 6 . 4  33.9 ± 12.6  0.59  0.068  26.9 ±3.5  3 1 . 0 ± 14.5  0.71  0.064  N o . Sessile  19.5 ± 0.71  10.0 ± 2 . 8  0.044  0.66  27.5 ± 17.7  12.4 ± 6 . 2  0.027  0.64  % Sessile  72.3 ± 6.4  66.1 ±  0.59  0.068  59.8 ± 22.2  64.2 ± 15.6  0.73  0.062  (%)  N u m b e r of Lesions No. Pedunculated  % Pedunculated  12.6 No. Distal  12.5 ± 2 . 1  5.5 ± 3 . 5  0.14  0.28  17.0 ± 8 . 5  8.3 ± 4 . 8  0.051  0.51  %  46.6 ± 10.3  37.9 ± 27.1  0.72  0.058  37.9 ± 7 . 8  41.6 ± 13.4  0.72  0.063  No. Proximal  10.0 ± 2 . 8  9.0 ± 4 . 2  0.81  0.054  18.0 ± 5 . 7  8.8 ± 4 . 7  0.027  0.64  % Proximal  37.4 ± 12.4  58.9 ± 22.7  0.36  0.11  41.3 ± 0.24  45.6 ± 17.3  0.74  0.061  No. Pelvic  1.5 ±2.1  0.0 ± 0 . 0  0.42  0.095  7.5 ± 0 . 7 1  0.50 ± 1.2  O.OO 01  1.0  % Pelvic  5.4 ± 7 . 6  0.0 ± 0.0  0.42  0.095  18.4 ± 7 . 3  2.2 ± 5 . 4  0.002 7  0.95  No Diaphyseal  2.5 ± 3 . 5  0.50 ±  0.51  0.078  1.5 ± 0 . 7 1  1.1 ± 1.4  0.69  0.066  Distal  0.71 % Diaphyseal  8.9 ± 12.6  3.1 ± 4 . 4  0.60  0.067  3.4 ± 0 . 5 9  7.8 ± 13.0  0.65  0.071  No. Flat Bone  2.5 ± 0 . 7 1  0.0 ± 0.0  0.038  0.72  8.0 ±0.0  0.67 ± 1.2  O.OO 01  1.0  % Flat Bone  9.2 ± 2 . 1  0.0 ± 0.0  0.026  0.85  19.3 ± 5 . 9  3.3 ± 5 . 5  0.002 7  0.95  No. Complex  2.5 ± 0 . 7 1  3.0 ± 1.4  0.69  0.059  9.5 ± 12.0  2.8 ± 2 . 6  0.060  0.47  % Complex  9.3 ± 3 . 1  20.5 ± 11.4  0.31  0.13  18.5 ± 21.9  13.6 ± 10.1  0.59  0.079  No. Simple  24.5 ± 2 . 1  12.0 ± 2 . 8  0.038  0.72  29.5 ± 7.8  16.3 ± 7 . 9  0.051  0.50  221  Table 8.7.7.1 Lesion Quality by Gene and Mutation Type (continued) Variable  E X T l  EXT 2  Missense  Missense  P-value  Power  EXT 1  EXT 2  P-  Nonsense  Nonsense  value  Power  (n=2)  (n=2)  (n=2)  (n=12)  % Simple  90.7 ±3.1  0.31  0.13  68.3 ±3.2  84.5 ± 10.5  0.057  0.48  No. Flared  3.5 ±0.71  79.5 ± 11.4 8.0 ±2.8  0.16  0.25  7.0 ± 6.4  13.0 ±3.3  0.059  0.55  32.3 ±24.5  No. Not Flared  23.5 ±2.1  52.7 ± 13.9 7.0 ± 1.4  0.001 9 0.14  0.96  % Flared  0.011  0.98  12.4 ±6.3  0.32  0.15  % Not Flared  86.9 ±3.3  0.059  0.55  67.7 ±24.5  0.14  0.29  No. Left  14.5 ± 0.71 53.7 ± 0.19 12.5 ± 0.71 46.3 ± 0.19  47.3 ± 13.9 10.5 ±2.1  0.13  0.31  25.5 ± 0.71 61.3 ± 17.3 18.0 ± 12.7 38.7 ± 17.3 27.5 ±4.9  9.6 ±6.1  0.96  69.6 ±7.6  0.097  0.38  64.5 ± 8.6  46.5 ± 7.5  4.5 ±0.71  0.0077  0.99  16.0 ±8.5  9.8 ±3.4  0.002 1 0.009 3 0.072  30.4 ±7.6  0.097  0.38  35.5 ±8.6  53.5 ±7.5  0.009 3  0.82  % Left No. Right %  Right  222  0.29  0.82 0.43  Table 8.7.7.1 Lesion Quality by Gene and Mutation Type (continued) Variable  EXTl Splice Site  P-value  5.0 ± 0 . 0  EXT 2 Splice Site (n=2) 5.0 ± 0 . 0  18.3 ± 5 . 9 5.7 ± 3 . 5  20.0 ± 7 . 1 6.5 ± 6.4  0.79 0.86  0.055 0.052  18.0 ± 0 . 0 4.0 ± 0 . 0  18.0 ± 7 . 6 5.7 ± 2 . 1  32.5 ± 0 . 7 1 2.0 ± 1.4  0.082 0.12  0.43 0.32  36.0 ± 0.0 1.0 ± 0 . 0  20.0 ± 6 . 2 13.0 ± 4 . 6  10.0 ± 1.4 12.0 ± 2 . 8  0.12 0.81  0.32 0.054  9.0 ± 0 . 0 4.0 ± 0 . 0  44.0 ± 7 . 8 17.9 ± 6 . 1 33.9 ± 0 . 9 8  37.0 ± 7 . 1 20.8 ± 9 . 8 27.8 ± 15.8  0.39 0.71 0.52  0.11 0.060 0.082  36.0 ± 0 . 0 26.4 ± 7.6 39.2 ± 12.3  L a r g e (%) Average Number of Lesions No. Pedunculat ed  47.2 ± 6 . 3 29.3 ± 8.3  50.5 ± 7 . 1 25.5 ± 10.6  0.62 0.68  0.068 0.063  34.3 ± 8.5 11.0 ± 0 . 0  7.7 ± 3 . 1  5.0 ± 1.4  0.35  0.12  8.3 ± 6 . 7  %  26.3 ± 7.5  22.7 ± 14.9  0.74  0.058  45.5 ± 0 . 0  Pedunculat ed N o . Sessile % Sessile No. Distal % Distal  18.0 61.8 11.0 37.5  20.0 ± 12.7 74.5 ± 18.9 10.5 ± 2 . 1 43.2 ± 9 . 6 9.5 ± 6.4  0.81 0.30 0.89 0.53  0.054 0.14 0.052 0.080 0.11  15.3 ± 8 . 1 54.5 ± 0.0 7.3 ± 5 . 0 18.2 ± 0 . 0  35.1 ± 10.4 2.0 ± 2 . 8  0.15  0.28 0.050  54.5 ± 0 . 0  1.7 ± 1 . 2  6.1 ± 8 . 6 2.5 ± 0 . 7 1  0.93 0.44  0.051 0.097  1.3 ± 1.5 0.0 ± 0 . 0 2.0 ± 1.0  6.9 ± 6 . 9  10.1 ± 1.4  0.59  0.072  27.3 ± 0.0  2.7 ± 1.5  2.0 ± 2 . 8  0.75  0.058  1.3 ± 1.5  8.6 ± 3 . 8  6.1 ± 8 . 6  0.66  0.064  0.0 ± 0.0  No. Complex  3.3 ± 2 . 1  2.5 ± 0 . 7 1  0.64  0.066  2.3 ± 1.5  % Complex  10.5 ± 4 . 8  10.1 ± 1.4  0.93  0.051  18.2 ± 0 . 0  (n=3) Lesion Rankl % Rank 1 Lesion Rank 2 % Rank 2 Lesion Rank 3 % Rank 3 Lesion Rank 4 % Rank 4 S m a l l (%) Medium  Power  EXT 2 FS  (n=3) 2.0 ± 0 . 0  (%)  No. Proximal % Proximal No. Pelvic % Pelvic No Diaphyseal  % Diaphyseal No. Flat Bone % Flat Bone  ±4.6 ±2.9 ±4.0 ±8.4  15.0 ± 6 . 0 50.1 ± 7 . 2 2.0 ± 1.0 6.5 ± 1.7  0.39  223  11.7 ± 5.5  Table 8.7.7.1 Lesion Quality by Gene and Mutation Type (continued) EXT 2 Splice Site  Variable  EXT 1 S p l i c e Site  (n=3)  (n=2)  No. Simple % Simple No. Flared % Flared No. Not Flared % Not Flared No. Left % Left No. Right % Right  23.0 ± 5 . 3 79.6 ± 8 . 8  2  P-value  Power  EXT FS  22.5 ± 10.6  0.95  0.050  21.3 ± 13.1  87.1 ± 5 . 4 2.0 ± 1.4  0.37 0.34  81.8 ± 0 . 0 8.0 ± 8 . 0  7.3 ± 2 . 5 23.5 ± 9 . 2  0.31 0.38  0.12 0.13 0.14 0.12  9.1 ± 0 . 0 10.0 ± 0 . 0  59.4 ± 36.2  92.7 ± 2.5  0.31  0.14  90.9 ± 0 . 0  15.3 53.3 14.3 47.7  13.0 ± 2 . 8 53.3 ± 11.1 12.5 ± 7 . 8 4 6 . 7 ± 11.1  0.45 0.99 0.77 0.91  0.094 0.050 0.056 0.051  9.7 ± 4 . 9 36.4 ± 0 . 0 14.0 ± 8 . 2 63.6 ± 0 . 0  13.7 ± 13.9 40.6 ± 36.2 15.7 ± 7 . 8  ±3.1 ±5.8 ±5.5 ± 6.9  (n=3)  224  Table 8.7.7.2. Limb Alignment by Gene and Mutation Type Variable  1. Carpal Slip Right 2. Carpal Slip Left 3. Radial Inclination Right 4. R a d i a l Inclination Left 5. U l n a r Shortening Right 6. U l n a r Shortening Left 7. R a d i a l B o w Right 8. Radial B o w Left 9. R a d i a l Head Dislocation R 10. R a d i a l Head Dislocation L 11 E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N.S. Angle Right 16. Femoral N.S. Angle Left 17. Femoral M . A . Right 18. Femoral M . A . Left 19. Sharp's Right  Normal  EXTl  EXT 2  P-  Powe  EXT 1  Values  MS  MS  value  r  N S (n=2)  EXT 2 N S (n=12)  value  5 ± 2mm  (n=2) 5.5 ± 4 . 9  0.49  0.083  5.0  1.8 ± 4 . 5  0.51  0.093  0.37  0.11  7.0 ± 1.4  3.0 ± 4 . 2  0.22  0.21  28.5 ± 12.0  (n=2) 2.5 ± 0.71 1.5 ± 0.71 24.5 ± 4.9  0.71  0.059  29.0  24.5 ± 4.0  0.32  0.15  28.0 ± 8.5  26.0 ± 2.8  0.78  0.055  28.5 ± 9.2  27.0 ± 4.7  0.71  0.064  -1.0 ± 2.8  -1.0±2.8  0.050  -8.0  -2.0 ± 5.0  0.28  0.17  -2.5 ± 4.9  -2.5 ± 3.5  0  1.5 ± 4 . 9  -0.83 ± 4 . 9  0.68  0.067  9.0 ± 4 . 2  11.0± 1.4  0.068  11.0  6.9 ± 2 . 4  0.12  0.32  9.5 ± 1.4  9.5 ± 0.71 1 dislocatio n 1 dislocatio n -13.5 ± 0.71 -9.5 ± 2 . 1  0.050  20.0 ± 15.6 0  7.0 ± 2 . 1  0.0048  0.89  1 dislocati on 2.0  0  -1.9 ± 14.5  0.80  0.056  -3.5 ± 4.9 -5.5 ± 7.8 -9.5 ± 3.5 146.0 ± 7.1  -7.2 ± 13.7  0.72  0.063  -5.7 ± 9 . 7  0.98  0.050  -3.6 ± 11.0  0.48  0.10  141.8 ± 6 . 2  0.39  0.13  5.0 ± 4 . 2 21° ± 2 °  0 ± 1 mm  10° ± 5 °  0  0  9° ± 3 °  7° ± 2 ° valgus  135°±5°  0°±5° varus  35° ± 4°  -23.0 ± 1.4 -10.5 ± 10.6  0.59  0.014  0.97  0.91  0.051  P-  Power  0  -8.5 ± 12.0 4.0 ± 2 . 