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Genotype-phenotype correlations in hereditary multiple exostoses in British Columbia 2003

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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 this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) 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 ii Table of Contents iii ListofTables viii List of Figures ix Acknowledgements xi Chapter I Background 1 1.1 Osteochondroma (Exostosis) 1 1.1.1 definition 1 1.1.2 features 1 1.1.1.1 radiology 1 1.1.1.2 gross pathology 2 1.1.1.3 microscopic pathology... 4 1.1.1.4 clinical 5 1.2 Hereditary Multiple Exostoses 8 1.2.1 definition 8 1.2.2 demographic features 8 1.2.3 genetics and molecular biology 10 1.2.3.1 general information 10 1.2.3.2 physiologic function 14 1.2.3.3 EXT gene products 20 1.2.4 Mutations 22 1.2.4.1 EXT 1 mutation summary 29 1.2.4.2 EXT 2 mutation summary 29 1.2.5 Phenotyping 30 1.2.5.1 Schmale's findings 30 1.2.5.2 Porter's findings 32 1.2.5.3 Genotype-phenotype correlations 33 1.2.5.3.1 Carroll 33 1.2.5.3.2 Francannet.... 35 1.3 Proj ect rationale 36 1.4 Hypothesis 39 1.5 Objective 39 Chapter II Methods and Materials 40 2.1 Ethical Approval 40 2.2 Protocol Overview 41 2.3 Subject Recruitment 42 2.3.1 Subject identification 42 2.3.2 Pedigree accumulation 42 iii 2.4 Genotype 42 2.4.1 Sample collection 42 2.4.2 DNA extraction 43 2.4.3 Gene Assignment - Highly Polymorphic Repeats 44 2.4.3.1 Marker Selection 44 2.4.3.2 PCR 46 2.4.3 3 PAGE 46 2.4.3.4 Hybridization 47 2.4.3.5 Visualization 48 2.4.3.6 Exclusion Analysis 48 2.4.4 EXT 1 and EXT 2 amplification 48 2.4.5 DNA sequencing 51 2.4.6 Mutation identification 52 2.4.7 Segregation Analysis 54 2.5 Phenotype 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 63 2.6.1. Genotype 63 2.6.2 Phenotype 63 2.6.3 Genotype-phenotype correlation 64 Chapter III Results 66 3.1 Subject recruitment 66 3.1.1. Subject identification 66 3.1.2 Family pedigrees 68 3.2 Genotype 68 3.2.1 Highly Polymorphic Repeats 68 3.2.2 Mutation identification 81 3.3 Phenotype 82 iv 3.3.1 Phenotype data 82 3.3.2 Range of motion 82 3.3.3 Pearson Correlation matrix 83 3.4 Genotype-Phenotype Correlations 83 3.4.1 Gene versus phenotype 88 3.4.2 Gene and gender versus phenotype 89 3.4.3 Gene and mutation type versus phenotype... 90 3.4.4 Gene and severity versus phenotype 91 3.4.5 Gene and mutation location versus phenotype 91 3.4.6 Gender versus phenotype 92 3.4.7 Mutation type versus phenotype 93 3.4.8 Mutation severity versus phenotype 93 3.4.9 Mutation location versus phenotype 93 3.4.10 Gender and severity versus phenotype 94 3.4.11 Gender and mutation type versus 95 phenotype Chapter IV Discussion 96 4.1 Subject recruitment 96 4.2 Genotpye 96 4.3 Phenotype 103 4.3.1 Lesion Quality 103 4.3.2 Limb Alignment 107 4.3.3 Limb segments and percentile height 108 4.3.4 Intra-family variability 110 4.4 Genotype-phenotype correlation 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). 147 8.4.2 EXT 2 translation 158 8.5 Genotyping 161 8.5.1 HPR markers 161 8.5.2 HPR sequences 162 8.6 Data 163 8.6.1 Family pedigrees 163 8.6.2 STR gels 173 8.6.3 Phenotype data 179 8.6.3.1 Core data 179 8.6.4.1.1 Lesion quality 179 8.6.4.1.2 Limb alignment 181 8.6.4.1.3 Limb segments and percentile height 186 8.6.3.2 Pearson Correlation matrix 188 8.7 Genotype-Phenotype Correlation tables 202 8.7.1. Gene 202 8.7.1.1 Lesion Quality 202 8.7.1.2 Limb alignment 203 8.7.1.3 Limb segments and percentile 204 height 8.7.2 Gender 205 8.7.2.1 Lesion Quality 205 8.7.2.2 Limb alignment 206 8.7.2.3 Limb segments and percentile 207 height 8.7.3 Mutation Type 208 8.7.3.1 Lesion Quality 208 8.7.3.2 Limb alignment 208 8.7.3.3 Limb segments and percentile 210 height 8.7.4 Mutation severity 211 8.7.4.1 Lesion Quality 211 8.7.4.2 Limb alignment 212 8.7.4.3 Limb segments and percentile 213 height 8.7.5 Mutation location 214 8.7.5.1 Lesion Quality 214 8.7.5.2 Limb alignment 215 8.7.5.3 Limb segments and percentile 216 height 8.7.6 Gene and gender 217 8.7.6.1 Lesion Quality 217 8.7.6.2 Limb alignment 219 8.7.6.3 Limb segments and percentile 220 height 8.7.7 Gene and mutation type 221 vi 8.7.7.1 Lesion Quality 221 8.7.7.2 Limb alignment 225 8.7.7.3 Limb segments and percentile 229 height 8.7.8 Gene and severity 231 8.7.8.1 Lesion Quality 231 8.7.8.2 Limb alignment 233 8.7.8.3 Limb segments and percentile 235 height 8.7.9 Gender and Severity 236 8.7.9.1 Lesion Quality 236 8.7.9.2 Limb alignment 238 8.7.9.3 Limb segments and percentile 240 height 8.7.10 Gender and Mutation Type 241 8.7.10.1 Lesion Quality 241 8.7.10.2 Limb alignment 243 8.7.10.3 Limb segments and percentile height 245 8.7.11 Gene and Location 249 8.7.11.1 Lesion Quality 249 8.7.11.2 Limb alignment 251 8.7.11.3 Limb segments and percentile height 253 vii List of Tables Table 1.1 Summary of Family Mutations 11 Table 1.2 Summary of Mutations Identified in the EXT 1 Gene 24-25 Table 1.3 Summary of Mutations Identified in the EXT 2 Gene 26 Table 1.4 Modified Functional Assessment Scale of the 32 Musculoskeletal Tumour Society (as per Schmale 1994) Table 2.1 Primer pair sequences used for EXT 1 50 Table 2.2 Primer pair sequences used for EXT 2 51 Table 3.1 Subject Recruitment 67 Table 3.2 Summary of STR Markers as per family and EXT gene 69 assignment for mutations identified in EXT 1 and EXT 2 Table 3.3 Mutations identified in each proband 81 Table 3.4 Breakdown of Genotype Features 84 Table 3.5 Summary of Results for Comparison between EXT 1 and 88 EXT 2 Genes Table 3.6 Summary of Results for remaining unvariant data 92 Table 3.7 Summary of Results for Comparison between Males and 94 Females Covariant Data viii List of Figures Figure 1.1 X-rays showing presence of an exostoses at the (a) distal femur (Wold 1990) and (b) proximal humerus 1 Figure 1.2 X-ray and CT scan showing location of an exostosis in relation to the parent bone 2 Figure 1.3 Gross pathology of with the xray of the same lesion in situ (Wold 1990) 3 Figure 1.4 X-rays showing a (a) pedunculated exostosis, (b) sessile exostosis (solitary osteochondroma subject) and (c) a lesion causing metaphyseal flaring 4 Figure 1.5 (a) Epiphyseal growth plate (Wheater 1987); (b) An osteochondroma at low magnification and (c) at high magnification (Wold 1990) 5 Figure 1.6 (a) X-rays showing exostosis tethering the growth plate in an affected ankle (b) A normal ankle is shown for comparison 7 Figure 1.7 (a) X-ray showing an exostosis causing deformity in the foream; (b) a normal forearm is shown for comparison 7 Figure 1.8 X-ray showing exostosis causing growth impedance 8 Figure 1.9 Alignment of EXT 1 and EXT 2 genes 13 Figure 1.10 Distribution of Mutations in the EXT 1 Gene 27 Figure 1.11 Distribution of Mutations in the EXT 2 Gene 28 Figure 1.12 Anatomical Distribution of Lesions (Schmale 1994). 31 Figure 2.1 Overview of materials and methods 41 Figure 2.2 HPR marker locations in relation to EXT 1, 2, and 3 45 Figure 2.3 Calculation of Lesion Size and Rank 58 Figure 2.4 Measurement of carpal slip 59 Figure 2.5 Measurement of radial inclination and ulnar shortening 59 Figure 2.6 Measurement of radial bowing 59 Figure 2.7 Radial head subluxation / dislocation 60 Figure 2.8 Measurement of the elbow joint angle 60 Figure 2.9 Measurement of the femoro-tibial anatomic angle 60 Figure 2.10 Measurement of the weight bearing axis, the femoral neck/shaft angle, and the femoral anatomic angle 61 Figure 2.11 Measurement of Sharp's acetabular angle 62 Figure 2.12 Measurement of fibular height. 62 Figure 2.13 Measurement of ankle j oint angle 62 Figure 3.1a EXT 1 and EXT 2 STR Markers for Family 1 70 Figure 3.1b Sequencher output for segregation analysis for Family 1 70 Figure 3.2a EXT 1 and EXT 2 STR Markers for Family 16 71 Figure 3.2b Sequencher output for segregation analysis for Family 16 71 Figure 3.3a EXT 1 and EXT 2 STR Markers for Family 18 72 Figure 3.3b Sequencher output for segregation analysis for Family 18 72 Figure EXT 1 STR Markers Family 6. 