8  -0.50 ± 4.9 0.50 ± 3.5  0.48  0.084  0.39  0.10  141.5 ± 9.2  142.5 ± 3.5  0.90  0.051  146.5 ± 2.1  139.0 ± 22.6  0.69  0.060  142.5 ± 0.71  137.9 ± 6 . 8  0.37  0.13  7.0 ± 1.4  2.0 ± 1.4  0.072  0.48  1.0 ± 5 . 7  -0.50 ± 5 . 9  0.75  0.060  6.0 ± 4 . 2  0.50 ± 3.5 46 (n=l)  0.29  0.14  -5.0 ± 2.8 37.5 ± 3.5  0.91 ± 6 . 0  0.21  0.22  41.1 ± 5 . 7  0.42  0.12  37.0 (n=l)  225  Table 8.7.7.2. L i m b Alignment by Gene and Mutation Type (continued) Variable  Normal  E X T l  EXT 2  P-  Values  MS  MS  value  Power  E X T l  EXT 2  P-  NS  N S (n=12)  value  Power  (n=2)  (n=2)  (n=2)  33.0 (n=l)  41.0 (n=l)  41.0± 8.5  40.8 ± 4.6  0.96  0.050  52.5 ± 0.71  55.0 ± 4.2  0.49  0.081  59.0 ± 7.1  49.9 ± 11.5  0.31  0.16  59.5 ± 10.6  35.5 ±  0.12  0.31  48.0 ±  49.5 ± 13.5  0.90  0.052  -6.5 ± 0.71  0.0  0.084  0.44  -26.0 ± 7.1  1.0 ± 10.5  0.005 5  0.89  1.5 ± 0.71  0.0  0.33  0.11  -20.5 ± 19.1  2.4 ± 11.1  0.03  0.61  52.5 ± 45.9  58.5 ± 3.5  0.87  0.052  61.5 ± 10.6  45.7 ± 2 4 . 3  0.40  0.12  26. % Weightbear Left  75.5 ± 7.8  58.5 ± 9.2  0.18  0.22  53.0 ± 5.7  47.9 ± 2 2 . 8  0.77  0.059  Parameters  111  14  15  9  20. Sharp's Left 21. Fibular Height Right  50 ± 10  22. Fibular Height Left 23. A n k l e Joint A n g l e Right  0 ° ± 5°  24. A n k l e Joint A n g l e Left 25. % Weightbear Right  50 ± 10  7.8  22.6  b e y o n d the n o r m a l range  226  Table 8.7.7.2. L i m b Alignment by Gene and Mutation Type (continued) Variable  Normal Values  1. Carpal Slip Right 2. Carpal Slip Left 3. R a d i a l Inclination Right 4. R a d i a l Inclination Left 5. U l n a r Shortening Right 6. Ulnar Shortening Left 7. R a d i a l B o w Right 8. R a d i a l B o w Left 9. R a d i a l Head Dislocation R 10. Radial Head Dislocation L 11 E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N.S. Angle Right  5 ± 2mm  21° ± 2 °  0 ± 1 mm  10° ± 5 °  9° ± 3 °  7 ° ± 2° valgus  135° ± 5 °  16. Femoral N.S. Angle Left 17. Femoral M . A . Right 18. Femoral M . A . Left  0 ° ± 5° varus  19. Sharp's Right 20. Sharp's Left  35° ± 4°  EXTl Splice Site (n=3) 5.0 ± 2 . 6  EXT 2 Splice Site (n=2) 2.0 ± 0 . 0  P-value  Power  0.23  0.19  EXT 2 FS (n=3) 3.7 ± 1.2  3.0 ± 2 . 6  4.5 ± 2 . 1  0.56  0.076  3.7 ± 2 . 1  27.3 ± 0.58  27.5 ± 0 . 7 1  0.79  0.055  20.7 ± 9 . 5  33.7±2.1  24.5 ± 4.9  0.057  0.54  25.7 ± 7 . 6  1.0 ± 1.7  3.0 ± 1.4  0.27  0.16  -4.0 ± 6.2  7.0 ± 4 . 6  2.5±2.1  0.29  0.14  -4.7 ± 5.9  8.3 ± 1.2  8.5 ± 0 . 7 1  0.87  0.052  7.3 ± 1.5  13.5 ± 5 . 7  8.0 ± 0 . 0  0.28  0.15  9.3 ± 3 . 8  1 dislocation  0  1 dislocation  0  11.0 ± 12.2  -14.0 ± 2 . 8  0.073  0.46  -9.3 ± 10.0  0.33 ± 13.2  -17.5 ± 2 . 1  0.17  0.24  -7.3 ± 4.5  2.2 ± 2 . 0  -5.5 ± 0 . 7 1  0.016  0.89  -8.7 13.5  0.0 ± 9 . 9  -4.5 ± 0 . 7 1  0.59  0.82  -4.7 ± 5.7  142.3 ± 29.4  134.5 ± 17.7  0.76  0.057  135.7 ± 11.0  149.0 ± 18.2  137.0 ± 16.9  0.51  0.083  132.3 ± 6 . 8  10.8 ± 3 . 9  5.5 ± 3 . 5  0.29  0.14  -4.0 ± 8.0  -4.0 ± 8 . 5  0.0 ± 0 . 0  0.57  0.070  3.0 ± 3 . 6  40.3 ± 3 . 9  39.0 ± 4 . 2  0.79  0.054  42.7 ± 8 . 0  38.8 ± 1.8  38.5 ± 7 . 8  0.97  0.050  44.3 ± 5 . 5  227  Table 8.7.7.2. Limb Alignment by Gene and Mutation Type (continued) Variable  21. Fibular Height Right 22. Fibular Height Left 23. A n k l e Joint A n g l e Right 24. A n k l e Joint A n g l e Left 25. % Weightbear Right 26. % Weightbear Left Parameters b e y o n d the normal range  Values  EXTl Splice Site  50 ± 10  Normal  0°±5°  50 ± 10  P-value  Power  44.5 ± 7.8  EXT 2 Splice Site (n=2) 42.5 ± 20.5  0.91  0.051  62.2 ± 1.0  49.0 ± 12.7  56.0 ± 9 . 9  0.60  0.067  68.3 ± 7 . 6  3.0 ± 0 . 0  -1.5 ± 2 . 1  0.096  0.39  -13.0 ± 6 . 1  2.5 ± 0 . 7 1  -4.5 ± 0 . 7 1  0.010  0.99  -11.3 ± 5 . 9  69.0 ± 1.4  54.5 ± 4 . 9  0.058  0.56  36.3 ± 29.0  67.0 ± 9 . 9  50.5 ± 0 . 7 1  0.14  0.28  60.0 ± 20.2  14  9  (n=3)  EXT 2 FS  (n=3)  12  228  Table 8.7.7.3. Segment Lengths and Percentile Height by Gene and Mutation Type Variable  Total Leg LengthRight Upper Leg Right Lower Leg Right Total Leg Length Left Upper Leg Left Lower Leg Left Total Arm Length Right Upper Arm -Right Lower Arm -Right Total Arm Length Left Upper Arm -Left Lower Arm -Left Percentile Height  E X T l  EXT 2  P-  M S (n=2)  M S (n=2)  value  73.5 ± 14.1  75.0 ± 19.1  0.94  36.3 ±8.8  38.3 ± 10.3  30.0 ±6.4  Power  EXT 1  EXT 2  P-  N S (n=2)  N S (n=12)  value  0.050  80.5 ±2.1  86.7 ± 8.4  0.34  0.15  0.85  0.052  38.5 ±0.71  45.7 ±5.0  0.075  0.42  33.0 ±9.2  0.74  0.057  32.0 ± 1.4  35.5 ±3.2  0.17  0.25  72.3 ± 14.5  74.8 ± 18.7  0.90  0.051  80.0 ± 1.4  85.9 ±9.1  0.39  0.13  36.0 ±9.9  38.5 ±8.5  0.81  0.053  38.3 ± 1.1  44.6 ±5.0  0.11  0.35  29.0 ± 4.9  32.0 ±8.5  0.71  0.059  31.0± 1.4  37.2 ±4.9  0.12  0.33  44.0 ± 9.9  46.5 ± 11.3  0.84  0.053  43.0 ±0.0  51.6 ±5.4  0.051  0.51  26.5 ± 4.9  26.8 ± 6.0  0.97  0.050  28.0 ±0.0  31.1 ±3.8  0.29  0.17  20.3 ± 3.9  21.8 ±6.0  0.79  0.054  19.5 ±2.1  23.9±3.1  0.084  0.40  45.3 ± 10.3  45.3 ± 12.4  0.050  42.0 ± 1.4  52.0 ±5.3  0.025  0.66  27.5 ±7.8  25.3 ± 6.0  0.78  0.055  26.5 ±0.71  31.8 ± 4.3  0.12  0.32  22.0 ±3.5  22.8 ±7.4  0.91  0.051  17.5 ±3.5  24.5 ±2.7  0.0065  0.87  4.0 ± 1.4  39.0 ±33.9  0.28  0.14  3.0 ±0.0  54.3 ± 27.7  0.026  0.64  229  Power  Table 8.7.7.3. Segment Lengths and Percentile Height by Gene and Mutation Type (continued) EXT 1 EXT 2 Power EXT 2 P-value Variable Total L e g Length-Right Upper L e g - Right L o w e r L e g - Right Total L e g Length - Left Upper L e g - Left L o w e r L e g - Left Total A r m Length Right Upper A r m - Right L o w e r A r m - Right Total A r m Length Left Upper A r m - Left L o w e r A r m - Left Percentile Height  S p l i c e Site  S p l i c e Site  (n=3)  (n=2)  83.2 ± 6 . 9  83.5 ± 8 . 5  0.96  0.050  85.7 ± 4 . 3  41.7 ± 2 . 0  42.3 ± 6.7  0.89  0.051  42.3 ± 1.9  34.2 ± 3 . 8  34.5 ± 3 . 5  0.93  0.051  34.7 ± 2 . 0  82.5 ± 6.5  83.3 ± 8 . 1  0.92  0.051  84.8 ± 2.9  41.2 ± 2 . 4  42.8 ± 6.0  0.69  0.062  41.0 ± 0 . 9  FS (n=3)  35.7 ± 4 . 6  34.0 ± 4.2  0.71  0.060  36.7 ± 1.2  46.7 ± 4.5  49.8 ± 8 . 1  0.61  0.069  50.7 ± 4 . 1  27.7 ± 2 . 1  31.8±4.6  0.25  0.17  30.5 ± 2 . 2  22.3 ± 1.4  23.5 ± 3 . 5  0.63  0.068  23.5 ± 4 . 4  46.8 ± 3 . 5  51.3 ± 6 . 0  0.36  0.12  51.0±5.8  28.2 ± 2 . 0  32.8 ± 3 . 9  0.17  0.24  31.5 ± 1.8  20.2 ± 2.5  23.0 ± 2 . 8  0.32  0.14  23.0 ± 4 . 6  17.0 ± 19.3  25.0 ± 0 . 0  0.62  0.069  9.7 ± 7.6  230  8.7.8  Gene and Severity  Table 8.7.8.1 Lesion Quality by Gene and Severity EXTl  EXT 2  Severe (n=5) 9.6 ± 7 . 4 25.8 ± 11.6  Severe (n=17) 5.7 ± 4 . 4 26.5 ± 15.7  6.2 ± 3 . 3 17.0 ± 6 . 0 5.4 ± 2 . 1 16.4 ± 6 . 8 13.8 ± 3 . 5  4.7 ± 2 . 9 25.2 ± 12.1  EXTl  EXT 2  Mild (n=2) 8.0 ± 1.4 30.0 ± 7 . 1  Mild (n=2) 10.0 ± 1.4 66.5 ± 3.5  0.15 0.27 0.88 0.41 0.57 0.06 0 0.05 0  7.0 ± 2 . 