73 3.4a(i) Figure 3.4a EXT 2 STR Markers Family 6 73 (ii) Figure 3.5a EXT 1 and EXT 2 STR Markers Family 2 74 ix Figure 3.5b Sequencher output for segregation analysis for Family 2 74 Figure 3.6a EXT 1 and EXT 2 STR Markers Family 5 75 Figure 3.6b Sequencher output for segregation analysis for Family 5 75 Figure 3.7a EXT 1 and EXT 2 STR Markers Family 17 76 Figure 3.7b Sequencher output for segregation analysis for Family 17 76 Figure 3.8a EXT 1 and EXT 2 STR Markers Family 8 77 Figure 3.8b Sequencher output for segregation analysis for Family 8 77 Figure EXT 1 STR Markers for Family 4 78 3.9a(i) Figure 3.9a EXT 12 STR Markers for Family 4 78 (ii) Figure EXT 1 and EXT 2 STR Markers for Family 3 79 3.10a Figure Sequencher output for segregation analysis for Family 3 80 3.10b 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 Radio log ic 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 CT 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 Mic roscop ic 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 1- 12) (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 Ancestry Number of families studied E X T 1 Mutation MS or non- truncating mutations EXT 2 Mutations #MS or non- truncating mutations #of Unidentified Mutations # (%) # (%) # (%) # (%) Philippe et al., 1997 M i x e d 17 12 71 2 16.7 5 29.4 1 8.3 0 Wuyts et al., 1998 M i x e d 26 10 38.5 2 7.9 10 38.5 ~ 6 X u et al, 1998 Chinese 36 5 13.9 2 5.6 12 33.3 1 2.8 19 Seki et al., 2001 Japanese 43 17 39.5 ~ 6 13.9 1 2.3 20 Francannet et al., 2001 French 42 27 64.3 — 9 21.4 1 2.4 6 Gigante et al., 2001 Italian 9 4 44.4 ~ 3 33.3 ~ 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 W+ E S+ P + A+S I + CRM +CFD C EXT 2: FWPHSIESSND WNV EKRSIRDVPWRLPADSPIPERGDLSCRMHTCFDVYRCGF 98 EXT 1:-KKNGFKVYVYPQQK GEKIAESYQNILAAIEGSRFYTSDPSQACLFVLSLD 159 KN KVY+Y +K 1+ Y +L A l S +YT D ++ACLFV S+D EXT 2:NPKNKIKVYIYALKKYVDDFGVSVSNTISREYNELLMAISDSDYYTDDINRACLFVPSID 158 EXT 1: TLDRDQLSPQYVHNLRSKVQSLHLWNNGRNHLIFNLYSGTWPDYTEDVGFDIGQAMLAKA 219 L+++ L + + L W+ G NHL+FN+ G PDY + +A+LA EXT 2: VLNQNTLR IKETAQAMAQLSRWDRGTNHLLFNMLPGGPPDYNTALDVPRDRALLAGG 215 EXT 1: SISTENFRPNFDVSIPLFSK DHPRTGGERGFLKFNTIPPLRKYMLVFKGKRYLTG 274 ST +R +DVSIP++S D P 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 628 + T+ E+ F + VW+ FP+R+VGYP R H WD+ +W Y S+WTN+ SMVLTGAA EXT 2: MLTSDELQFGYEVWREFPDRLVGYPGRLHLWDHEMNKWKYESEWTNEVSMVLTGAAFYHK 602 EXT 1: XXXXXXXXXXPASLKNMVDQLANCEDILMNFLVSAVTKLPPIKVTQKKQYKETMMGQTSR 688 P +KN VD NCEDI MNFLV+ VT IKVT +K++K EXT 2: YFNYLYTYKMPGDIKNWVDAHMNCEDIAMNFLVANVTGKAVIKVTPRKKFKCPECTAIDG 662 EXT 1: ASRWADPDHFAQRQSCMNTFASWFGYMPLIHSQMRLDPVLFKDQVSILRKKYRDIERL 74 6 S D H +R C+N FAS FG MPL + R DPVL+KD K + +1 L EXT 2: LS—LDQTHMVERSECINKFASVFGTMPLKWEHRADPVLYKDDFPEKLKSFPN1GSL 718 Figure 1.9 Alignment of EXT 1 and EXT 2 genes. Identical amino acids are outlined in boxes. EXT 1 sequence from NCBI 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 Physio logic Func t ion 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 products and funct ion 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 c D N A change" E x o n Protein Change T y p e Reference 1 42delG 1 G15 FS Francannet et al . , 2001 2 7 9 C ^ A 1 Q 2 7 K M S D. Zaletayev, unpublished 3 118delC 1 F S H 4 0 F S Raskind et al. , 1998 4 174-176delC 1 F S P59 F S Philippe et al., 1997 5 2 0 4 G - » A 1 W 6 8 X N S Wuyts etal . , 1998 6 242-247insC 1 F S R 8 3 FS Wells etal . , 1997 7 248insC 1 R83 FS Francannet et al . , 2001 8 248-249delG 1 F S Q84 FS Wells etal . , 1997 9 2 5 0 C ^ T 1 Q 8 4 X N S Francannet et al . , 2001 10 3 3 1 A - » T 1 K 1 1 0 X N S X u et al. , 1999 11 352insC 1 V118 FS Francannet et al . , 2001 12 3 5 7 C - » A 1 Y 1 9 9 X N S Raskind, et al. , 1998 13 3 5 7 C - > G 1 N S Alvarez et a l . , 2003 14 388de lAG 1 F A S130 F S D . Zaletayev, unpublished 15 420ins4 1 F S S 1 4 1 FS Hecht etal . , 1997 16 456delC 1 F S L 1 5 3 F S D . Zaletayev, unpublished 17 458delTC 1 L153 FS Francannet et al . , 2001 18 460del2T 1 F154 FS Francannet et al . , 2001 19 477delTA 1 D160 FS Francannet et al . , 2001 20 490G->C 1 D146H M S Bovee etal . , 1999 21 515delA 1 H I 72 FS Francannet et al . , 2001 22 527del8 1 F S K 1 7 7 F S Hecht etal . , 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 W 2 0 0 X N S Wuyts etal . , 1998 26 600G->A 1 W 2 0 0 X N S Wuyts etal . , 1998 27 624ins5 1 F S F209 FS Wuyts etal . , 1998 28 651-664dell4 1 F S L 2 1 6 F S Seki etal . , 2001 29 679delC 1 R227 F S Francannet et al . , 2001 30 6 7 9 C - » T 1 R 2 2 7 X N S Seki et al. , 2001 31 703dell5 1 P L F S K d e l 5 A A d e l Bovee etal . , 1999 32 712delT 1 S238 FS Francannet et al . , 2001 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 F S G274 FS Seki etal . , 2001 36 8 3 8 A - » G 1 R280G M S Wuyts et al . , 1998, Raskind et al., 1998 37 840G->C 1 R280S M S Raskind et al., 1998 38 876-877insT 1 F S V292 FS D. Zaletayev, unpublished 39 943delGA 1 FS D315 M S D . Zaletayev, unpublished 40 947A->G 1 N316S M S Bovee etal . , 1999 41 1 0 1 6 G - » A 2 G339D M S Philippe et al., 1997 42 1018C->T 2 R340C M S Philippe et al., 1997 43 1 0 1 8 C ^ A 2 R340S M S Wuyts etal . , 1998 44 1 0 1 9 G ^ T 2 R340L M S Hecht et al . , 1997; Seki et al . , 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, SS - 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 cDNA change* Exon Protein Change Type Reference 45 1 0 1 9 G - » A 2 R 3 4 0 H M S Raskind et al. , 1998 (2 families); Sekit et al., 2001; A l v a r e z et a l . , 2003 46 1035- 1056+2del24 2 F S F345 SS Seki et al . , 2001 47 1 0 5 6 + G - » A Intron 2 SS Wells et al . , 1997 48 1091-1093delG 3 F S E365 F S Raskind et al. , 1998 49 1 1 2 2 G - » A 3 W 3 7 4 X N S Philippe et al. , 1997 50 1157T-KJ 3 L 3 8 6 X N S Seki et al . , 2001 51 1198-1199insA 4 FS D339 FS Seki et al . , 2001 52 1203-1204deIC 4 FS L402 F S Raskind et al . , 1998 53 1213-1216del4 4 423STOP F S Gigante et al. , 2001 53 1215del4 4 F S R405 F S Raskind et al. , 1998 (2 families)? 54 1215-1218del4 4 F S R405 F S Seki et al . , 2001 55 1370delT 4 T424 FS Francannet et al . , 2001 56 1320insT 5 441 S T O P FS Gigante et al. , 2001 57 1333-1334insG 5 F S N 4 4 6 FS Seki etal . , 2001 58 1376C->G 5 S459X N S Wuyts etal . , 1998 59 1409dell0 5 SS Park etal . , 1999 60 1 4 1 7 + l G ^ A Intron 5 SS Philippe et al. , 1997 61 1417+2del6 Intron 5 SS Wuyts etal . , 1998 62 1426-1431insC 6 FS S478 F S Hecht et al . , 1997, Raskind et al. , 1998 63 1431insT 6 F S S478 F S Wells etal . , 1997 64 1457C->T 6 A 4 8 6 V M S X u e t a l . , 1999 65 1468-1469insC 6 F S L 4 9 0 F S Seki et al . , 2001 66 1469delT 6 F S L490 F S 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 67 1474-1475delTC 6 F S L492 F S Seki etal . . 2001 68 1487C->T 6 P496L M S X u etal. . 1999 69 1568delT 7 L523 F S Francannet et al . , 2001 70 1642delA 8 621 S T O P F S Gigante et al. , 2001 71 1642delA 8 S548 FS Francannet et al . , 2001 72 1679-1680insC 8 F S V561 F S Wuyts etal . , 1998 73 1723G->C 8 SS Alvarez et al., 2003 74 1 7 4 5 G - » A 9 W 5 8 2 X N S Francannet et al . , 2001 75 1 7 4 4 G - » A 9 W 5 8 2 X N S Francannet et al . , 2001 76 1773delG 9 G591 F S Francannet et al . , 2001 77 I 7 7 6 C - » A 9 Y 5 9 2 X N S Francannet et al . , 2001 78 1784delGC 9 R595 F S Francannet et al., 2001 79 1797G->A 9 W 5 5 9 X N S Seki et al , 2001 80 1817G->A 9 W 6 0 6 X N S Wells etal . , 1997 81 1878del3 9 H627del 1 A A deletion Raskind et al. , 1998 82 1883+2T-»G 9 SS Seki etal . , 2001 83 1980delG 10 664STOP F S Gigante et al. , 2001 84 2053C->T 10 Q 6 8 5 X N S Raskind et al . , 1998 85 2 1 0 1 C - » T 11 R 7 0 1 X N S Seki e ta 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, FS - 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 cDNA change* Exon Protein Change Type Reference 1 67C->T 2 Q23X NS Wuyts et al., 1998 2 77-78insT 2 FSY26 FS Philippe et al., 1997 3 233delC 2 FS P78 FS Sekietal., 2001 4 239-244insG 2 FS G81 FS Raskind et al., unpublished 5 253T->C 2 C85R MS Parketal., 1999 6 302del56 2 FS K101 FS Raskind et al., unpublished 7 313A-»T 2 K105X NS Xuetal., 1999 8 315-316insG 2 FS VI06 FS Xu et al., 1999 9 319insGT 2 FSC107 FS Xuetal., 1999 10 374-443del70 2 FS 1126 FS Seki et al., 2001 11 449del4 2 FS A150 FS Stickens etal, 1996 12 455T-»G 2 L152R MS Xuetal., 1999 13 455del4 2 FS Alvarez et al., 2003 14 495delG 2 FSL165 FS Xuetal., 1999 15 514C-»T 2 Q172X NS Wuyts et al., 1998; Wuyts etal., 1996; Xuetal., 1999 16 537G->C 2 R180T MS Francannet et al., 2001 17 537-lG->A Intron 2 SS Seki et al., 2001 18 580G^T 3 G193X NS Francannet et al., 2001 19 605C^T 3 A202V MS Sekietal., 2001 20 624deIC 3 D208 FS Francannet et al., 2001 21 627-2A^G Intron 3 3' Splice Junction Gigante et al., 2001 22 629-63 linsC 4 FSL211 FS Xuetal., 1999 23 649-652delT 4 FSS218 FS Wuyts et al., 1998 24 666C-»G 4 Y222X NS Philippe et al., 1997 25 679G->A 4 D227N MS Philippet et al., 1997 (2 families); Alvarez et al., 2003 26 730G->T 4 NS Alvarez et al., 2003 27 751C->T 5 NS Alvarez et aL, 2003 28 772C->T 5 Q258X NS Francannet et al., 2001 29 812-814delC 5 FS A271 FS Wuyts etal., 1998 30 1079+G->T Intron 6 FS Q313 SS Wolf et al., 1998 31 1079+G^C Intron 6 SS Sekietal, 2001 32 1104insGA 7 E368 FS Francannet et al., 2001 33 1132C-»T 7 Q378X NS Raskind et al., unpublished 34 1139T-»C 7 I380T Gigante et al., 2001 35 1173+G-»A Intron 7 FS R360 SS Wuyts et al., 1998 (2 families); Wuyts etal., 1996 36 1173+G->T Intron 7 FS R360 SS Wuyts etal., 1998 37 1174G->A Intron 7 SS Alvarez et al., 2003 38 1188G->A 8 W396X NS Xuetal., 1999 39 1201C->T 8 Q401X NS Philippe et al., 1997; Xu et al., 1999 40 1234C-»T 8 Q412X NS Xu et al., (3 families) 41 1257T-»A 8 Y419X NS Francannet et al., 2001 42 1263 ins AT 8 FS A422 FS Wuyts etal., 1998 43 1669delC 11 FS R557 FS Seki et al., 2001 44 1726G-»A 11 E576K 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: MS - missense, NS - nonsense, FS - frameshift, SS - splice site; Blue font indicate missense or non-truncating mutations. 26 K-OKHZ EX-±Z+e88t OSU!69tl--99t'l Mr 0 6 101 3<-09Zei V<-9ZZU o w o H 3 R I — J * * 5 a CO a .2 2 OJ a u CO 09 11 V0I°PCT6 1 0I3PEU- suapeoz—I 1SUI/28-9Z9 0<-DC*9 9«-V8E9 9 9 I S P L 2 8 - 0 Z 9 maPS99-l99 V<-O009 V<-0669- OI6P16S-06S- ±9PP 6*9- VI»P 919—1 1Z l»P 09* 01I8P 99* VW88E 3SU3SU0N i « - v i e e 1 0« U ! 9 t z 0|8P9Zl-*Zt 0I8P8LI OiepZ* —• *su|0Z* W9Q* w<-ozse V1I3P i i * 0«-306» W-0 09Z 9|Bp6*Z-9*i V<-EI*0Z 27 Osui.£t-z-z*z aiisaonds V^SPIU asuasuoN asuasuoN it-sou ±|3PZ99"6t'9 _LVSU!£92i 0̂ -0999 . CO a o H fc w ' i •= i •a 2 o *•» c o c W O 2 « B> co (/) C CU CO CO CD CO O -S c " e « ,o 3 <u OJD J3 S 3 oisps6t> SJ ±<-099* O ^ I S S * I— OZiaPEŴZE 13SU!6t£ 0<-lE9Z osuitt-z-eez 3ieP££3 " I ~ l 28 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) Rat ing M o t i o n (% Strength Pa in Act iv i ty Deformity Total Motion of Normal Joint) Bowing of forearm Shortening of Forearm (cm) Varus - Valgus Angulat of Knee n Shortening of limb (cm) Excel . >90 5/5 None (no medication) N o restrict None None 0-5 None Good 6 0 - 9 0 4/5 M i d (medication occasionally) Restric. in recreational activities M i l d <1 6-10 <1 Fair 30 - <60 3/5 M o d . (medication weekly) Partial disability M o d . 1-2 11-20 1-3 Poor <30 1-2/5 Severe (narcotics or other medication daily) Total disability Severe >2 >20 >3 1.2.5.2 Por te r ' s F ind ings 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 Genotype-Phenotype Corre lat ions 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 developed from the 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 10th percentile was 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 and from the 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 • Informed consent obtained I Subject Identification • Pedigree delineated • Recruitment of family members I I Genotype • • D N A extraction from blood samples from al l participants Gene Assignment •Highly polymorphic repeats •Family members run together with controls •Assignment criteria (see section 2.4.3.1) M u t a t i o n Identification Phenotype f _L C l i n i c a l •Lesion count • L i m b alignment •L imb segments •Height (percentile) •Weight •Range o f motion R a d i o g r a p h i c •Lesion quality • L i m b alignment • E X T 1 or 2 amplified for each proband • E X T 1 18 primer pairs (11 exons) • E X T 2 16 primer pairs (14 exons) D N A Sequencing •Mutation identification •Confirmation o f true mutation (literature review, controls, type o f mutation, change in m R N A ) f Segregation Analys is •Sequence exons o f all affected and available family members Genotype Defined Phenotype Defined Genotype - Phenotype Analysis • Gene vs. Phenotype • Gender vs. Phenotype • Mutat ion 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 Corre lat ions 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 blood 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 re- suspended; 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 Tris- EDTA 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 - 2 D11S1355 D11S903 D19S216 D19S221 D19S226 D11S5547 12 D11S1319 D11S1313 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 EXT 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 (with 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 (po lyacry lamide 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 dH20, 500pl ammonium perphospate(APS), 50pl Temed). The 0.4mm 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 an 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 Visual izat ion 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 Exclus ion Analys is 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 MgCl2), 200pM dNTP, 0.5uM primer (see 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 Primer Name Exon Sequence Length (bp) Temp (°C) 1-1 E X T 1-ex l a E X T l - e x l b 1 C A G G C G G G A A G A T G G C G G A C T G G C 7 / C C G G C T G 7 / G G C T CCTCGATGCCC 212 58 1-2 E X T l - e x l c E X T l - e x l d 1 T G C T C T C A G C T G G C T C T T G T C T C G G A A T C C T C G T T T T C C A A T T G A T C C C 201 55 1-3 E X T l - e x l e E X T 1-ex If 1 C G G A G C C T C T G C G C C C C T T C G T T C C C r a G ^ 4 r G 7 T TTGGTAACTTTCGGCG 232 55 1-4 E X T l - e x l g E X T l - e x l h 1 C G T A T A C C C A C A G C A A A A A G G G G C 4 7 T G 7 T C C 4 C AAGTGGAGACTCTCG 209 55 1-5 E X T l - e x l i E X T l - e x l f 2 1 C C A G T T G T C A C C T C A G T A T G T G C G G C 7 T 7 U G C C 4 GCATCGCCAGG 168 55 1-6 E X T l - e x l k E X T 1-ex 11 1 CCTGACTACACCG AGG ACGGGTGTCTGA TCCTA T CCCTG 237 55 1-7 E X T l - e x l m E X T l - e x l j 1 GGTATTCAAGGGGAAGAGGT ACggaccaaggCCgg cagagccc 231 55 1-8 EXTl-ex2a EXTl-ex2b 2 ccccacattcgcaatgagtcgagaggtgataatgttaaaccc 225 55 1-9 EXTl-ex3a EXTl-ex3b 3 cgatAXggaacagcttcgXcXggacgggggcagcaataatctgc 224 55 1-10 EXTl-ex4a EXTl-ex4b 4 gtgcattctctttgttttacagctgagagaagtgtataaagg 239 55 1-11 EXTl-ex5a EXTl-ex5b 5 cctttccaaatatcatcaggcatcttcagggtaaacaagggc 237 55 1-12 EXTl-ex5a EXTl-ex5c 5 cctttccaaatatcatcaggccattttgcaatgctctgctctg 237 55 1-13 EXTl-ex6a EXTl-ex6b 6 gcmccagcgcttcattaggcctggagctggagcaggcagggg 210 55 1-14 EXTl-ex7a EXTl-ex7b 7 ggcgtacataaatacatcctaccccccaaggctccacagtggttcc 189 56 1-15 EXTl-ex8a EXTl-ex8b 8 caagactctgaagttacctctttcccggtgactgcctgaacagcccaacc 204 58 1-16 EXTl-ex9a EXTl-ex9b 9 cattgttgattgcttgtttggccgtaaagtctgtaagagacatgtcc 235 55 1-17 E X T 1-ex 10a E X T 1-ex 10b 10 cttgtcatcatgigataatggcccgagtgaagcaaggaagaggg 259 55 1-18 E X T l - e x l l a E X T l - e x l l b 11 ccttgcacttctctcatattatccCCTCAAAGTCGCTCAATGTCTC GG 230 55 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 MgCl2; Accession Number: U67356-U67368 (Wuyts 1998) 50 Table 2.2 Primer pair sequences used for E X T 2 Primer Pair Primer Name Exon Sequence Length (bp) Temp C Q 2-1 EXT2-ex2a EXT2-ex2A8 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 338 56 2-2 EXT2-ex2A26 EXT2-ex2A25 2 GACAGTCCCATCCCAGAGCGGGGAGGGAACAA AACAGACAGG 249 56 2-3 EXT2-ex2A4 EXT2-ex2b 2 ACTACACTGATGACAlCAACCGccctttagttCCCtg agggcc 176 55 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 E X T 2 - e x l l a E X T 2 - e x l l b 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 re- centrifuged. 