8 25.5 ± 9.2 4.0 ± 1.4 15.0 ± 5 . 7 8.0 ± 1.4  26.5 ± 14.8  0.012  0.08 0 0.08 5 0.75  6.2 ± 4 . 5  0.19  26.6 ± 5 . 6  30.8 ± 14.3  0.53  13.8 ± 7 . 2  0.065  65.5 ± 15.3  0.55  No. Distal  21.8 ± 10.8 61.0± 11.3 13.4 ± 6 . 1  8.4 ± 4 . 5  0.057  %  Distal  37.6 ± 7 . 1  39.8 ± 13.7  0.75  No. Proximal % Proximal  16.2 ± 5 . 4  9.4 ± 4 . 7 45.2 ± 15.3  0.013 0.85 0.0024 0.017 0.77  Variable  Lesion R a n k 1 % Rank 1 Lesion R a n k 2 % Rank 2 Lesion R a n k 3 % Rank 3 Lesion R a n k 4 % Rank 4 S m a l l (%) M e d i u m (%) L a r g e (%) Average Number of Lesions No. Pedunculated  % Pedunculated N o . Sessile % Sessile  Pvalue  Pow er  0.16 0.93  0.28 0.05 1  2.2 ± 1.9 9.8 ± 7 . 0 8.2 ± 5 . 1 38.3 ± 18.6  0.34 0.16 0.0043 0.079 0.035 0.76  26.9 ± 14.2  0.97  32.8 ± 13.4  0.59  38.3 ± 17.9  0.57  20.9 ± 9.6  9.2 ± 3 . 2  41.0± 10.1 26.6 ± 13.7 29.3 ± 9.9 43.4 ± 14.5 35.0 ± 11.8  46.6 ± 6 . 9  No. Pelvic % Pelvic No Diaphyseal  4.2 ±3.1 11.3 ± 7.5 1.6 ± 0 . 8 9  0.82 ±1.5 3.2 ± 5 . 7 1.4 ± 1.3  % Diaphyseal  5.5 ± 5 . 3  8.7 ± 11.9  0.58  No. Flat Bone % Flat Bone No. Complex % Complex  4.8 ±3.1 12.9 ± 7 . 1 5.8 ± 7 . 1 13.7 ± 12.3 25.6 ± 6 . 5 75.1 ± 8 . 9  0.94 ±1.4 3.9 ± 5 . 7 2.6 ± 2 . 2 13.0 ± 8 . 9  0.0007 0.0081 0.11 0.89  17.9 ± 8 . 9 85.4 ± 9.4  0.090 0.042  No. Simple % Simple  Pvalue  Power  0.29 0.022  0.14 0.88  0.096 0.10 0.096 0.17 0.038 0.072  0.39 0.37 0.39 0.24 0.72  29.5 ± 3 . 5  1.0 ± 0 . 0 6.5 ± 0 . 7 1 1.0 ± 0 . 0 6.5 ± 0 . 7 1 3.0 ± 0 . 0 20.0 ± 1.4  32.8 ± 4 . 5  64.3 ± 10.1  0.057  0.57  33.8  14.0  0.0007  1.0  21.4 ± 10.1  0.72  0.058  27.0 ± 1.4  15.0 ± 1.4  0.014  0.97  0.24  7.5 ± 2 . 1  5.0 ± 1.4  0.29  0.14  0.09 2 0.45  27.6 ± 6.4  33.9 ± 12.6  0.59  0.068  19.5 ± 0 . 7 1  10.0 ± 2 . 8  0.044  0.66  0.08 7 0.47 0.06 1 0.75  72.4 ± 6.4  66.1 ± 12.6  0.59  0.068  ±0.71  ±0.0  0.48  12.5 ± 2 . 1  5.5 ± 3 . 5  0.14  0.28  46.6 ± 10.3  37.9 ± 2 7 . 1  0.72  0.058  10.0 ± 2 . 8 37.4 ± 12.4  9.0 ± 4 . 2  0.81 0.36  0.054  58.9 ± 2 2 . 7  0.93 0.70 0.05 9  1.5 ± 2.1 5.4 ± 7 . 6 2.5 ± 3 . 5  0.0 ± 0 . 0 0.0 ± 0 . 0  0.42 0.42  0.50 ± 0 . 7 1  0.51  0.095 0.095 0.078  0.08 3 0.98 0.81 0.34 0.05 2 0.38 0.54  8.9 ± 12.6  3.1 ± 4 . 4  0.60  0.067  2.5 ± 0 . 7 1 9.2 ± 2 . 1  0.0 ± 0 . 0 0.0 ± 0 . 0 3.0 ± 1.4 20.5 ± 11.4  0.038  0.72  0.026 0.69 0.31  0.85 0.059 0.13  12.0 ± 2 . 8 79.5 ± 11.4  0.038  0.72  0.31  0.13  0.05 4  231  2.5 ± 0 . 7 1 9.3 ± 3 . 1 24.5 ± 2 . 1 90.7 ± 3 . 1  0.11  Table 8.7.8.1 Lesion Quality by Gene and Severity (continued) Variable  No. Flared  EXT 1  EXT 2  P-  Pow  E X T l  EXT 2  P-  Severe  Severe  value  er  Mild  Mild  value  (n=5)  (n=17)  (n=2)  (n=2)  18.4 ±  6.6 ± 6.3  0.0068  0.83  3.5 ± 0 . 7 1  8.0 ± 2 . 8  0.16  0.25  28.5 ± 2 2 . 9  0.11  0.33  13.0 ± 3.3  52.7 ± 13.9  0.059  0.55  14.1 ± 6 . 9  0.51  0.09  23.5 ± 2 . 1  7.0 ± 1.4  0.011  0.98  86.9 ± 3 . 3  47.3 ± 13.9  0.059  0.55  Power  11.8 % Flared  48.9 ± 29.3  No. Not F l a r e d  16.6 ± 8 . 5  6 % Not Flared  51.1 ±  70.8 ± 2 3 . 0  0.13  0.31  29.3 No. Left  20.2 ±7.4  10.0 ± 5 . 5  0.0031  0.91  14.5 ± 0 . 7 1  10.5 ± 2 . 1  0.13  0.31  % Left  57.8 ± 8 . 5  46.3 ± 8 . 1  0.012  0.75  53.7 ± 0 . 1 9  69.6 ± 7 . 6  0.097  0.38  No. Right  15.0 ± 5 . 8  10.9 ± 4 . 8  0.12  0.32  12.5 ± 0 . 7 1  4.5 ± 0 . 7 1  0.0077  0.99  % Right  42.8 ± 9.3  53.7 ± 8.1  0.019  0.68  46.3 ± 0 . 1 9  30.4 ± 7 . 6  0.097  0.38  232  Table 8.7.S1.2. Limb A ignmenlt by Gene and Severity Variable  1. Carpal SlipR 2. Carpal SlipL 3. Radial Inclination R 4. Radial Inclination L 5. U l n a r Shortening R 6. U l n a r Shortening L 7. Radial BowR 8. Radial B o w Left 9. Radial Head Dislocation R 10. Radial Head Dislocation L 11. E l b o w Joint R 12. E l b o w Joint L 13. Femoral A.A. R  Normal  EXT 1  EXT 2  P-  Values  Severe  Severe  value  5 ± 2mm  21° ± 2 °  0 ± 1 mm  10° ± 5 °  9 ° ± 3°  7° ± 2 ° valgus  14. Femoral A.A. L 15. Femoral N.S. Angle R  135° ± 5 °  16. Femoral N.S. Angle L 17. Femoral M.A. R 18. Femoral M.A. L 19. Sharp's Right 20. Sharp's Left  0° ± 5° varus  35° ± 4°  Power  E X T l  EXT 2  P-  Mild  Mild  value  (n=2) 2.5 ± 0.71 1.5 ± 0.71 24.5 ± 4.9 26.0 ± 2.8 -1.0 ± 2.8  Power  (n=5)  (n=17)  5.0 ± 2.2 4.6 ± 2.9 27.8 ± 0.96 31.6 ± 5.6 -1.3 ± 4.7  2.2 ± 3 . 8  0.17  0.26  (n=2) 5.5 ± 4 . 9  3.3 ± 3 . 7  0.48  0.10  5.0 ± 4 . 2  24.2 ± 5 . 2  0.19  0.23  26.5 ± 4.9  0.063  0.45  28.5 ± 12.0 28.0 ± 8 . 5  -1.8 ± 5.1  0.85  0.054  -1.0±2.8  4.8 ± 5.1  -0.59 ± 5 . 1  0.050  0.50  -2.5 ± 4 . 9  -2.5 ± 3.5  9.0 ± 1.6 16.1 ± 9.5 1  7.1 ± 2 . 1  0.12  0.33  9.0 ± 4 . 2  7.5 ± 2 . 4  0.0020  0.94  9.5 ± 1.4  0  0  11.0 ± 1.4 9.5 ± 0.71 1  2  0  0  1  8.8 ± 10.9 -1.2± 9.9 -0.90 ± 5.9 -3.8 ± 8.9  -4.6 ± 13.3  0.079  0.41  0.97  0.24  0.20  0.91  0.051  -6.2 ± 9.4  0.25  0.19  0.48  0.084  -3.9 ± 9.4  0.99  0.050  -13.5 ± 0.71 -9.5 ± 2.1 -0.50 ± 4.9 0.50 ± 3.5  0.014  -8.4 ± 11.9  -23.0 ± 1.4 -10.5 ± 10.6 -8.5 ± 12.0 4.0 ± 2 . 8  0.39  0.10  143.8 ± 21.2  139.8 ± 8 . 4  0.53  0.092  141.5 ± 9.2  142.5 ±3.5  0.89  0.051  146.4 ± 13.4  136.8 ± 7 . 8  0.053  0.49  146.5 ± 2.1  139.0 ±22.6  0.69  0.060  5.9 ± 6.9 -4.5 ± 5.2 38.9 ± 3.4  -0.46 ± 6.4  0.099  0.36  7.0 ± 1.4  0.072  0.48  1.2 ± 5 . 2  0.066  0.45  6.0 ± 4 . 2  0.29  0.14  41.1 ± 5 . 7  0.47  0.11  37.0 (n =  41.1 ± 5 . 0  0.65  0.071  1) 33.0 ( n = l )  2.0 ± 1.4 0.50 ± 3.5 46.0 (n=l) 41 (n=l)  39.9 ±5.2  233  0.49  0.083  0.37  0.11  0.71  0.059  0.78  0.055 0.050  0.050  0.59  0.068 0.050  Table 8.7.8 .2. Limb A ignmenlt by Gene and Severity (continued) Variable  21. Fibular Height R 22. Fibular Height L 23. A n k l e Joint A n g l e R 24. A n k l e Joint A n g l e L 25. % Weightbear R 26. % Weightbear L N u m b e r of  Normal  E X T l  EXT 2  P-  Values  Severe  Severe  value  (n=5)  (n=17)  51.8 ± 10.3 48.5 ± 15.0  51.2 ± 12.3  0.94  0.051  53.8 ± 13.9  0.51  0.095  -11.5 ±  -1.9 ± 10.4  0.17  0.26  50 ± 1 0  0°±5°  Power  17.2  50 ± 10  EXT 1  EXT 2  P-  Mild  Mild  value  (n=2)  (n=2)  52.5 ± 0.71 59.5 ± 10.6 -6.5 ± 0.71  55.0 ± 4.2  0.49  0.081  35.5 ± 7.8 0.0  0.12  0.31  Power  (n=l)  -9.0 ± 17.3  -1.0 ± 10.9  0.26  0.19  1.5 ± 0 . 7 1  0.0 (n=l)  65.3 ± 7.5  45.0 ± 2 3 . 2  0.11  0.35  52.5 ± 45.9  58.5 ± 3.5  0.87  0.052  60.0 ± 10.4  50.5 ± 20.6  0.39  0.13  75.5 ± 7 . 8  58.5 ± 9.2  0.18  0.22  15  14  11  7  parameters that fall b e y o n d the normal range  234  Table 8.7.8.3. Segment Lengths and Percentile Height by Gene and Severity Variable  Total L e g Length-Right Upper L e g Right Lower Leg Right Total L e g Length - Left Upper L e g Left Lower Leg Left Total A r m Length - Right Upper A r m Right Lower A r m Right Total A r m Length - Left Upper A r m Left Lower A r m Left Percentile Height  EXTl Severe (n=5) 82.1 ± 5 . 