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 obtained from available films; the data collected included lesion quality (count, size sidedness, complexity, location, and metaphyseal flaring) 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 Les ion 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 al ignment 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 and L i m b Lengths 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 Les ion qual i ty 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. Lesion size was calculated and ranked. First the protrusion ratio (A) was obtained by dividing the protrusion distance o f the lesion (bony stalk) (a) by the native bone width (b). The height ratio (B) was obtained by dividing lesion height (bony cap long axis) (c) by (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), > 75% (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 Size < 25% 1 small 26 - 50% 2 medium 51-75% 3 medium >75% 4 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 al 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 a l i g n m e n t 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 +/- 2mm (Keats 1990) The ulnar displacement in 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 jo ining 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) The distance between the distal surface o f the ulna and the radius. 2.5.2.2.4 R a d i a l bowing - expected normal value equals 10 +/- 5° (Green 1993) The angle subtended between the long mid-axis o f the forearm ( A - B ) and the maximal radial deviated point o f the radius' diaphysis (C - D ) . F i g u r e 2.5 Measurement o f radial inclination and ulnar shortening F i g u r e 2.6 Measurement o f radial bowing 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) . Figure 2.7 Radial head subluxation / dislocation 2.5.2.2.6 E l b o w joint angle - normal range equals females 10+/-2 0 valgus, males 8+/-2 0 valgus (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 Femoro- t ib ia l anatomic 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 2.5.2.2.9 Femoro- t ib ia l mechanical angle - normal value equals 0 +/- 5° (Hsu et al. 1990) The angle subtended by 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 ) . Weight-bear ing axis - normal equals 50 +/- 10% (Hsu e ta 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 weight- bearing axis is where this line crosses the knee joint and is expressed as a percentage o f the total t ibial joint surface. The distance in millimetres from the lateral tibial-joint-line border to the weight-bearing line is divided by the total joint width and expressed as a percentage. Numbers greater than 50% are in varus and those less than 50% are in valgus. 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. 2.5.2.2.10 F e m o r a l neck/shaft angle - normal equals 135 +/- 5° (Pettersson and Ringertz 1991) The angle subtended by 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 2.5.2.2.11 S h a r p ' s Ace tabular 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 jo ining the tip to the lateral edge o f the acetabulum ( A - B ) . F i g u r e 2.11 Measurement o f Sharp's Acetabular angle. 2.5.2.2.12 F i b u l a r height - 50 +/- 10% (described in this study) Expressed as a percentage o f the distance from the proximal tibial joint line to the proximal tip o f the fibula ( A ) over the distance from the proximal tibial joint line to the proximal 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 joint a n g l e - n o r m a l range equals 0 + / - 5 ° valgus (Hsu e ta 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 1500th base pair or late after the 1500th basepair in either EXT 1 or 2. 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 I D Pos i t ion Affected B l o o d I D Pos i t ion Affected B l o o d Family 1 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 G M no yes 5-4 S no yes Family 2 5-5 G M no yes 2-1 P yes yes 5-6 no yes 2-2 B yes yes Family 6 2-3 S no yes 6-1 P yes yes 2-4 F yes yes 6-2 step B yes yes 2-5 M no yes 6-3 M yes yes 2-6 G M no yes 6-4 F 6 - 2 no yes 2-7 G F no yes 6-5 F 6 - 1 no Family 3 Family 7 3-1 G M yes yes 7-1 P yes yes 3-2 P yes yes 7-2 M yes yes 3-3 S no yes 7-3 G F 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 G M no yes 3-7 step S no yes Family 8 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 Family 16 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 G M yes yes 3-18 M yes yes Family 17 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 G F yes yes 3-24 S no no 17-6 G M no yes Family 4 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; B- brother; 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 Summary of STR Markers as per family and EXT gene assignment for mutations identified in EXT 1 and EXT 2 Family Exclusion Analysis Mutation Gene Location of EXT1 EXT 2 Found Sequenced Mutation 1 NI NI Yes EXT 1 EXT 1 exon 2 16 NI NI Yes EXT 1 EXT 1 exon 8 18 No Yes Yes EXT 1 EXT 1 exon 1 6 No No No EXT 1 and EXT 2 None found 2 Yes No Yes EXT 2 EXT 2 exon 4 5 Yes No Yes EXT 2 EXT 2 exon 4 17 Yes No Yes EXT 2 EXT 2 exon 2 8 No No Yes EXT 2 EXT 2 exon 7 4 Yes No No EXT 1 and EXT 2 None found 3 NI NI Yes EXT 1 and EXT 2 EXT 2 exon 5 69 ID: 1-3 ID: 1-2 EXT1 D8S555 1, 3 EXT 2 D11S903 c, c o 2, 4 a, b ID: 1-1 EXT1 D8S555 1, 4 EXT 2 D11S903 c, a Exclusion analysis: not informative (NI) Figure 3.1a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 1 1-1 proband affected 1-2 mother unaffected 1-3 father affected 62.1-1 • 1.SB #34 of 170 - Figure 3.1b - Sequencer output for segregation analysis for Family 1. Mutation location: EXT 1 exon 2. 70 ID: 16-5 E X T 1 D8S555 1, 3 EXT 2 D11S903 b, c ID: 16-2 EXT 1 D8S555 2, EXT 2 D11S90 c, ID: 16-1 E X T 1 D8S555 2, 1 EXT 2 D11S90 c, ID: 16-3 o 1, 4 c, a ID: 16.4 0 1, ? c, Exclusion analysis: not informative (NI) Figure 3.2a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 16. 16-1 proband affected 16-1 control unaffected 16-4 sister unaffected 16-5 - 3 3 . 1 6 - 1 - 1 . 1 S A 0 1 3 O o f 161 « C A A C A G A G f B l T A A G A A C C C A C A R C R C" R G B T A A" G A A C C A A C A A C A G A R f d T A A G A A C C C i H C H_ R C R C L R T l fl B G A A C C C l • 0 8 . 1 6 S - 1 . 1 5 A #85 o f 2G8 - A A C A A C A G A R B I T W A G A A C C C A A R C A R C R G A R N I UJ R G H fl C C C R Figure 3.2b - Sequencer output for segregation analysis for Family 16. Mutation location: EXT 1 exon 8 71 ID: 18-2 ID: 18-3 EXT1 D8S555 I, 2 EXT 2 D11S90 a, d 6 o 2, 3 c, c IDID848-1 Cannot exclude EXT 1 because affected father passed marker 2 to the affected child and marker 1 to the unaffected child. Exclude EXT 2 because marker a was passed to an affected and an unaffected child. E X T l D8S555 2, 3 1, 2 EXT2 D11S90 a, c a, c Figure 3.3a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 18. 18-1 proband affected 18-2 father affected 18-4 brother unaffected 18-3 mother unaffected 28.18.1 -MAKWofM 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 HETETRCBTH TBS C C f l C H C C f i R HH RC C C C H G H H H f C A A A G T C T A C G T A T A C C H A C A E C A A A A A G G G G A C I i i n c u e { I I I I H fit C l S M M - U H B M l n ' - - • . " C y A [ •_ 57.18-3-mm of 1 « C A A A G T C T A C G T A T A C C • A C A G C A A A A A G G G G A . i T t i n 1111 f t J T l 111 f I 11 1 1 i i 1 1 1 Figure 3.3b- Sequencer output for segregation analysis for Family 18. Mutation location: EXT 1 exon 1 72 ID: 6-3 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 ID: 6-2 ID: 6-4 Exclusion analysis: not informative (NI) 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 D11S903 2, D11S905 1, D11S903 3, ID: 6-1 ID: 6-4 ID: 6-2 Exclusion analysis: not informative (NI) D11S903 2, 4 D11S905 1, 2 D11S903 3, 3 1, 4 3, 3 2, 3 Figure 3.4 a(ii) EXT 2 STR Markers. Pedigree for Family 6. 73 ID: 2-4 EXT1 D8S555 1, 3 EXT 2 D11S903 a, b ID: 2-3 EXT 1 D8S555 3, 2 EXT 2 D11S903 a, d I, c, ID: 2-1 ID: 2-5 ID: 2-2 1, 2 b, c Exclude EXT 1 because marker 3 from the affected father was passed to an affected and an unaffected child. Cannot exclude EXT 2 because marker b from the affected father was only passed onto both affected children. Figure 3.5a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 2. 2-3 sister unaffected 2-1 proband 2-4 father affected 2-2 brother affected 2-5 mother unaffected - 2-SA #133 of 163 • A G G T G G A T C T T C r A B l A E A A A G G A C C A G G 6 T A A R C G I C G H C C C f l T R G f l f l R G C R C C R G G G T R R • 47.2-2 - 2.5A #132 of 163 • I T G G A T C T T C f ' f A G A A A G G A C C A G G I ~ G G R T T T ^ f T C C R ! f f G R H fl G G R C C~R G G i Figure 3.5b - Sequencer output for segregation analysis for Family 2. Mutation location: EXT 2 exon 4. 74 IE): 5-1 EXT 1 D8S555 2, 2 EXT 2 D11S903 a, d ID: 5-3 EXT1 D8S555 2, 1 EXT 2 D11S903 d, a I, a- ID: 5-2 ID: 5-4 2, a. Can exclude EXT 1 because the affected mother passed marker 2 to an unaffected and an affected child. Cannot exclude EXT 2 because the affected mother passed an undistinguishable marker a to an unaffected and an affected child. Figure 3.6a - EXT 1 and EXT 2 STR Marker. Pedigree for Family 5. 5-2 mother unaffected 5-3 proband affected 5-1 father affected 5-3 proband affected reverse read 5-4 sister unaffected 40 5-2 2 5B #81 of 181 T s T • • A T G C A A G G • 37.5-1 - 2.5A #71 of 155 « C T A 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 ( J T C A G C A T T C C G G C R R G C C T H C N R T G T C f l G C R T T C I Figure 3.6b - Sequencer output for segregation analysis for Family 5. Mutation location: EXT 2 exon 4. 75 ID: 17-5 ID: 17-6 EXT 1 D8S555 1, 4 EXT 2 D11S903 d, d EXT1 D8S555 3, 1 EXT 2 D11S903 a, d o 1, 2 b, d ID: 17-2 EXT 1 D8S555 4, 1 EXT 2 D11S903 d, b ID: 17-1 ,—I—, ID: 17-3 ID: 17-4 1, 4 c, b 1, 1 c, b 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. 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 ID: 8-2 EXT 1 D8S555 I, 2 EXT 2 D11S903 b, b Exclusion analysis: not informative (NI) ID: 8-1 EXT1 D8S555 1, 1 EXT 2 DUS903 b, a 2, b. ID: 8-3 Figure 3.8a - EXT 1 and EXT 2 STR Markers. Pedigree for Family 8. 8-1 proband affected 8-3 brother unaffected 8-2 mother affected 8-4 father unaffected A T G C A G A •33.8-1 -2.8A#191 of 251 • G A C A G B T A A G A G G 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 • 34.8-2 - 2.8B #106 of 144 • A C A G l M T A A G A G 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 D8S555 1 3 2 3 D8S592 1 5 4 6 65CA 1 5 1 8 46 1 3 1 2 D8S522 9 8 6 4 D8S85 D8S547 D8S555 D8S592 65CA 46 D8S522 ID: 4-3 o ID: 4-4 Figure 3.9a (i) EXT 1 STR Markers. Pedigree for Family 4. ID: 4-2 ID: 4-1 D11S903 2 D11S905 1 D11S1313 1 D11S903 2 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 D11S903 marker 1 from the affected mother was passed to an affected child while marker 3 was passed to an unaffected child. ID: 4-3 D11S903 2 D11S905 1 D11S1313 1 D11S903 2 ID: 4-4 Figure 3.9a (ii) EXT 2 STR Markers. Pedigree for Family 4. 78 o • • N "a -o . — -a f ro o • o — u •a l-H "O •o ro "a I Vi f-i 0 •a H CN H X W T3 bo • • o •O <N bo H X W « OS s- 00 00 Q s ro xo wo On 00 ~, 00 ~ - i Q Q ro wo Ov wo Co CO 00 ~» Q Q 79 3-21 Aunt unaffected 3-8 Mother affected 3-3 cousin unaffected 3-6 proband affected 3-15 cousin affected 3-7 cousin unaffected 3-21 24af»o>232 • 3 * 2 -6» »13 o t2*0 H H W W f f f H f i H iii 11 H I t Hf £1 Iff t f H H 111 til H H 1 • 3-6 2-6a *28 of 233 • tt 1 i Hit i*ttl t i l 1 til iii 11 ill • 3-15 2 « a »17 of 221 • ' T 6 T A 6 T C N C G B l A A T A t T T C C T C 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 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 Amino Acid Change Type Unique 1 1 G1019A 2 RtoH Arginine to Histidine Basic to Basic Missense No 2 2 G730T 4 EtoX Glutamic acid to Stop Nonsense Yes 3 2 C751T 5 QtoX Glutamine to Stop Nonsense Yes 4 2 ? - - - - 5 2 G679A 4 D to N Aspartic acid to Asparagines Acidic to uncharged polar Missense No 6 1 9 - - - Yes 8 2 G1174A 7 - Splice Site Yes 16 1 G1723C 8 - Splice Site Yes 17 2 455del4 2 Premature Stop at 1293 Frameshift Yes 18 1 C357G 1 YtoX Tyrosine to Stop Nonsense 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 Feature N u m b e r of subjects Distr ibut ion of ages at time of study F a m i l y 4 F a m i l y 6 E X T 1 7 9, 14, 14,44,47, 55, 72 - 3 E X T 2 9 7, 7, 10, 11, 14, 14, 15, 15, 16,31,36, 38, 39, 44, 45,46,47, 70, 74 3 M a l e 14 10, 11, 14, 14, 14, 15, 15, 39,44,44, 45, 47, 55, 73 - 2 Female 12 7, 7, 9, 14, 16, 31, 36, 38, 46,47, 70, 72 3 1 M S 4 7, 9, 39, 47 N S 14 7, 10, 14, 14, 14, 15, 15, 36, 38, 44,46, 47, 55, 70 F S 3 16,45, 73 SS 5 11, 14,31,44, 72 M i l d 4 7, 9, 39, 47 Severe 22 7, 10, 11, 14, 14, 14, 14, 15, 15, 16,31, 36, 38, 44, 44,45, 46, 47, 55, 70, 72, 73 E a r l y 19 7, 7, 10, 14, 14, 14, 15, 15, 16, 36, 38, 39, 44, 45, 46, 47, 55, 70, 73 Late 7 9, 11, 14,31,44,47, 72 E X T 1 M a l e 4 14,44, 47, 55 E X T 1 Female 3 9, 14, 72 E X T 1 Severe 2 14, 14, 44, 55, 72 E X T 1 M i l d 5 9, 47 E X T 1 M S 2 9, 47 E X T 1 SS 3 14, 44, 72 E X T 1 N S 2 14, 55 E X T 2 M a l e 10 10, 11, 14, 14, 15, 15,39,44,45, 73 E X T 2 Female 9 7, 7, 16,31,36,38,46, 47, 70, E X T 2 Severe 17 7, 10, 11, 14, 14, 15, 15, 16,31,36,38, 44, 45, 46, 47, 70, 73 E X T 2 M i l d 2 7,39 E X T 2 M S 2 7,39 E X T 2 SS 2 11,31 E X T 2 N S 12 7, 10, 14, 14, 14, 15, 15, 36, 38, 46, 47, 70 Males severe 12 10, 11, 14, 14, 14, 15, 15, 44, 44, 45, 55, 73 Males mi ld 2 39, 47 Males M S 2 39, 47 Males N S 8 10, 14, 14, 14, 15, 15,44,55 Males SS 2 11,44 Males F S 2 45, 73 Females severe 10 7, 14, 16, 31, 36, 38, 46,47, 70,72 Females mi ld 2 7,9 Females M S 2 7,9 Females N S 6 7, 36, 38, 46,47, 70 Females SS 3 14,31,72 Females F S 1 16 84 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 non- truncating (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 EXT 1 and EXT 2 Genes E X T 1 vs. E X T 2 Comparisons # lesions % Pelvic % Flatbone % Flared Limb Alignment % Height E X T 1 vs. E X T 2 (Appendix 8.7.1) 1>2 p < 0.01 power .82 1>2 p < 0.01 power .68 1 >2 p<0.01 power .91 n/s 1>2 (17 vs 10) n/s 1<2 p < 0.01 power .8 Gene and Gender (Appendix 8.7.6) 1 M > 1 F > 2 M > 2 F p < 0.01 gene and gender 1 M > 1 F > 2 M > 2 F p<0.01 gene 1 M > 2 M > 1 F > 2 F n/s 1 M > 2 M > 1 F > 2 F p < 0.02 gender 1 M > 2 M > 1 F > 2 F (16 > 13 > 10 > 8) 1 F < 1 M < 2 M < 2 F p < 0 . 0 1 gene Gene and Mutation Type (Appendix 8.7.7) I N S > ISS > 1 N S > 2 N S > 2SS > 2 M S > 2FS n/s 1 N S > 1 M S > 1 S S > 2 S S > 2 N S > 2 M S > 2FS n/s 1 N S > I M S > ISS > 2 S S > 2 N S > 2 M S > 2FS n/s 1 N S > 1 S S > 2 M S > (15) (14) (14) 2FS > I M S > 2 N S > (12) (11) (9) 2SS (9) 1 N S > 1 M S > 2 F S > ISS > 2 S S > 2 M S > 2 N S n/s Gene and Severity (Appendix 8.7.8) 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) 1M< IS < 2M<2S n/s Gene and Mutation location (Appendix 8.7.11) 1 E > 2 E p< 0.0021 power .95 1 E > 1L 1 E > 2 E p < 0.001 power .99 1 E > 1L 2 E < 2 L 1 E > 2 E p < 0.001 power .99 1 E > 1L 2 E < 2 L 1 E > 1L 1 E = 1 L > 2 E > 2 L (15 = 15 > 13 > 10) 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 3.0%> (p- value 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, 40th percentile and EXT 2 females at the 45th percentile. This was significant for gene (p- 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 (p- values 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 3rd percentile, followed by EXT 1 late mutations at the 11.8th percentile, then EXT 2 late mutations at the 25th percentile and finally EXT 2 early mutations at the 44.6th percentile. Table 3.6 Summary of Results for remaining unvariant data Compar i sons # lesions % Pelvic % Flatbone % F l a r e d L i m b Al ignment % Height Males vs. Females (Appendix 8.7.2) M > F p < 0.01 F > M (12 vs 9) n/s M S vs. N S vs. SS vs. F S (Appendix 8.7.3) M S > SS = F S > N S (13 > 12= 12> 11) n/s F S < M S < SS < N S p<0.01 Severe vs. M i l d (Appendix 8.7.4) Severe = M i l d (11 vs 11) n/s M i l d < Severe n/s Early vs. Late (Appendix 8.7.5) Early = M i l d (11 vs 11) n/s Late < Ear ly 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 9.7th percentile and nonsense mutation subjects, represented by 3 families, were the tallest, 51.3rd percentile (p-value 0.048). 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 M a l e and Female Compar i sons # lesions % Pelvic % Flatbone % F l a r e d L i m b Al ignment % Height 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 > F s (10) (9) M m smaller than the rest n/s Gender and Mutation Type (Appendix 8.7.10) M ns > F ns > M ss > M ns > F ss > F fs > M fs > F ms > F ns M ns > F ns > M ns > M ss > M ms > M fs > F ms > F ns > F ss > F fs n/s F fs > M ss > (16) (14) F ss > M fs > (13) (12) M ms > F ms > (11) (11) M ns > F ns (9) (9) F fs < M fs < M ms < F ss < F ms < M ss < M ns < F ns 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 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, 10th percentile. 