2  EXT 2 Severe (n=17)  Pvalue  Power  86.1 ± 7 . 5  0.28  0.18  40.4 ± 2.3  44.7± 4.8  0.071  0.43  36.3 ± 8 . 8  33.3 ± 3 . 0  35.2 ± 2 . 9  0.22  0.21  81.5 ± 4 . 8  85.4 ± 7.9  0.31  0.16  40.0 ± 2 . 4  43.8 ± 4 . 7  0.10  33.8 ± 4 . 2  36.7 ± 4 . 4  45.2 ± 3 . 8  Pvalue  Power  0.94  0.050  0.85  0.052  30.0 ± 6 . 4  EXT 2 Mild (n=2) 75.0 ± 19.1 38.3 ± 10.3 33.0 ± 9 . 2  0.74  0.057  74.8 ± 18.7 38.5 ± 8 . 5  0.89  0.051  0.36  72.3 ± 14.5 36.0 ± 9 . 9  0.81  0.053  0.21  0.23  29.0 ±4.9  32.0 ± 8 . 5  0.71  0.059  51.2 ± 5 . 2  0.026  0.63  44.0 ± 9.9  0.84  0.053  27.8 ± 1.5  31.1 ± 3.5  0.057  0.48  26.5 ± 4 . 9  46.5 ± 11.3 26.8 ± 6 . 0  0.97  0.050  21.2±2.1  23.8 ± 3 . 1  0.11  0.35  20.3 ± 3.9  21.8 ± 6 . 0  0.79  0.054  44.9 ± 3 . 7  51.7±5.1  0.012  0.76  27.5 ± 1.7  31.9 ± 3.8  0.023  0.65  45.3 ± 10.3 27.5 ± 7 . 8  45.3 ± 12.4 25.3 ± 6 . 0  0.78  0.055  19.1 ± 2 . 9  24.1 ± 2 . 9  0.0034  0.90  22.0 ± 3 . 5  22.8 ± 7.4  0.91  0.051  11.4 ± 15.6  42.9 ± 29.6  0.035  0.57  4.0 ± 1.4  39.0 ± 33.9  0.28  0.14  235  EXT 1 Mild (n=2) 73.5 ± 14.1  0.050  8.7.9  Gender and Severity  Table 8.7.9.1 Lesion Quality by Gender and Severity Variable  Males  Females  P-  Severe  Severe  value  Power  Males  Mild  (n=2)  Females  P-value  Power  Mild  (n=12)  (n=10)  8.7 ± 6 . 2  4.2 ± 2.3  0.044  0.52  10.0 ± 1.4  8.0 ± 1.4  0.29  0.14  % Rank 1  27.8 ± 16.6  24.6 ± 12.4  0.63  0.075  52.0 ± 24.0  44.5 ± 24.6  0.79  0.054  Lesion R a n k  5.8 ± 3 . 3  4.2 ± 2 . 6  0.24  0.19  3.0 ± 2 . 8  5.0 ± 5 . 7  0.69  0.059  19.7 ± 9 . 2 3.9 ± 2 . 4  27.8 ± 12.7 1.7 ± 1.7  0.099 0.025  0.36 0.64  12.5 ± 9.2 3.0 ± 2 . 8  19.5 ± 17.7 2.0 ± 1.4  0.67 0.69  0.061 0.059  % Rank 3  12.5 ± 6 . 7  9.9 ± 8 . 2  0.44  0.11  12.5 ± 9.2  9.0 ± 2 . 8  0.66  0.062  Lesion R a n k  11.6 ± 5.1  7.0 ± 4 . 6  0.040  0.54  5.0 ± 2 . 8  6.0 ± 4 . 2  0.81  0.054  % Rank 4  40.0 ± 16.2  37.6 ± 18.4  0.74  0.062  23.0 ± 5 . 7  26.5 ± 7 . 8  0.66  0.062  S m a l l (%)  28.6 ± 15.0  24.6 ± 12.5  0.51  0.096  53.7 ± 2 5 . 0  43.4 ± 19.4  0.69  0.060  Medium  Lesion R a n k  (n=2)  1  2 % Rank 2 Lesion R a n k  3  4  29.6 ± 8 . 9  35.0 ± 15.9  0.32  0.15  24.3 ± 14.2  23.8 ± 13.5  0.97  0.050  L a r g e (%)  38.4 ± 16.5  40.7 ± 18.5  0.76  0.060  15.1 ± 1.3  32.8 ± 6.0  0.056  0.58  Average  29.9 ± 11.6  17.1 ± 6 . 9  0.0061  0.84  21.0 ± 7.1  21.0 ± 9 . 9  8.3 ± 5 . 3  5.2 ± 2 . 2  0.097  0.34  5.0 ± 1.4  7.5 ± 2 . 1  0.29  0.14  30.0 ± 14.4  33.2 ± 12.6  0.64  0.073  24.0 ± 1.4  37.5 ± 7 . 6  0.13  0.29  (%)  N u m b e r of  0.050  Lesions No. Pedunculated  % Pedunculated N o . Sessile  19.6 ± 8 . 9  10.9 ± 5 . 2  0.014  0.74  16.0 ± 5 . 7  13.5 ± 7 . 8  0.75  0.056  % Sessile  64.8 ± 15.2  6 0 . 2 ± 11.1  0.49  0.099  75.9 ± 1.4  62.5 ± 7.6  0.13  0.29  No. Distal  11.7 ± 5 . 2  7.0 ± 4 . 1  0.032  0.59  8.5 ± 2 . 1  9.5 ± 1.5  0.88  0.051  %  39.7 ± 14.2  38.8 ± 10.6  0.88  0.053  36.3 ± 24.8  48.2 ± 12.6  0.61  0.067  Distal  No. Proximal  13.6 ± 5 . 5  7.8 ± 3 . 9  0.011  0.76  12.0 ± 0 . 0  7.0 ± 1.4  0.038  0.72  % Proximal  46.5 ± 8.5  43.9 ± 2 0 . 3  0.73  0.062  60.6 ± 20.4  3 5 . 7 ± 10.1  0.26  0.16  No. Pelvic  2.3 ± 2 . 9  0 . 8 0 ± 1.1  0.16  0.28  0.0 ± 0.0  1.5 ± 2 . 1  0.42  0.095  % Pelvic  5.9 ± 7 . 7  4.0 ± 6 . 1  0.55  0.087  0.0 ± 0.0  5.4 ± 7 . 6  0.42  0.095  No  1.2 ± 1.1  1.8 ± 1.3  0.24  0.20  0.50 ± 0 . 7 1  2.5 ± 3 . 5  0.51  0.078  Diaphyseal % Diaphyseal  5.6 ± 8 . 6  12.8 ± 14.0  0.22  0.22  3.1 ± 4 . 4  8.9 ± 12.6  0.60  0.067  No. Flat Bone  2.4 ± 3 . 0  1.1 ± 1.4  0.22  0.21  1.0 ± 1.4  1.5 ± 2.1  0.81  0.054  % Flat Bone  6.4 ± 7.6  5.4 ± 6 . 5  0.75  0.061  3.8 ± 5 . 4  5.4 ± 7 . 6  0.84  0.052  No. Complex  3.8 ± 2 . 5  2.0 ± 1.3  0.091  0.38  2.5 ± 0 . 7 1  3.0 ± 1.4  0.69  0.059  % Complex  13.7 ± 8 . 4  11.3 ± 5 . 8  0.51  0.095  12.0 ± 0 . 6 8  17.9 ± 15.2  0.64  0.064  No. Simple  23.9 ± 9 . 0  14.6 ± 5 . 7  0.010  0.78  18.5 ± 6 . 4  1 8 . 0 ± 11.3  0.96  0.050  % Simple  84.4 ±10.1  83.6 ± 6 . 4  0.84  0.054  87.9 ± 0 . 6 8  82.1 ± 15.2  0.64  0.064  No. Flared  14.3 ± 9 . 3  3.2 ± 3 . 2  0.0018  0.94  7.0 ± 4 . 2  4.5 ± 2 . 1  0.53  0.075  % Flared  46.1 ± 24.2  17.4 ± 16.8  0.0049  0.87  38.9 ± 3 3 . 3  26.8 ± 2 2 . 7  0.71  0.058  No. Not  15.6 ± 8 . 7  13.6 ± 4 . 9  0.53  0.091  14.0 ± 11.3  16.5 ± 12.0  0.85  0.052  Flared  236  Table 8.7.9.1 Lesion Quality by Gender and Severity (continued) Variable  % Not  Males  Females  P-  Severe  Severe  value  (n=12)  (n=10)  53.9 ± 24.2  81.3 ± 17.9  0.0075  0.82  Power  Males  Mild  (n=2)  Females  P-  Mild  value  Power  (n=2) 61.1 ± 3 3 . 3  73.2 ± 2 2 . 7  0.71  0.058  Flared No. Left  15.6 ± 7 . 8  8.4 ± 4.2  0.017  0.70  13.0 ± 1.4  12.0 ± 4 . 2  0.78  0.055  % Left  47.6 ± 8.3  48.9 ± 9 . 1  0.75  0.061  64.4 ± 14.9  58.9 ± 7 . 6  0.69  0.060  No. Right  14.3 ± 5 . 3  8.8 ± 3 . 4  0.0095  0.80  8.0 ± 5 . 7  9.0 ± 5 . 7  0.88  0.051  % Right  52.4 ± 8 . 3  51.4 ± 9.1  0.82  0.055  35.6 ± 14.9  41.1 ± 7 . 6  0.69  0.060  237  Table 8.7.9.2 Limb Alignment by Gender and Severity Variable  Males  Females  P-  Mild  Mild  value  (n=2) 6.0 ± 4.2  (n=2) 2.0 ± 0 . 0  0.31  0.13  0.050  5.0 ± 4 . 2  1.5 ± 0 . 7 1  0.37  0.11  0.72  0.063  32.5 ± 6.4  20.5 ± 0 . 7 1  0.12  0.33  26.7 ± 4 . 9  0.48  .10  31.0 ± 4 . 2  23.0 ± 1.4  0.13  0.31  -2.5 ± 5 . 1  -0.90 ± 4.9  0.49  0.098  -3.0 ± 0 . 0  1.0 ± 0 . 0  0.30 ± 5 . 7  0.92 ± 5.5  0.79  0.057  -5.5 ± 0.71  0.50 ± 0 . 7 1  0.014  0.97  7.4 ± 2 . 3  7.7 ± 2 . 1  0.77  0.059  12.0 ± 0 . 0  8.0 ± 2 . 8  0.18  0.22  8. R a d i a l B o w Left  10.3 ± 6 . 9  8.6 ± 4 . 5  0.52  0.094  8.8 ± 0 . 3 5  10.3 ± 0 . 3 5  0.051  0.61  9. R a d i a l Head Dislocation R  1 dislocation  0  0  1 dislocation  10. R a d i a l Head Dislocation L  1 dislocation  1 dislocation  0  1 dislocation  -1.4 ± 15.1  -2.9 ± 12.9  0.81  0.056  -17.5 ± 6.4  -19.0 ± 7 . 1  0.84  0.052  -8.4 ± 12.6  -4.9 ± 10.9  0.49  0.099  -5.5 ± 3 . 5  -14.5 ± 4 . 9  0.17  0.23  1.6 ± 7.1  -0.11 ± 6 . 6  0.58  0.082  5.5 ± 3 . 5  3.5 ± 3 . 5  0.63  0.065  -5.5 ± 8 . 5  -1.9 ± 9 . 7  0.38  0.13  2.5 ± 0 . 7 1  2.0 ± 5 . 7  0.91  0.051  1 4 1 . 0 ± 8.8  140.4 ± 15.4  0.91  0.051  140.0 ± 7.1  144.0 ± 5.7  0.59  0.068  138.4 ± 6 . 9  139.7 ± 12.9  0.77  0.059  135.0 ±  150.0 ± 7 . 1  0.39  0.10  1. Carpal SlipR  Normal  Males  Females  P-  Values  Severe  Severe  value  5 ± 2mm  (n=12) 2.9 ± 4 . 4  2.6 ± 3 . 0  0.84  0.054  3.6 ± 3 . 6  3.6 ± 3 . 6  0.99  25.3 ± 3 . 4  24.5 ± 6.2  28.4 ± 5 . 9  2. Carpal SlipL 3. R a d i a l Inclination R 4. R a d i a l Inclination L 5. U l n a r Shortening R  21° ± 2 °  0 ± 1 mm  6. U l n a r Shortening L 7. R a d i a l BowR  11. E l b o w Joint R  10° ± 5 °  9° ± 3 °  12. E l b o w Joint L 13. Femoral A.A. R  7° ± 2 °  135° ± 5 °  16. Femoral N.S. Angle L 17. Femoral M.A. R  17.7 0°±5° varus  18. Femoral M.A. L 19. Sharp's  (n=10)  35° ± 4°  1.6 ± 7.1  -0.11 ± 6 . 