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 10th percentile, the females with splice site, 12th percentile, and the rest were greater than the 30th percentile. 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 (21st versus 27th percentile average for the others). No other features of the 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 hetero- oligomeric 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 metaphyseal flaring), 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 39th 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. The first method looks at the data from a 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 mal- alignment 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 twenty- four abnormal alignments with the range between nine and eighteen. The severity of the mal- alignment 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 27th percentile (33rd if including family 4 and 6). The range however was from the 3rd (5 subjects, 3 males and 2 females) to greater than the 85th (3 subjects, 1 male and 2 females) percentile. Short stature is defined as less than or equal to the 3rd percentile, therefore rather than classifying HME 108 as a pathologic short stature or dwarfism it would better be described as having a propensity for stature below the 30th percentile, i.e. growth impedance. 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 37th percentile. In the lower extremity they were both below the 51st percentile. In this regard, with respect to these 32 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 mal- alignment 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 less fit a normal distribution. Percentile height was 48.6 for the family with a range between eighteen and ninety (males 43rd, females 51st). Limb segment shortening was in 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 50th percentile, some are greater than the 85th percentile and none are below the 3rd percentile in these studies. On balance HME does not 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 CHILDREN'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 in the research procedures. Sincerely yours, Nevio Cimoiai, M D , FRCPC 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 WOMEN'S HOSPITAL A N D HEALTH 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 WITH T f . f i t / « W « I T > I V D W r j S M COLUMt. AND r « e a.c. moKMKH msrmvrc rot> cwtxm*ir*<• SVOMIWS W^ITH 137 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 Richard D. Beachamp, MD. FRCSC H. Michael Bail. MD, FRCSC Kenneth L. B. Brown, MD, FRCSC Christopher Reilly, MD, FRCSC Bonita J. Sawatzy, Ph.D., Research Sharon A Secord. BSc.N.. Nursing Associate Telephone: (604) 875-3187 Facsimile Line: (604) 375-2275 Letter of Information Project: Establ ish ing the Genet i c Prof i le of Mult iple Hereditary Exos toses in Fami l ies of Brit ish Co lumb ia Investigators: Dr. C . A lvarez, Dr. S . Tredwel l , Dr. M. Hayden , Os teochondromas, a l so known a s exos toses , a re benign bone tumors, wh ich ar ise near growth plates at the end of long bones or on flat bones. They c a n occur a s solitary les ions a s in solitary os teochondromas ( S O C ) or in mult iples a s seen in Mult iple Hereditary Exos toses (MHE) . Osteochondromas do not usual ly c a u s e symptoms but on occas ion c a n cause mechanica l problems due to their s i ze and o r location by caus ing pa in , nerve compress ion o r deformity. L e s s commonly , they c a n c a u s e asymmetr ical growth of the long bones result ing in limb malal ignment or l imb length discrepancy. A very rare compl icat ion of os teochondromas, particularly in M H E is the transformation of the benign les ion into a malignant one. This however is an exceptional ly rare occur rence and usual ly occurs after skeletal maturity. It is known that M H E is an inherited condit ion 95% of the time but may a lso occur sporadical ly. O n the other hand, solitary osteochondromas are thought to be random occur rences. In M H E , 3 principal g e n e s have been identified. Repor ted in the literature to date, is that most fami l ies with M H E have a n abnormali ty identified in one of these 3 principal genes . N o study has been done to confirm whether patients with solitary osteochondromas or patients without a family history of os teochondromas have similar genet ic changes . T h e purpose of this study is to establ ish the genet ic make-up of fami l ies with M H E , patients with multiple les ions but no family history, and patients with solitary osteochondromas. Th is entails identifying patients with M H E and S O C . Th i s will occu r a s the patient presents to a regular cl inic v is i t Dr. A lva rez wi l l b e introduced to interested patients and their parent(s) and a brief d iscuss ion about the project will occur. If the patient and their direct family a re interested they will be entered into the study. This will involve interviewing the patients and their direct family. This interview will take about 1 hour. W e are interested in British Columbia's Children's Hospital 4 4 8 0 Oak Street, Vancouve r , BC V 6 H 3 V 4 Phone: {604) 875 -2345 A part of Children's & Women's Health Centre of British Columbia An taAtmie htclit, crmrt djillsud Ki th 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 > apr110 1021 gcagaagcgal tt gggatc aattggaaaa cgaggalttc c agegtgeaca tttccccccg < ex Id > gatgecaa ct ccagcatcta caaaggcaag aagtgccgca tggagtcctg apr 211 —» 1081 cttcgatttc accctttgca agaaaaaegg cttcaaagtc ta gecgaaa < 1141 agggg agaaa ate > ; «/"•( 1201 ctacacctcg gaccccagd c aggegtgect ctttgtc < apr 210 >J egtatace cacagcaaaa < ex Ig gttaccaaaa cattctag eg gc catcgagg gctccaggtt ex If J 109 - ctg agtctggata ctttagacag 142 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 aga ccagttg tcacctcagt atgtgc acaa tttgagatcc aaagt gcaga gtctccactt ex li ex li gtggaacaatg gtaggaatc atttaatttt tad tttatat tccggcactt gg c ctgacta apr107 ex Ik cacc gaggac | tggggtttg acatcggcca ggcgatgctg gccaaagcca gcatcagta < apr 209 » tgaaaacttc cgacccaact ttgatgtttc tattcccctc ttttctaagg atcatcccag gacaggaggg gagagggggt ttttgaag get ggtattc aagg ggaagaggtac ex lm * tt caacaccatc cctcctc apr 108 apr207- tca ggaagtacat < .  vw« >. i . ctga c aggge tagga tcagacacq a ggaatgeett ex 11 atatcaegtc cataaegggg aggacgttgt gctcctcacc acctgcaagc atggcaaaga ctggcaaaag cacaaggatt ctcgctgtga cagagacaac accgagtatg a gaagtatga exon 2 ttatcgggaa atgetgeaca atgccacttt ctgtctggtt cctcgtggtc gcaggcttgg gtccttcaga ttcctggagg etttgeag gc tgcctgcgtc cctgtgatgc tcagcaatgg exon 3 atgggagttg ccattctctg aagtgattaa ttggaaccaa gctgccgtca taggegatga gagattgtta ttacag attc cttctacaat caggtctatt catcaggata aaatcctagc exon 4 acttagacag cagacacaat tcttgtggga ggcttatttt tcttcagttg agaagattgt attaactaca ctagag atta ttcaggacag aatattcaag cacatatcac gtaacagttt exon 5 aatatggaac aaacatcctg gaggattgtt cgtactacca cagtattcat cttatctggg agattttcct tactactatg ctaatttag g tttaaagece ccctccaaat teactgeagt exon 6 catccatgcg gtgacccccc tggtctctca gtcccagcca gtgttgaagc ttctcgtggc tgcagccaag tcccagtact gtgcccag at catagttcta tggaattgtg acaagcccct exon 7 accagccaaa caccgctggc ctgccactgc tgtgcctgtc gtcgtcattg aaggagagag caag gttatg ageagcegtt ttctgcccta cgacaacatc atcacagacg ccgtgctcag exon 8 ccttgacgag gacacggtgc tttcaacaac agag gtggat ttcgccttca cagtgtggca exon 9 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 > apr215 gagtgg gggaggggaa gcaagaaggg atgggggtca 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 Q A K K R Y F I L L S A G S 1 ATG CAG GCC AAA AAA CGC TAT TTC ATC CTG CTC TCA GCT GGC TCT C L A L L F Y F G G L Q F R A 16 TGT CTC GCC CTT TTG TTT TAT TTC GGA GGC TTG CAG TTT AGG GCA C L A L L F Y F G G L Q F R A 31 TCG AGG AGC CAC AGC CGG AGA GAA GAA CAC AGC GGT AGG AAT GGC L H H P s P D H F W P R F P D 46 TTG CAC CAC ccc AGT CCG GAT CAT TTC TGG CCC CGC TTC CCG GAG A L R P F V P W D Q L E N E D 61 CCT CTG CGC CCC TTC GTT CCT TGG GAT CAA TTG GAA AAC GAG GAT S S V H I S P R Q K R D A N S 76 TCC AGC GTG CAC ATT TCC CCC CGG CAG AAG CGA GAT GCC AAC TCC S I Y K G K K C R M E S C F D 91 AGC ATC TAC AAA GGC AAG AAG TGC