6  0.58  0.082  5.5 ± 3 . 5  3.5 ± 3 . 5  0.63  0.065  -1.1 ± 5 . 8  1.4 ± 5 . 2  0.32  0.15  3.5 ± 7 . 8  3.5 ± 0 . 0  0.94  0.050  39.3 ± 3 . 7  42.3 ± 6.6  0.21  0.22  Data not  Data not  Right 20. Sharp's Left 2 1 . Fibular Height R 22. Fibular Height L  Power  valgus  14. Femoral A.A. L 15. Femoral N.S. Angle R  Power  50 ± 10  available  available  41.3 ± 4 . 8  40.5 ± 5.3  0.69  0.066  Data not available  Data not available  54.4 ± 10.3  48.0 ± 12.8  0.22  0.21  55.5 ± 3 . 5  52.0 ± 0 . 0  0.29  0.14  52.4 ± 11.2  53.1 ± 17.3  0.92  0.051  48.5 ± 26.2  46.5 ± 7 . 8  0.93  0.050  238  Table 8.7.9.2 Limb Alignment by Gender and Severity (continued) Variable  23. A n k l e  Normal  Males  Females  P-  Values  Severe  Severe  value  (n=12)  (n=10)  -3.9 ±  -3.8 ± 8 . 3  0.99  0.050  -2.9 ± 8.9  0.94  46.1 ± 19.7  0.56  0 ° ± 5°  Joint A n g l e R  15.7  24. A n k l e  -2.4 ±  Joint A n g l e L  15.5  25. %  50 ± 10  52.1 ±  Weightbear R  25.5  26. %  51.8 ±  Weightbear L  17.5  N u m b e r of  10  Power  Males  Females  P-  Mild  Mild  value  Power  (n=2)  (n=2)  -7.0 ± 4.3  -7.0 (n=l)  0.58  0.065  0.051  0.50 ± 0 . 7 1  2.0 ( n = l )  0.33  0.11  0.085  70.5 ± 20.5  40.5 ±  0.35  0.11  0.95  0.050  28.9 53.1 ± 2 1 . 7  0.88  9  parameters that fall b e y o n d the normal range  239  0.052  66.5 ± 20.5  67.5 ± 3 . 5  11  12  Table 8.7.9.3. Variable  Total L e g Length-Right  Males  Females  Severe  Severe  (n=12) 86.5 ± 5 . 9  (n=10) 83.8 ± 8 . 6  P-value  0.39  Power  0.13  Males  Females  P-  Mild  Mild  value  Power  (n=2) 86.0 ±  (n=2) 62.5 ± 1.4  0.013  0.98  3.5 Upper L e g - Right  43.3 ± 4 . 3  44.2 ± 5 . 3  0.69  0.066  44.0 ± 2.1  30.5 ± 0 . 7 1  0.013  0.97  L o w e r L e g - Right  35.3 ± 2 . 9  34.2 ± 3 . 1  0.39  0.13  37.0 ±  26.0 ± 0 . 7 1  0.049  0.62  3.5 Total L e g Length - Left  85.9 ± 6 . 6  82.9 ± 8 . 5  0.37  0.13  85.3 ±  61.8 ± 0 . 3 5  0.014  0.97  3.9 43.8 ± 1.1  30.8 ± 2 . 5  0.021  0.90  Upper L e g - Left  42.5 ± 4.0  43.5 ± 5.2  0.62  0.076  L o w e r L e g - Left  36.8 ± 4 . 7  35.2 ± 4.2  0.43  0.12  35.3 ± 3.9  25.8 ± 0 . 3 5  0.075  0.47  Total A r m Length Right  50.0 ± 5 . 5  49.6 ± 5.7  0.86  0.054  52.8 ± 2.5  37.8 ± 1.1  0.016  0.95  Upper A r m - Right  31.0±3.3  29.5 ± 3.5  0.28  0.17  30.5 ± 0.71  22.8 ± 0.35  0.0052  1.0  L o w e r A r m - Right  23.4 ± 3 . 3  22.9 ± 2.9  0.71  0.065  24.5 ± 2.1  17.5 ± 0 . 0  0.043  0.67  Total A r m Length Left  50.5 ± 5 . 9  49.8 ± 5 . 4  0.78  0.058  53.3 ± 1.1  37.3 ± 1.1  0.0044  1.0  Upper A r m - Left  31.3 ± 3 . 8  30.4 ± 4 . 2  0.62  0.076  31.3 ± 2.5  21.5 ± 0 . 7 1  0.033  0.77  L o w e r A r m - Left  23.0 ± 3 . 9  22.9 ± 3 . 2  0.92  0.051  26.3 ± 2.5  18.5 ± 1.4  0.062  0.54  Percentile Height  36.0 ± 2 8 . 8  35.5 ± 3 2 . 9  0.97  0.050  10.0 ± 7.1  33.0 ± 4 2 . 4  0.53  0.076  240  8.7.10 Gender and Mutation Type Table 8.7.10.1 Lesion Quality by Gender and Mutation Type Variable  Lesion R a n k % Rank 1 Lesion R a n k % Rank 2 Lesion R a n k % Rank 3 Lesion R a n k % Rank 4  1 2 3 4  S m a l l (%) M e d i u m (%) L a r g e (%) Average Number of Lesions No. Pedunculated  Males Missense (n=2) 10.0 ± 1.4 52.0 ± 24.0 3.0 ± 2 . 8 12.5 ± 9 . 2 3.0 ± 2 . 8 12.5 ± 9 . 2 5.0 ± 2 . 8 23.0 ± 5 . 7 53.7 ± 2 5 . 0 24.3 ± 14.2 15.1 ± 1.3 21.0 ± 7.1  Females Missense (n=2) 8.0 ± 1.4 44.5 ± 27.6 5.0 ± 5 . 7 19.5 ± 17.7 2.0 ± 1.4 9.0 ± 2 . 8 6.0 ± 4 . 2 26.5 ± 7 . 8 43.4 ± 19.4 23.8 ± 13.5 32.8 ± 6 . 0 21.0 ± 9 . 9  P-value  Power  P-value  Power  0.025 0.37 0.41  0.65 0.13 0.12  —  0.026 0.020 0.32 0.018 0.91 0.38 0.20 0.78 0.0050  0.64 0.69 0.16 0.72 0.051  0.060 0.050 0.58 0.050  15.7 ± 6 . 9 3.8 ± 2 . 3 11.9 ± 7 . 4 11.0 ± 4.8 38.9 ± 19.6 33.1 ± 15.8 26.5 ± 7 . 7 35.9 ± 19.8 30.4 ± 12.1  EXT 2 Nonsense (n=6) 3.3 ± 2 . 5 25.2 ± 16.1 3.7 ± 1.6 29.2 ± 12.9 1.0 ± 1.1 7.7 ± 7 . 8 4.8 ± 3 . 1 37.6 ± 2 2 . 9 25.2 ± 16.0 36.4 ± 18.8 39.1 ± 2 2 . 2 12.8 ± 3.1  0.29 0.79 0.69 0.67  0.14 0.054  0.69 0.66 0.81 0.66 0.69 0.97 0.056  0.059 0.061 0.059 0.062 0.054 0.062  Males Nonsense (n=8) 10.8 ± 6 . 7 33.7 ± 17.7 4.9 ± 3 . 2  0.13 0.23 0.058 0.89  5.0 ± 1.4  7.5 ± 2 . 1  0.29  0.14  8.5 ± 5 . 6  4.3 ± 0.82  0.098  0.37  %  24.0 ± 1.4  37.5 ± 7 . 6  0.13  0.29  29.8 ± 14.1  39.1 ± 14.5  0.36  0.13  Pedunculated N o . Sessile % Sessile No. Distal % Distal No. Proximal % Proximal No. Pelvic % Pelvic No Diaphyseal % Diaphyseal  16.0 ± 5 . 7 75.9 ± 1.4 8.5 ± 7 . 8 36.3 ± 24.8 12.0 ± 0 . 60.6 ± 20.4 0.0 ± 0.0 0.0 ± 0.0 0.50 ± 0 . 7 1 3.1 ± 4 . 4  13.5 ± 7 . 8 62.5 ± 7.6 9.5 ± 2 . 1 48.2 ± 12.6 7.0 ± 1.4 35.7 ± 10.1  0.75 0.13 0.88 0.61 0.038 0.26 0.42 0.42 0.51 0.60  0.056 0.29 0.051 0.067 0.72 0.16 0.095 0.095 0.078 0.067  19.8 ± 9 . 3 64.8 ± 16.8 13.4 ± 4 . 9 46.1 ± 12.1 13.1 ± 5 . 4 43.2 ± 8 . 7 2.4 ± 3 . 5 5.9 ± 8 . 9 0.75 ± 0.89 1.7 ± 2 . 4  0.0093 0.23 0.0013 0.084 0.015 0.87 0.19 0.49 0.20 0.11  0.82 0.19 0.98 0.39 0.74 0.052 0.24 0.098 0.23 0.34  1.0 ± 1.4 3.8 ± 5 . 4  1.5 ± 2 . 1 5.4 ± 7 . 6 3.0 ± 1.4  0.81 0.84  0.054 0.052 0.059  2.6 ± 3 . 6  7.7 ± 2 . 4 53.2 ± 6 . 1 4.5 ± 2 . 1 34.4 ± 10.7 6.2 ± 2 . 9 45.5 ± 29.2 0.33 ± 0.82 2.8 ± 6 . 8 1.7 ± 1.6 17.4 ± 19.3 0.50 ± 0 . 8 4 4.1 ± 6 . 9 1.0 ± 0 . 6 3 11.3 ± 7 . 4  0.18 0.55 0.061  0.24 0.086 0.47  0.46  0.10  11.2 ± 2 . 5  0.0051 0.89  0.89 0.052  0.0019 0.0067 0.26  0.96 0.86  No. Flat Bone % Flat Bone No. Complex % Complex No. Simple % Simple No. Flared % Flared No. Not Flared % Not Flared N o . Left % Left No. Right % Right  2.5 ± 0 . 7 1 12.0 ± 0 . 6 8 18.5 ± 6 . 4 87.9 ± 0 . 6 8 7.0 ± 4.2 38.9 ± 3 3 . 3 14.0 ± 12.0 61.6±33.3 1 3 . 0 ± 1.4 64.4 ± 14.9 8.0 ± 5 . 7 35.6 ± 14.9  1.5 5.4 2.5 8.9  ± 2.1 ±7.6 ±3.5 ± 12.6  17.9 ± 15.2 1 8 . 0 ± 11.3 82.1 ± 15.2 4.5 ± 2 . 1 26.8 16.5 73.2 12.0  ± 22.7 ± 12.0 ±22.7 ±4.2  58.9 ± 7 . 6 9.0 ± 5 . 7 41.1 ± 7 . 6  0.064  6.7 ± 8 . 9 5.8 ± 5 . 5 15.9 ± 9 . 8  0.96 0.64  0.050 0.064  23.5 ± 8 . 5 84.1 ± 9 . 8  0.53 0.71  0.075 0.058 0.052 0.058  15.3 ± 7 . 7 50.9 ± 18.2  0.69 0.64  0.85 0.71 0.78 0.69 0.88 0.69  0.055 0.060 0.051 0.060  241  15.1 49.0 17.1 52.0  ±8.6 ± 18.2 ±8.5 ±6.8  13.3 ± 4 . 5 47.9 ± 6 . 8  83.1 ± 9 . 7 2.2 ± 2 . 3 17.1 ± 2 0 . 5 10.7 ± 3 . 6 82.9 ± 2 0 . 5 5.5 ± 1.9 42.6 ± 7 . 1 7.3 ± 1.2 57.4 ± 7 . 1  0.0067 0.0068 0.081 0.0087 0.081  0.19 0.86 0.86 0.41 0.83 0.41  Table 8.7.10.1 Lesion Quality by Gender and Mutation Type (continued) Variable  Lesion R a n k 1 % Rank 1 Lesion R a n k 2 % Rank 2 Lesion R a n k 3 % Rank 3 Lesion R a n k 4 % Rank 4 S m a l l (%) M e d i u m (%) L a r g e (%) Average Number of Lesions No. Pedunculated % Pedunculated N o . Sessile % Sessile No. Distal % Distal No. Proximal % Proximal No. Pelvic % Pelvic No Diaphyseal  Males Splice Site (n=2) 5.0 ± 0 . 