CGC ATG GAG TCC TGC TTC GAT F T L c K K N G F K V Y V Y P 106 TTC ACC CTT TGC AAG AAA AAC GGC TTC AAA GTC TAC GTA TAC CCA Q Q K G E K I A E S Y Q N I L 121 CAG CAA AAA GGG GAG AAA ATC GCC GAA AGT TAC CAA AAC ATT CTA A A I E G S R F Y T S D P S Q 136 GCG GCC ATC GAG GGC TCC AGG TTC TAC ACC TCG GAC CCC AGC CAG A C L F V L S L D T L D R D Q 151 GCG TGC CTC TTT GTC CTG AGT CTG GAT ACT TTA GAC AGA GAC CAG L S P Q Y V H N L R s K V Q S 166 TTG TCA CCT CAG TAT GTG CAC AAT TTG AGA TCC AAA GTG CAG AGT L H L w N N G R N H L I F N L 181 CTC CAC TTG TGG AAC AAT GGT AGG AAT CAT TTA ATT TTT AAT TTA Y S G T W P D Y T E D V G F D 15 30 45 60 75 90 105 120 135 150 165 180 195 144 196 t a t t c c ggc a c t t g g c c t gac t a c a c c gag gac g t g ggg t t t gac 210 I G Q A M L A K A S I S T E N 211 a t c ggc cag gcg a t g c t g gcc a a a gcc agc a t c a g t a c t gaa aac 225 F R P N F D V S I P L F S K D 226 t t c c g a c c c aac t t t g a t g t t t c t a t t c c c c t c t t t t c t aag g a t 240 H P R T G G E R G F L K F N T 241 c a t c c c agg a c a gga ggg gag agg ggg t t t t t g aag t t c a a c a c c 255 I P P L R K Y M L V F K G K R 256 a t c c c t c c t c t c agg aag t a c a t g c t g g t a t t c aag ggg aag agg 27 0 Y L T G I G S D T R N A L Y H 271 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 285 Y L T G I G S D T R N A L Y H 28 6 g t c c a t aac ggg gag gac g t t g t g c t c c t c a c c a c c t g c aag c a t 300 G K D W Q K H K D S R C D R D 301 ggc a a a gac t g g c a a aag c a c aag g a t t c t cgc t g t gac a g a gac 315 N T E Y E K Y D Y R E M L H N 316 aac 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 330 A T F C L V P R G R R L G S F 331 gcc a c t t t c t g t c t g g t t c c t c g t g g t cgc agg c t t ggg t c c t t c 345 R F L E A L Q A A C V P V M L 34 6 a g a t t c c t g gag g c t t t g cag g c t gcc t g c g t c c c t g t g a t g c t c 360 S N G W E L P F S E V I N W N 361 agc 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 aac 375 Q A A V I G D E R L L L Q I P 37 6 c a a g c t gcc g t c a t a ggc g a t gag a g a t t g t t a t t a cag a t t c c t 390 S T I R S I H Q D K I L A L R 391 t c t a c a a t c agg t c t a t t c a t cag g a t a a a a t c c t a g c a c t t a g a 4 05 Q Q T Q F L W E A Y F S S V E 4 06 cag 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 t c t t c a g t t gag 420 K I V L T T L E I I Q D R I F 421 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 cag gac a g a a t a t t c 4 35 K H I S R N S L I W N K H P G 4 36 aag c a c a t a t c a c g t a a c a g t t t a a t a t g g a a c a a a c a t c c t gga 4 50 G L F V L P Q Y S S Y L G D F 451 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 4 65 P Y Y Y A N L G L K P P S K F 4 66 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 a a a t t c 4 80 T A V I H A V T P L V S Q S Q 4 81 a c t g c a g t c a t c c a t gcg g t g a c c c c c c t g g t c t c t cag t c c cag 4 95 P V L K L L V A A A K S Q Y C 4 96 c c a g t g t t g aag c t t c t c g t g g c t g c a gcc aag t c c cag t a c t g t 510 A Q I I V L W N C D K P L P A 511 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 g c c 525 K H R W P A T A V P V V V I E 52 6 a a a c a c cgc t g g 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 54 0 G E S K V M S S R F L P Y D N 541 gga gag agc aag g t t a t g agc agc c g t t t t c t g c c c t a c gac aac 555 I I T D A V L S L D E D T V L 55 6 a t c a t c a c a gac gcc g t g c t c agc c t t gac gag gac a c g g t g c t t 57 0 S T T E V D F A F T V W Q S F 571 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 cag agc t t c 585 P E R I V G Y P A R S H F W D 58 6 c c t gag agg a t t g t g ggg t a c c c c gcg c g c agc c a c t t c t g g g a t . 600 N S K E R W G Y T S K W T N D 601 aac t c t aag 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 615 Y S M V L T G A A I Y H K Y Y 616 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 c a c a a a t a t t a t 630 H Y L Y S H Y L P A S L K N M 631 c a c 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 64 5 145 V D Q L A N C E D I L M N F L 64 6 GTG GAC CAA TTG GCC AAT TGT GAG GAC ATT CTC ATG AAC TTC CTG 660 V S A V T K L P P I K V T Q K 661 GTG TCT GCT GTG ACA AAA TTG CCT CCA ATC AAA GTG ACC CAG AAG 675 K Q Y K E T M M G Q T S R A S 67 6 AAG CAG TAT AAG GAG ACA ATG ATG GGA CAG ACT TCT CGG GCT TCC 690 R W A D P D H F A Q R Q S C M 691 CGT TGG GCT GAC CCT GAC CAC TTT GCC CAG CGA CAG AGC TGC ATG 7 05 N T F A S W F G Y M P L I H S 706 AAT ACG TTT GCC AGC TGG TTT GGC TAC ATG CCG CTG ATC CAC TCT 720 Q M R L D P V L F K D Q V S I 721 CAG ATG AGG CTC GAC CCC GTC CTC TTT AAA GAC CAG GTC TCT ATT 735 L R K K Y R D I E R L 736 TTG AGG AAG AAA TAC CGA GAC ATT GAG CGA CTT TGA 146 Appendix 8.4 EXT 2 8.4.1 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 la 1 tcctccggcg gcggccgcgc tttcagcatc ttggtaccca cctgttctcc tagccaacct 61 tcgcccccag tccgctcctt cctttcctcc tgcgacccgc cctccgccct ccgcggcgac 121 ccctcccttc ctgctgccac cttcccgcca g l̂ cacaggga tctgattcct cccaggggga exon la 181 tgtcctgcgc ctcagggtcc ggtggtggcc tgcggcatcc cttgcggtgc cagaagccgt 241 gggacgag y gt acggaagggg ccaggggcat gtaaggccgg ggactgggtg gtcgggggcg intron la 301 tgtcaggccg gggactgggt gaccgggaac tagatggccg ggggcgtgtt a E X T 2 exon lb 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 I • ctctcccctg gtgacce g gagtgtgaggaagaggctgtct gtgtcattat gtgtgcgtcg 121 ex 2a r r exon 2 181 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 ex2A4 > *<—-—ex 2A25 atcg atgtgcttaa ccagaacaca 661 ctgcgcatca aggagacagc acaagegatg gcccagctct. ctag < 721 cagcccagcc cccaggagat acttgagt gtatct cacactcata intron 2 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 tcatal ^ ex 3a ^ gttga cacattaatt ctccca catt ttaaattttt tgacad gtgg gatcgaggta ~* 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 agaaaggacĉ 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 <——' intron 5 U 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 ctccag agca tctgtggttg taccagaaga aaagatgtca gatgtgtaca *• exon 7 481 gtattttgea gagcatcccc caaagacaga ttgaagaaat gcagagaSag Igtaagaggcc 541 aagtcttggg gaggtgacat gggtggtacc gaaatggtgg cctt intron 7 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 361 ctcacttaaa ad agcattat tttttttata g gcccggtgg ttctgggaag y I ^. exon 8 L<—ex 8a c tacttcca 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 cagâ 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 Igg ttttacacag tgtgttt lata tgtttaatat tacttcctat gaetgettgt intron 10 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 Igtct̂ gc gggaatttcc tgaccggttg gtgggttacc ->• exon 11 421 cgggtcgtct gcatctctgg gaccatgaga tgaataagtg gaagtatgag tctgagtgga 481 cgaatgaagt gtccatggtg ctcactgggg cagcttttta tcacatiglgta agggggcgca —̂I intron 11 lagaat gatacacatt ttatttgacc caatttaatt 541 gtcctlggcaa ggtgacaaaa ctgag 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 catatĝ 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 attttattl̂ t ccttgacact gacagccagĝ  tatgtttttg tcctcctctg gcag gtaacc ex 13a exon 13 361 ccacgaaaga aattcaagtg tcctgagtgc acagccatag atgggctttc actagaccaa 421 acacacatgg tggagag gta agtgagcctc caaccaaaag I gcgccttag cctctgatct 481 eta i f ^ intron 13 ex 13b Jtttcctg 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 Igtcê 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 ac ggcatgc acccacctaa cccacj 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 gttgtgggaa ttgtgacccc catcccaagg ggatgccaaa 1321 atttctctca ttcttttggt ataaacttaa cattagccag ggaggttctg gctaacgtta 1381 aatgctgcta tacaactgct ttgcaacagt tgctggtata tttaaatcat taaatttcag 8.4.2 EXT 2Translation M C A S V K Y N I R G P A L I 1 A T G T G T G C G T C G G T C A A G T A T A A T A T C C G G G G T C C T G C C C T C A T C 15 P R M K T K H R I Y Y I T L F 16 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 T A T A T C A C C C T C T T C 30 S I V L L G L I A T G M F Q F 31 T C C A T T G T C C T C C T G G G C C T C A T T G C C A C T G G C A T G T T T C A G T T T 45 W P H S I E S S N D W N V E K 46 T G G C C C C A T T C T A T C G A G T C C T C A A A T G A C T G G A A T G T A G A G A A G 60 R S I R D V P V V R L P A D S 61 C G C A G C A T C C G T G A T G T G C C G G T T G T T A G G C T G C C A G C C G A C A G T 75 P I P E R G