0 14.5 ± 0 . 7 1 10.0 ± 1.4 29.0 ± 5 . 7 5.5 ± 3 . 5 15.5 ± 9 . 2 14.0 ± 0 . 0 40.5 ± 2 . 1 13.8 ± 0 . 0 36.2 ± 4 . 0 47.7 ± 3 . 3 34.5 ± 2 . 1 5.5 ± 2 . 1 15.8 ± 5 . 2 25.5 ± 4 . 9 74.5 ± 18.9 11.5 ± 0 . 7 1 33.5 ± 4 . 1 17.5 ± 4 . 9 50.4 ± 11.2 3.5 ± 0 . 7 1 10.2 ± 2.7 2.0 ± 1.4  % Diaphyseal No. Flat Bone % Flat Bone No. Complex % Complex No. Simple % Simple  5.9 ± 4 . 5 3.5 ± 0 . 7 1 10.2 ± 2 . 7 4.0 ± 1.4 11.5 ± 3 . 4 27.5 ± 3 . 5 80.2 ± 15.2  No. Flared % Flared No. Not Flared % Not Flared No. Left % Left No. Right  16.0 ± 18.4 44.8 ± 50.5  % Right  Females Splice Site (n=3) 5.0 ± 0 . 0 22.0 ± 5 . 2 3.3 ± 2 . 3 20.3 ± 11.1 3.3 ± 2 . 1 16.3 ± 7 . 6 11.7 ± 4 . 7 41.7 ± 10.6 22.6 ± 6 . 7 28.3 ± 10.2 49.0 ± 8.0 23.3 ± 7 . 6 7.3 ± 3.2 30.9 ± 5 . 1 14.3 ± 4 . 2 61.8 ± 2 . 9 10.3 ± 4 . 2 43.9 ± 7 . 9 9.7 ± 5 . 0 39.9 ± 10.5 1.0 ± 1.0 3.8 ± 3 . 3 2.0 ± 1.0 9.7 ± 6 . 1 1.7 ± 2.1  Pvalue  Power  Males F S (n=2)  —  —  0.15 0.038 0.39 0.44 0.92  0.27 0.67 0.11 0.098 0.051 0.076 0.051 0.23 0.11 0.053 0.27  4.0 ± 2 . 8 17.4 ± 0 . 9 2  0.56 0.89 0.18 0.39 0.85 0.15 0.54 0.049 0.071 0.30 0.73 0.19 0.19 0.36 0.058 0.11  —  5.8 ± 6 . 3 2.3 ± 1.5 9.5 ± 3 . 9 19.7 ± 6 . 4 84.2 ± 0.84  0.51 0.33 0.44 0.31 0.61 0.23 0.65  18.5 ± 16.3 55.2 ± 5 0 . 5 16.5 ± 2 . 1 47.7 ± 3 . 2 18.0 ± 0 . 0  4.3 ± 4 . 9 15.6 ± 13.7 19.0 ± 2 . 6 84.4 ± 13.7 13.0 ± 2 . 6 57.0 ± 6 . 1 10.7 ± 5 . 5  0.34 0.38 0.96 0.38 0.22 0.15 0.17  52.3 ± 3 . 2  44.0 ± 7.9  0.27  0.079 0.59 0.47 0.14  5.0 ± 1.4 26.4 ± 13.7 3.0 ± 2 . 8 11.5 ± 3 . 5 11.5 ± 10.6 44. ± 11.9 25.8 ± 10.7 35.3 ± 14.5 39.0 ± 3 . 8 23.5 ± 17.7  10.0 41.7 1.0 4.2 6.0 25.0 27.7 47.0 25.0 24.0  P-value  Power  0.55 0.060 0.21 0.53 0.67 0.34 0.75 0.41 0.91 0.63 0.21 0.99  0.069 0.59 0.18 0.071 0.058 0.11 0.055 0.090 0.051 0.061 0.19 0.050  0.62 0.021 0.67 0.021 0.41 0.074 0.97 0.91 0.21 0.14  0.062 0.99 0.058 0.99 0.091 0.49 0.050 0.050 0.18 0.28 0.050  15.0 ± 17.3 0.50 ± 0 . 7 1  4.0 16.7 20.0 83.3 12.0 50.0 12.0 50.0 3.0 12.5 2.0 4.0 3.0  1.4 ± 1.9 1.5 ± 0 . 7 1 10.5 ± 10.9 22.0 ± 18.4  12.5 4.0 16.7 20.0  89.5 ± 10.9 9.0 ± 11.3 28.2 ± 26.9  0.19 0.27 0.24  71.9 ± 2 6 . 9 8.5 ± 6.4 36.2 ± 0 . 1 9 15.0 ± 11.3  83.3 6.0 25.0 15.0 62.5 12.0 50.0 12.0  0.73 0.011 0.86  0.16  63.8 ± 0 . 1 9  50.0  0.011  0.058 0.22 0.23 0.12 0.53 0.35 0.050 0.084 0.13 0.098 0.14 0.069 0.19 0.065 0.13 0.11 0.050 0.11  242  10.5 ± 7 . 8 44.9 ± 0.75 13.0 ± 9 . 9 55.1 ± 0 . 7 5 5.0 ± 4 . 2 20.2 ± 2.9 11.5 ± 7 . 8 50.9 ± 5 . 2  Females FS (n=l) 7.0 29.2  0.50 ± 0 . 7 1 1.4 ± 1.9 2.0 ± 1.4  14.5 ± 6.4  0.69 0.21 0.14 0.21 0.72 0.94 0.72 0.86 0.94 0.96 0.82  0.057 0.18 0.28 0.18 0.055 0.050 0.055 0.051 0.050 0.050 0.052 0.055 1.0 0.051 1  Table 8.7. 0.2. Limb Alignment by Gender and Mutation Type  Variable  1. Carpal Slip Right 2. Carpal Slip Left 3. R a d i a l Inclination Right 4. R a d i a l Inclination Left 5. Ulnar Shortening Right 6. Ulnar Shortening Left 7. R a d i a l B o w Right 8. R a d i a l B o w Left 9. R a d i a l Head Dislocation R 10. R a d i a l Head Dislocation L 11 E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N . S . A n g l e Right 16. Femoral N . S . A n g l e Left 17. Femoral M . A . Right 18. Femoral M . A . Left 19. Sharp's Right 20. Sharp's Left  Normal  Males  Females  P-  Values  Missense  Missense  value  5± 2mm  21° ± 2 °  0±1 mm  10° ± 5 °  7° ± 2 ° valgus  135°± 5°  0°±5° varus  35° ± 4°  21. Fibular Height Right 22. Fibular Height Left  50 ± 10  23. A n k l e Joint A n g l e Right 24. A n k l e Joint A n g l e Left  0°±5°  Males  EXT 2  P-  Nonsense  Nonsense  value  (n=8)  Power  (n=2)  (n=2)  6.0 ± 4 . 2  2.0 ± 0 . 0  0.31  0.13  1.8 ± 5 . 2  (n=6) 2.3 ± 3 . 9  0.88  0.052  5.0 ± 5 . 2  1.5 ± 0 . 7 1  0.37  0.11  3.8 ± 4 . 4  3.3 ± 4 . 2  0.86  0.053  32.5 ± 6.4  20.5 ± 0 . 7 1  0.12  0.33  24.5 ± 3 . 1  25.3 ± 5 . 1  0.74  0.061  31.0±4.2  23.0 ± 1.4  0.13  0.31  28.5 ± 6 . 0  25.5 ± 3 . 1  0.29  0.17  -3.0 ± 0 . 0  1.0 ± 0 . 0  ~  -4.6 ± 5.3  0.18  0.25  -5.5 ± 0.71 12.0 ± 0 . 0  0.50 ± 0 . 7 1  0.014  0.97  1.1 ± 5 . 1  -0.50 ± 4.3 -1.2 ± 4 . 4  0.39  0.12  8.0 ± 2 . 8  0.18  0.22  7.4 ± 2 . 8  6.9 ± 2 . 4  0.73  0.062  8.8 ± 0 . 3 5  10.3 ± 0.35  0.051  0.61  10.9 ± 8 . 4  6.2 ± 1.5  0.20  0.23  0  1 dislocation 1 dislocation -19.0 ± 7 . 1  0  0 0  0.84  0.052  1 dislocation 1.1 ± 14.9  0.46  0.10  -14.5 ± 4 . 9  0.17  0.23  -9.3 ± 13.1  0.39  0.13  1.5 ± 2 . 1  -10.5 ± 9 . 2  0.21  0.19  -5.8 ± 10.9  -4.8 ± 13.3 -3.1 ± 12.5 -5.5 ± 8 . 3  0.95  0.050  2.5 ± 0 . 7 1  2.0 ± 5 . 7  0.91  0.051  -7.4 ± 7.9  0.24  0.20  140.0 ± 7.1  144.0 ± 5 . 7  0.59  0.068  144.6 ± 6 . 3  -0.50 ± 12.6 139.3 ± 5.0  0.12  0.33  135.5 ± 17.6 5.5 ± 3 . 5  150.0 ± 7 . 1  0.39  0.10  139.4 ± 5 . 3  137.5 ± 8.2  0.61  0.076  3.5 ± 3 . 5  0.63  0.065  1.4 ± 6 . 3  -2.3 ± 4.8  0.27  0.18  3.5 ± 7 . 8  3.0 ± 0 . 0  0.94  0.050  -0.71 ± 6 . 2  0.83 ± 6.2  0.66  0.069  Data not available Data not available 55.5 ± 3 . 5  Data not available Data not available 52.0 ± 0.0  39.1 ± 3 . 8  42.5 ± 7.2  0.28  0.18  40.6 ± 4 . 8  41.2 ± 5 . 4  0.83  0.055  0.29  0.14  51.9 ± 11.1  0.80  0.056  48.5 ± 26.2  46.5 ± 7.8  0.93  0.050  48.3 ± 10.3  0.76  0.059  -7.0 ( n = l )  -3.0 ± 4 . 2  0.58  0.065  -2.9 ± 18.8  50.3 ± 12.5 50.8 ± 19.9 -3.5 ± 7.2  0.94  0.051  2.0 ( n = l )  0.50 ± 0 . 7 1  0.33  0.11  -0.57 ± 18.5  -1.8 ± 9 . 2  0.89  0.052  0 9° ± 3 °  Power  -17.5 ± 6.4 -5.5 ± 3 . 5  243  Table 8.7. 0.2. Limb Alignment by Gender and Mutation Type (continued) Variable  25. % Weightbear  Normal  Males  Females  P-  Values  Missense  Missense  value  50 ± 10  (n=2)  (n=2)  70.5 ±  40.5 ± 28.9  Power  0.35  0.11  20.5  Males  EXT 2  P-  Nonsense  Nonsense  value  Power  (n=8)  (n=6)  55.9 ±  39.1 ± 18.7  0.21  0.22  45.3 ± 23.9  0.61  0.076  25.2  Right 26. %  66.5 ±  Weightbear  20.5  67.5 ± 3 . 5  0.95  0.050  51.6 ± 19.5  Left Parameters b e y o n d the n o r m a l range  244  Table 8.7.1 0.2. Lim b Alignment by Gender and Mutal ion Type (continued) Females P-value Power Males Females Variable Normal M a l e s Splice Site Splice Site FS FS Values (n=2) (n=3) (n=2) (n=l) 1. Carpal Slip Right 2. Carpal Slip Left 3. Radial Inclination Right 4. Radial Inclination Left 5. Ulnar Shortening Right 6. Ulnar Shortening Left 7. Radial B o w Right 8. Radial B o w Left 9. R a d i a l Head Dislocation R 10. R a d i a l Head Dislocation L 11 E l b o w Joint Right 12. E l b o w Joint Left 13. Femoral A . A . Right 14. Femoral A . A . Left 15. Femoral N.S. Angle Right 16. Femoral N . S . A n g l e Left 17. Femoral M . A . Right  5 ± 2mm  21° ± 2 °  0 ± 1 mm  10° ± 5 °  9° ± 3 °  7° ± 2 ° valgus  135°±5°  Pvalue  Power  5.0 ± 4 . 2  3.0 ± 1.0  0.46  0.093  4.0 ± 1.4  3.0  0.67  0.058  4.0 ± 1.4  3.3 ± 3 . 1  0.79  0.055  2.5 ± 0 . 7 1  6.0  0.15  0.25  27.5 ± 0 . 7 1  27.3 ± 0 . 5 8  0.79  0.055  25.5 ± 6.4  11.0  0.31  0.12  30.0 ± 2 . 8  30.0 ± 7 . 9  -  0.050  26.5 ± 10.6  24.0  0.88  0.051  2.0 ± 2 . 8  1.7 ± 1.5  0.87  0.052  -0.50 ± 2 . 1  -11.0  0.15  0.25  4.5 ± 4 . 9  5.7 ± 4 . 7  0.81  0.054  -3.5 ± 7 . 8  -7.0  0.78  0.053  8.0 ± 1.4  8.7 ± 0 . 5 8  0.49  0.086  6.5 ± 0 . 7 1  9.0  0.21  0.18  9.5 ± 2 . 1  12.5 ± 6 . 5  0.59  0.072  8.5 ± 4.9  11.0  0.75  0.054  1 dislocation  0  0  0  1 dislocation  0  3.5 ± 2 1 . 9  -0.67 ± 16.6  0.82  0.054  -15.0 ± 2 . 8  2.0  0.13  0.30  -3.5 ± 2 1 . 9  -9.0 ± 10.4  0.72  0.059  -9.5 ± 3 . 5  -3.0  0.37  0.099  -0.50 ± 6 . 4  -1.2 ± 4 . 5  0.89  0.051  -8.5 ± 19.1  -9.0  0.99  0.050  3.8 ± 10.9  -5.5 ± 0 . 8 7  0.21  0.20  -7.0 ± 5 . 7  0.0  0.49  0.075  138.0 ± 12.7  140.0 ± 3 1 . 2  0.94  0.050  129.5 ± 3 . 5  148.0  0.15  0.26  144.5 ± 6.4  144.0 ± 2 3 . 3  0.98  0.050  128.5 ± 2 . 1  140.0  0.14  0.27  8.0 ± 0 . 0  8.3 ± 7.4  0.097  0.050  -4.0 ± 11.3  -4.0  -  0.050  245  Table 8.7.1 0.2. Lim b Alignment by Gender and Mutal ion Type (continued) Females P-value Power Males Females Variable Normal M a l e s S p l i c e Site S p l i c e Site FS FS Values 18. Femoral M . A . Left 19. Sharp's Right 20. Sharp's Left 2 1 . Fibular Height Right 22. Fibular Height Left 23. A n k l e Joint A n g l e Right 24. A n k l e Joint A n g l e Left 25. % Weightbear Right 26. % Weightbear Left  35° ± 4°  50 ± 10  0°±5°  50 ± 10  P-  Power  value  (n=2)  (n=3)  (n=2)  (n=l)  -5.0 ± 7 . 1  1.0 ± 1.4  0.36  0.11  1.5 ± 3 . 5  6.0  0.49  0.076  39.3 ± 3 . 9  38.8 ± 3 . 7  0.91  0.051  38.5 ± 4 . 9  51.0  0.29  0.13  42.0 ± 2.8  36.8 ± 3 . 5  0.19  0.22  43.0 ± 7 . 1  47.0  0.72  0.055  55.0 ± 2 . 8  39.0 ± 11.0  0.15  0.27  62.8 ± 0 . 3 5  61.0  0.15  0.25  59.5 ± 4 . 9  49.0 ± 9.0  0.24  0.18  63.9 ± 1.3  77.0  0.075  0.49  16.0 ± 2 2 . 6  1.0 ± 3 . 5  0.31  0.14  -9.5 ± 0 . 7 1  -20.0  0.052  0.66  8.5 ± 17.7  0.0 ± 4.4  0.45  0.095  -8.0 ± 1.4  -18.0  0.11  0.35  63.5 ± 17.7  65.3 ± 6.4  0.87  0.052  39.5 ± 40.3  30.0  0.88  0.051  45.5 ± 7.8  61.3 ± 12.1  0.21  0.20  52.5 ± 2 1 . 9  75.0  0.56  0.067  Parameters b e y o n d the n o r m a l range  246  Table 8.7.10.3. Segment Lengths and Percentile Height by Gender and Mutation Type Males Females P-value Power EXT 2 Males P-value Power Variable Total L e g Length-Right Upper L e g Right Lower Leg Right Total L e g Length -Left U p p e r L e g - Left Lower Leg Left Total A r m Length - Right Upper A r m Right Lower A r m Right Total A r m Length - Left Upper A r m Left Lower A r m Left Percentile Height  Missense (n=2) 86.0 ± 3.5 44.0 ± 2.1 37.0 ± 3.5 85.3 3.9 43.8 1.1 35.3 3.9 52.8 2.5 30.5 0.71 24.5 2.1 53.3 1.1 31.3 2.5 26.3 2.5 10.0 7.1  ± ± ± ± ± ± ± ± ± ±  Missense (n=2) 62.5 ± 1.4 30.5 ± 0.71 26.0 ± 0.71 61.8 ± 0.35 30.8 ± 2.5 25.8 ± 0.35 37.8 ± 1.1 22.8 ± 0.35 17.5 ± 0.0 37.3 ± 1.1  0.013  0.98  Nonsense (n=8) 86.9 ± 6 . 4  0.013  0.97  44.2 ± 4 . 7  45.3 ± 6.4  0.73  0.062  0.049  0.62  35.3 ± 3 . 4  34.5 ± 3 . 3  0.66  0.069  0.014  0.97  86.7 ± 7 . 6  83.0 ± 10.3  0.45  0.11  0.021  0.90  43.3 ± 4.7  44.3 ± 6 . 1  0.71  0.064  0.075  0.47  37.1 ± 5 . 3  35.3 ± 5 . 2  0.53  0.089  0.016  0.95  50.4 ± 6 . 2  50.3 ± 6.0  0.96  0.050  0.0052  1.0  3 1 . 7 ± 3.6  29.3 ± 3 . 5  0.23  0.20  0.04  0.67  23.3 ± 3 . 7  23.2 ± 3 . 1  0.94  0.051  0.0044  1.0  50.5 ± 6 . 8  50.7 ± 5 . 6  0.96  0.050  21.5 ± 0.71 18.5 ± 1.4  0.033  0.77  31.5 ± 4 . 5  30.4 ± 4.7  0.67  0.068  0.062  0.54  23.1 ± 4 . 4  24.0 ± 2 . 7  0.68  0.067  33.0 ± 42.4  0.53  0.076  43.4 ± 3 1 . 9  51.7 ± 33.3  0.65  0.071  247  Nonsense (n=6) 84.4 ± 10.4  0.59  0.078  Table 8.7.10.3. Segment Lengths and Percentile Height by Gender and Mutation Type (continued) Males Females P-value Power Males Females P-value Variable  Powe  S p l i c e Site  S p l i c e Site  FS  FS  (n=2)  (n=3)  (n=2)  (n=l)  83.3 ± 8 . 1  83.3 ± 7 . 1  0.99  0.050  88.0 ± 2 . 1  81.0  0.23  0.17  40.5 ± 4.2  42.8 ± 3 . 8  0.57  0.075  42.8 ± 2 . 5  41.5  0.75  0.054  34.8 ± 3 . 9  34.0 ± 3 . 6  0.84  0.053  35.8 ± 1.1  32.5  0.24  0.16  82.0 ± 6 . 4  83.3 ± 7 . 4  0.85  0.053  86.5 ± 0.0  81.5  O.0001  1.0  40.3 ± 2.5  42.8 ± 4 . 3 34.8 ± 3 . 8 49.5 ± 6.9  0.51 0.93 0.50  0.084 0.051 0.085  41.5 ± 0 . 0 37.0 ± 1.4 53.0 ± 0 . 7 1  40.0 36.0 46.0  <0.0001 0.67 0.078  -  35.3 ± 6 . 0 45.5 ± 2 . 1 27.8 ± 1.1  30.3 ± 4 . 5  0.50  0.085  31.8 ± 0 . 3 5  28.0  0.073  0.50  Lower A r m Right  21.3 ± 0 . 3 5  23.8 ± 2 . 3  0.22  0.19  26.0 ± 1.4  18.5  0.14  0.27  Total A r m Length -Left Upper A r m - Left L o w e r A r m - Left Percentile Height  47.3 ± 0.35  49.5 ± 6.3  0.66  0.064  53.8 ± 4 . 6  45.5  0.38  0.097  29.3 ± 1.1 20.0 ± 1.4 32.0 ± 9 . 9  30.5 ± 4 . 8 22.2 ± 3 . 3 12.3 ± 11.4  0.75 0.46 0.14  0.057 0.092 0.28  32.3 ± 1.8 25.5 ± 2 . 1 10.5 ± 10.6  30.0 18.0 8.0  0.49 0.21 0.88  0.076 0.18 0.051  Total L e g LengthRight Upper L e g Right Lower Leg Right Total L e g Length -Left Upper L e g - Left L o w e r L e g - Left Total A r m Length - Right Upper A r m Right  248  r  0.058 0.47  8.7.11 Gene and Mutation Location  Table 8.7.11.1 Lesion Quality by Gene and Mutation Location Variable  EXT 1  EXT 2  P-  Early  Early  value  Power  EXT 1  EXT 2  P-  Late  Late  value  (n=2)  (n=17)  6.2 ± 1.8 23.0 ± 8.4 6.2 ± 2.9 21.0± 8.2 5.0 ± 1.9 18.0 ± 5.9 11.0 ± 4.3 38.2 ± 9.8 23.9 ± 9.5  5.0 ± 0 . 0  0.41  0.11  20.0 ± 7 . 1  0.69  0.065  6.5 ± 6.4  0.93  0.051  32.5 ± 0 . 7 1  0.12  0.33  2.0 ± 1.4  0.10  0.36  10.0 ±1.4  0.13  0.30  12.0 ± 2 . 8  0.78  0.057  37.0 ± 7 . 1  0.88  0.052  20.8 ± 9 . 8  0.71  0.062  33.8 ± 0.78 38.9 ± 14.2  27.8 ± 15.8  0.36  0.13  50.5 ± 7 . 1  0.34  0.14  Power  (n=2)  (n=17)  Lesion R a n k 1  16.5 ± 7 . 8  6.3 ± 4 . 9  0.019  0.69  % Rank 1  37.0 ± 7 . 1  33.2±21.1  0.81  0.056  Lesion R a n k 2  7.0 ± 4 . 2  3.5 ± 2 . 2  0.076  0.42  % Rank 2  15.5 ± 4 . 9  21.2 ± 12.9  0.55  0.086  Lesion R a n k 3  5.0 ± 2 . 8  1.9 ± 1.9  0.058  0.48  % Rank 3  11.0 ± 2 . 8  9.5 ± 7 . 4  0.79  0.058  Lesion R a n k 4  15.0 ± 1.4  6.5 ± 4 . 4  0.018  0.70  % Rank 4  36.5 ± 14.8  35.9 ± 2 0 . 2  0.97  0.050  S m a l l (%)  39.7 + 10.4  31.9 ±18.5  0.58  0.082  M e d i u m (%)  22.5 ±15.3  31.2 ±14.2  0.42  0.12  L a r g e (%)  37.8 ± 25.7  34.8 ± 18.2  0.84  0.054  Average  43.5 ± 13.4  18.3 ± 8 . 6  0.0021  0.95  28.4 ± 6.1  25.5 ± 10.6  0.65  0.068  11.5 + 2.1  6.2 ± 4 . 5  0.13  0.31  7.6 ± 2.4  5.0 ± 1.4  0.22  0.19  26.9 ±3.5  32.2 ±13.8  0.61  0.077  22.7 ± 14.9  0.60  0.074  27.5 ± 17.7 59.8 ± 22.2  12.6 ± 6 . 1  0.015  0.73  26.8 ± 6.3 18.6 ± 3.4  20.0 ± 12.7  0.81  0.055  64.5 ± 14.5  0.68  0.067  66.1 ± 6.9 11.6 ± 3.1  74.5 ± 18.9  0.38  0.12  10.5 ± 2 . 1  0.68  0.065  41.1 ± 9.3 13.0 ± 5.2  43.2 ± 9 . 6  0.80  0.055  9.5 ± 6 . 4  0.48  0.095  44.9 ± 10.6 1.8± 1.3 6.1 ± 4.0  35.1 ± 10.4  0.31  0.15  2.0 ± 2 . 8  0.89  0.052  6.1 ± 8 . 6  0.99  0.050  Number of Lesions No. Pedunculated  % Pedunculated N o . Sessile % Sessile No. Distal  17.0 ± 8 . 5  7.8 ±4.6  0.023  0.66  %  Distal  37.9 ± 7 . 8  39.5 ± 15.2  0.88  0.052  No. Proximal  18.0 ± 5 . 7  9.4 ± 4 . 6  0.024  0.65  % Proximal  48.0 ± 16.1  0.58  0.083  No. Pelvic  41.3 ± 0.24 7.5 ± 0 . 7 1  0.59 ± 1.2  <0.001  1.00  %  18.4 ± 7 . 3  1.8 ± 4 . 9  <0.001  0.99  Pelvic  249  Table 8.7.11.1 Lesion Quality by Gene and Mutation Location (continued) Variable  No Diaphyseal  E X T l  EXT 2  P-  Early  Early  value  (n=2)  (n=17)  1.5 ± 0 . 7 1  1.2 ± 1.3  0.74  Power  0.062  EXT 1  EXT 2  P-  Late  Late  value  (n=2)  (n=17)  2.0 ±  2.5  Power  ±0.71  0.76  0.058  10.1 ± 1.4  0.71  0.062  2.0 ± 2 . 8  0.68  0.065  6.1 ± 8 . 6  0.50  0.090  2.5  ±0.71  0.69  0.063  10.1 ± 1.4  0.98  0.050  2 2 . 5 ± 10.6  0.83  0.054  87.1 ± 5 . 4  0.67  0.065  2 . 0 ± 1.4  0.41  0.11  7.3 ± 2 . 5  0.36  0.13  23.5 ± 9 . 2  0.49  0.093  92.7 ±2.5  0.36  0.13  13.0 ± 2 . 8  0.36  0.13  5 3 . 2 ± 11.1  0.97  0.050  12.5 ± 7 . 8  0.80  0.055  4 6 . 7 ± 11.1  0.94  0.050  2.0 % Diaphyseal  3.4 ± 0 . 5 9  7.9 ± 12.1  0.61  0.076  7.8 ± 8.1  No. Flat Bone  8.0 ± 0 . 0  0.71 ± 1.2  <0.001  1.00  2.6 ± 1.1  % Flat Bone  19.3 ± 5 . 9  2.6 ± 5 . 1  O.001  0.99  8.8 ± 2.9  No. Complex  9.5 ± 12.0  2.7  ±2.2  0.023  0.66  3.0 ± 1.6  % Complex  18.5 ± 2 1 . 9  14.2  ±9.6  0.61  0.078  10.0 ± 3.8  No. Simple  29.5 ± 7 . 8  16.7  ±8.5  0.059  0.47  23.6 ± 3.9  % Simple  68.3 ± 3 . 2  84.5 ± 9.9  0.038  0.56  84.0 ± 8.8  No. Flared  25.5 ±  7.4 ± 6 . 1  O.001  0.98  0.71 % Flared  11.3  61.3 ±  33.5  ±24.1  0.14  0.29  17.3 No. Not Flared  18.0±  11.5 ± 5.9  0.21  0.22  18.8± 7.1  38.7 ±  66.5 ± 2 4 . 1  0.14  0.29  17.3 No. Left  29.6 ± 29.8  12.7 % Not Flared  9.6 ±  70.4 ± 29.8  27.5 ± 4.9  9.7 ± 5 . 4  O.001  0.99  15.0 ± 2.2  % Left  6 4 . 5 ± 8.6  4 8 . 2 ± 10.9  0.060  0.46  53.5 ± 4.1  No. Right  16.0 ± 8 . 5  9.9 ± 4 . 8  0.13  0.31  13.6 ± 4.0  % Right  35.5  ±8.6  51.8 ± 10.9  0.060  0.46  47.1 ± 4.9  250  Table 8.7. 1.2. Limb Alignment by Gene and Mutation Location Variable  1. Carpal SlipR 2. Carpal SlipL 3. Radial Inclination R 4. Radial Inclination L 5. Ulnar Shortening R 6. U l n a r Shortening L 7. Radial BowR 8. Radial B o w Left 9. Radial Head Dislocation R 10. Radial Head Dislocation L 11. E l b o w Joint R 12. E l b o w Joint L 13. Femoral A.A. R 14. Femoral A.A. L 15. Femoral N.S. Angle R  Normal  E X T l  EXT 2  P-  Values  Early  Early  value  5 ± 2mm  (n=2) 5.0  2.2 ± 3 . 8  0.49  2.9 ± 3 . 6  21° ± 2 °  7.0 ± 1.4 29.0  0 ± 1 mm  E X T l  EXT 2  P-  Late  Late  value  (n=2) 5.2 ± 3 . 1  (n=17)  0.099  0.15  0.29  3.8 ± 3 . 0  23.8 ± 5 . 2  0.94  0.14  27.8 ± 6 . 1  28.5 ± 9.2 -8.0  26.6 ± 4 . 8  0.64  0.073  31.4±5.5  -2.3 ± 4.8  0.27  0.18  0.20 ± 2.2  1.5 ± 4.9  -1.2 ± 5 . 0  0.49  0.10  11.0  7.4 ± 2 . 5  0.18  20.0 ± 15.6 0  7.7 ± 2 . 5  0.0019  1  1  1  9° ± 3 °  2.0  -4.6 ± 13.3  0.64  0.073  -7.5 ± 11.5  0.64  0.072  7° ± 2 ° valgus  -3.5 ± 4.9 -5.5 ± 7.8 -9.5 ± 3.5  -5.6 ± 9 . 7  0.99  -3.3 ± 9 . 5  146.0 ± 7.1  10° ± 5 °  135° ± 5 °  16. Femoral N.S. Angle L 17. Femoral M.A. R 18. Femoral M.A. L  0° ± 5° varus  19. Sharp's Right 20. Sharp's Left  35° ± 4 °  Power  (n=17)  Power  2.0 ± 0.0 4.5 ± 2.1 27.5 ± 0.71 24.5 ± 4.9 3.0 ±1.4  0.23  0.19  0.78  0.057  0.95  0.050  0.18  0.24  0.16  0.26  3.2 ± 6 . 6  2.5 ± 2.1  0.89  0.052  0.25  8.6 ± 2 . 3  0.96  0.050  0.95  11.9 ± 4 . 6  0.31  0.15  1  8.5 ± 0.71 8.0 ± 0.0 1  1  1  0.092  0.20  0.22  0.62  0.072  0.38  0.13  1.6 ± 7 . 5  0.33  0.14  140.8 ± 6 . 9  0.33  0.15  142.0 ± 21.3  -14.0 ± 2.8 -17.5 ±2.1 -5.5 ± 0.71 -4.5 ± 0.71 134.5 ± 17.7  0.49  0.050  -2.6 ± 20.5 -4.0 ± 12.3 -2.1 ± 8 . 5  0.68  0.065  142.5 ± 0.71  137.1 ± 8 . 6  0.39  0.13  148.0 ± 13.0  137.0 ± 16.9  0.39  0.12  1.0 ± 5.7 -5.0 ± 2.8  -0.84 ± 5.9  0.69  0.067  8.9 ± 3 . 2  0.30  0.15  1.3 ± 5 . 3  0.12  0.32  1.0 ± 7 . 9  0.88  0.052  37.5 ± 3.5  41.7±5.9  0.35  0.14  38.5 ± 3 . 0  5.5 ± 3.5 0.0 ± 0.0 39.0 ± 4.2  0.87  0.052  41.0± 8.5  41.5 ± 4 . 7  0.90  0.052  37.6 ± 3 . 3  38.5 ± 7.8  0.84  0.053  251  Table 8.7. 1.2. Limb Alignment by Gene and Mutation Location (continued) Variable  Normal Values  2 1 . Fibular Height R 22. Fibular Height L 23. A n k l e Joint A n g l e R 24. A n k l e Joint A n g l e L 25. % Weightbear R 26. % Weightbear L Number of parameters that fall b e y o n d the normal range  50 ± 10  0°±5°  50 ± 10  EXTl Early (n=2) 59.0 ± 7.1 48.0 ± 22.6 -26.0 ± 7.1  EXT 2 Early (n=17) 52.7 ± 10.8  Pvalue  Power  0.44  51.2 ± 15.1  EXT 2 Late (n=17) 42.5 ± 20.5  Pvalue  Power  0.11  EXT 1 Late (n=2) 49.4 ± 5.9  0.47  0.096  0.79  0.058  54.6 ± 9 . 8  0.87  0.052  -1.9 ± 10.8  0.0085  0.82  5.0 ± 15.8  56.0 ± 9.9 -1.5 ± 2.1  0.61  0.073  -20.5 ± 19.1  -0.50 ± 11.2  0.041  0.55  5.8 ± 8 . 5  -4.5 ± 0.71  0.17  0.25  61.5 ±10.6  45.5 ± 2 3 . 4  0.36  0.14  63.8 ± 25.4  54.5 ± 4.9  0.65  0.068  53.0 ± 5.7  51.5 ± 2 0 . 9  0.92  0.051  65.0 ± 15.9  50.5 ± 0.71  0.28  0.17  252  Table 8.7.11.3. Segment Lengths and Percentile Height by Gene and Mutation Location Variable  Total L e g Length-Right Upper L e g Right Lower L e g  EXT 1  EXT 2  P-  Early  Early  value  (n=2)  (n=17)  80.5 ± 2 . 1  85.1 ± 9 . 4  0.51  0.096  38.5 ± 0 . 7 1  44.2 ± 5.6  0.18  32.0 ± 1.4  35.0 ± 3 . 7  80.0 ± 1.4  Power  EXT 1  EXT 2  P-  Late  Late  value  Power  (n=2)  (n=17)  83.5 ± 8 . 5  0.63  0.070  0.25  79.3 ± 10.1 39.5 ± 5 . 5  42.3 ± 6.7  0.59  0.075  0.28  0.18  32.5 ± 4 . 8  34.5 ± 3 . 5  0.62  0.071  84.4 ± 9.7  0.54  0.090  83.3 ± 8 . 1  0.58  0.076  38.3 ± 1.1  43.3 ± 5 . 2  0.20  0.23  78.4 ± 10.3 39.1 ± 5 . 9  42.8 ± 6 . 0  0.49  0.091  31.0 ± 1.4  36.5 ± 4 . 9  0.15  0.29  33.0 ± 5 . 5  34.0 ± 4 . 2  0.83  0.054  43.0 ± 0 . 0  50.8 ± 5 . 7  0.077  0.41  45.6 ± 6 . 1  49.8 ± 8 . 1  0.48  0.094  28.0 ± 0 . 0  30.5 ± 3 . 8  0.39  0.13  27.2 ± 2 . 9  31.8 ± 4 . 6  0.16  0.26  19.5 ± 2 . 1  23.6 ± 3 . 4  0.13  0.32  21.5±2.5  23.5 ± 3 . 5  0.42  0.11  42.0 ± 1.4  51.0±6.2  0.059  0.47  46.2 ± 5 . 8  51.3 ± 6 . 0  0.35  0.13  26.5 ± 0 . 7 1  30.9 ± 4 . 5  0.19  0.24  27.9 ± 4 . 2  32.8 ± 3 . 9  0.22  0.21  17.5 ± 3 . 5  24.0 ± 3 . 4  0.021  0.67  20.9 ± 2 . 7  23.0 ± 2 . 8  0.39  0.12  3.0 ± 0 . 0  44.6 ± 3 0 . 1  0.074  0.42  11.8 ± 15.4  25.0 ± 0 . 0  0.30  0.15  Right  Total L e g Length - Left Upper L e g Left Lower L e g Left Total A r m Length - Right Upper A r m Right Lower A r m Right Total A r m Length - Left Upper A r m Left  Lower A r m Left Percentile Height  253  

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