D L S C R M H T C 76 C C C A T C C C A G A G C G G G G G G A T C T C A G T T G C A G A A T G C A C A C G T G T 90 F D V Y R C G F N P K N K I K 91 T T T G A T G T C T A T C G C T G T G G C T T C A A C C C A A A G A A C A A A A T C A A G 105 V Y I Y A L K K Y V D D F G V 106 G T G T A T A T C T A T G C T C T G A A A A A G T A C G T G G A T G A C T T T G G C G T C 120 S V S N T I S R E Y N E L L M 121 T C T G T C A G C A A C A C C A T C T C C C G G G A G T A T A A T G A A C T G C T C A T G 135 A I S D S D Y Y T D D I N R A 136 G C C A T C T C A G A C A G T G A C T A C T A C A C T G A T G A C A T C A A C C G G G C C 150 C L F V P S I D V L N Q N T L 151 T G T C T G T T T G T T C C C T C C A T C G A T G T G C T T A A C C A G A A C A C A C T G 165 R l K E T A Q A M A Q L S R W 166 C G C A T C A A G G A G A C A G C A C A A G C G A T G G C C C A G C T C T C T A G G T G G 180 D R G T N H L L F N M L P G G 181 G A T C G A G G T A C G A A T C A C C T G T T G T T C A A C A T G T T G C C T G G A G G T 195 1561 agattagcca cagtttgggc tttagccaca acatatgtcc ccaaaacaca aaatacataa 158 P P D Y N T A L D V P R D R A 196 C C C C C A G A T T A T A A C A C A G C C C T G G A T G T C C C C A G A G A C A G G G C C 210 L L A G G G F S T W T Y R Q G 211 C T G T T G G C T G G T G G C G G C T T T T C T A C G T G G A C T T A C C G G C A A G G C 225 Y D V S I P V Y S P L S A E V 226 T A C G A T G T C A G C A T T C C T G T C T A T A G T C C A C T G T C A G C T G A G G T G 240 D L P E K G P G P R Q Y F L L 241 G A T C T T C C A G A G A A A G G A C C A G G T C C A C G G C A A T A C T T C C T C C T G 255 S S Q V G L H P E Y R E D L E 256 T C A T C T C A G G T G G G T C T C C A T C C T G A G T A C A G A G A G G A C C T A G A A 270 A L Q V K H G E S V L V L D K 271 G C C C T C C A G G T C A A A C A T G G A G A G T C A G T G T T A G T A C T C G A T A A A 2850 C T N L S E G V L S V R K R C 286 T G C A C C A A C C T C T C A G A G G G T G T C C T T T C T G T C C G T A A G C G C T G C 300 H K H Q V F D Y P Q V L Q E A 301 C A C A A G C A C C A G G T C T T C G A T T A C C C A C A G G T G C T A C A G G A G G C T 315 T F C V V L R G A R L G Q A V 316 A C T T T C T G T G T G G T T C T T C G T G G A G C T C G G C T G G G C C A G G C A G T A 330 L S D V L Q A G C V P V V I A 331 T T G A G C G A T G T G T T A C A A G C T G G C T G T G T C C C G G T T G T C A T T G C A 345 D S Y I L P F S E V L D W K R 346 G A C T C C T A T A T T T T G C C T T T C T C T G A A G T T C T T G A C T G G A A G A G A 360 A S V V V P E E K M S D V Y S 361 G C A T C T G T G G T T G T A C C A G A A G A A A A G A T G T C A G A T G T G T A C A G T 375 I L Q S I P Q R Q I E E M Q R 376 A T T T T G C A G A G C A T C C C C C A A A G A C A G A T T G A A G A A A T G C A G A G A 390 Q A R W F W E A Y F Q S I K A 391 C A G G C C C G G T G G T T C T G G G A A G C G T A C T T C C A G T C A A T T A A A G C C 405 I A L A T L Q I I N D R I Y P 406 A T T G C C C T G G C C A C C C T G C A G A T T A T C A A T G A C C G G A T C T A T C C A 420 Y A A I S Y E E W N D P P A V 421 T A T G C T G C C A T C T C C T A T G A A G A A T G G A A T G A C C C T C C T G C T G T G 435 K W G S V S N P L F L P L I P 436 A A G T G G G G C A G C G T G A G C A A T C C A C T C T T C C T C C C G C T G A T C C C A 450 P Q S Q G F T A I V L T Y D R 451 C C A C A G T C T C A A G G G T T C A C C G C C A T A G T C C T C A C C T A C G A C C G A 465 V E S L F R V I T E V S K V P 466 G T A G A G A G C C T C T T C C G G G T C A T C A C T G A A G T G T C C A A G G T G C C C 480 S L S K L L V V W N N Q N K N 481 A G T C T A T C C A A A C T A C T T G T C G T C T G G A A T A A T C A G A A T A A A A A C 495 159 P P E D S L W P K I R V P L K 496 C C T C C A G A A G A T T C T C T C T G G C C C A A A A T C C G G G T T C C A T T A A A A 510 V V R T A E N K L S N R F F P 511 G T T G T G A G G A C T G C T G A A A A C A A G T T A A G T A A C C G T T T C T T C C C T 525 Y D E I E T E A V L A I D D D 526 T A T G A T G A A A T C G A G A C A G A A G C T G T T C T G G C C A T T G A T G A T G A T 540 I I M L T S D E L Q F G Y E V 541 A T C A T T A T G C T G A C C T C T G A C G A G C T G C A A T T T G G T T A T G A G G T C 555 W R E F P D R L V G Y P G R L 556 T G G C G G G A A T T T C C T G A C C G G T T G G T G G G T T A C C C G G G T C G T C T G 570 H L W D H E M N K W K Y E S E 571 C A T C T C T G G G A C C A T G A G A T G A A T A A G T G G A A G T A T G A G T C T G A G 585 W T N E V S M V L T G A A F Y 586 T G G A C G A A T G A A G T G T C C A T G G T G C T C A C T G G G G C A G C T T T T T A T 600 H K Y F N Y L Y T Y K M P G D 601 C A C A A G T A T T T T A A T T A C C T G T A T A C C T A C A A A A T G C C T G G G G A T 615 I K N W V D A H M N C E D I A 616 A T C A A G A A C T G G G T A G A T G C T C A T A T G A A C T G T G A A G A T A T T G C C 630 M N F L V A N V T G K A V I K 631 A T G A A C T T C C T G G T G G C C A A C G T C A C G G G A A A A G C A G T T A T C A A G 645 V T P R K K F K C P E C T A I 646 G T A A C C C C A C G A A A G A A A T T C A A G T G T C C T G A G T G C A C A G C C A T A 660 D G L S L D Q T H M V E R S E 661 G A T G G G C T T T C A C T A G A C C A A A C A C A C A T G G T G G A G A G G T C A G A G 675 C I N K F A S V F G T M P L K 676 T G C A T C A A C A A G T T T G C T T C A G T C T T C G G G A C C A T G C C T C T C A A G 690 V V E H R A D P V L Y K D D F 691 G T G G T G G A A C A C C G A G C T G A C C C T G T C C T G T A C A A A G A T G A C T T T 705 P E K L K S F P N I G S L 706 CCT GAG AAG CTG AAG AGC TTC CCC AAC ATT GGC AGC TTA TGA 160 Appendix 8.5 Genotyping Appendix 8.5.1 Short Tandem Repeats (STR) markers M a r k e r name A03/04 85 547 A01/02 905 13 216 221 Chromosome 8 8 85 11 11 11 19 19 Gene E X T 1 E X T 1 E X T 1 E X T 2 E X T 2 E X T 2 E X T 3 E X T 3 D-number D8S555 D8S85 D8S547 D11S9 03 D11S905 D11S1313 D19S216 D19S221 Gene symbol Z24446 N/A Z24154 Z16529 Z16575 Z23608 Z16743 Z17017 Heterozygote Frequency 75.0 % 78.9 % 7 1 . 4 % 82.1% 71.4% 89.3% 81.5% 89.29% # of alleles 5 6 6 8 10 5 10 Al l e l e frequencies 1 - .464 2 - . 2 1 4 3 - .107 4 - .036 5 - .089 6 - .018 7 - .071 1 - .012 2 - .332 3 - . 1 8 8 4 - .250 5 - . 2 1 9 1 - .054 2 - . 1 0 7 3 - .321 4 - .464 5 - . 0 1 8 6 - .036 1 - .125 2 - . 4 1 1 3 - .036 4 - . 1 6 1 5 - .196 6 - .071 1 - .143 2 - .214 3 - .411 4 - .107 5 - .054 6 - . 0 1 8 7 - .036 8 - . 0 1 8 1 - .018 2 - . 1 2 5 3 - .232 4 - . 0 7 1 5 - .089 6 - . 1 4 3 7 - .054 8 - . 1 9 6 9 - .054 10-7018 1 - .259 2 - . 3 1 5 3 - .241 4 - . 1 3 0 5 - .056 1 - .232 2 - .089 3 - .089 4 - .071 5 - .071 6 - .179 7 - .107 8 - .125 9 - .018 1 0 - .018 Size of fragments 1 - .177 2 - . 1 7 3 3 - .167 4 - . 1 6 9 5 - .165 6 - .175 7 - .171 1 - .083 2 - . 0 8 1 3 - .079 4 - .075 5 - .073 1 - .193 2 - . 1 9 1 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 - . 2 1 0 4 - .226 5 - .208 6 - .228 7 - . 2 1 2 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 1 - .207 2 - .209 3 - .201 4 - .195 5 - .197 6 - .199 7 - .205 8 - .203 9 - .211 1 0 - .191 P C R T e m p 58°C 58°C 60°C 59°C 60°C 58°C 6 0 ° C 60°C 161 8.5.2 Short Tandem Repeats (STR) Primer Sequences M a r k e r N a m e Sequence A03/04 caagatggattcaaagccaaa cattcctaaggagggttcca 85 agctatcatcaccctataaaat ccttgcccatcacttacac 547 tttaaaatgcatgtggccttc tacacacagcctcatggctc A01/02 caacacttcgatgttccttcc agctgagagcgcatgtataa 905 tctcctgtccctcacacaca acaggggccaaataggtttc 13 taacgatttncaacgtctaagc gggaattttgacttcatatgca 216 ggagacctctggctaggta aggtacttagttactgactttg 221 gagcaagactctgactcaac 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- 1:1 -o l:2 GH 1-4 1:3 11:4 11:5 11:6 Blood 11:1 GH 1-3 -o ll:2 GH 1-2 Blood Cells 111:1 GH 1-1 F a m i l y 1 163 m- 1:1 "0 l:2 ll:3 NI2-7 •A" • • ll:4 ll:7 NI 2-6 D- 11:1 - o ll:2 If - O L> 111:10 111:11 D- lll:6 111:12 5 6± lll:8 lll:7 IV:4 IV:5 IV:6 IV:7 IV:8 IV:9 IV:10 IV:11 IV:12 111:1 NI 2-4 III2 NI2-5 IV: 1 NI2-2 ^ Blood C«IIs ^ B IV:2 NI2-1 IV:3 NI2-3 F a m i l y 2 164 o - 11:1 HO 3-1 1 Blood Blood 111:12 HO 3-5 lll:3 HO 3-13 111:14 HO 3-14 111:15 HO 3-9 IV:3 HO 3-2 IV:4 IV:5 IV:6 HO 3-3 HO 3-6 HO 3-7 ^ | ^ Blood ^ B b o d Blood ^ [^B lll:4 HO 3-8 111: 5 H 0 3- IV:7 IV:8 IV:9 HO 3-16 HO 3-15 HO 3-17 IV:10 HO 3-11 IV:11 HO 3-12 T^Btoo. IV:12 Hoi 3-10 —o 112 11:4 AT IV: 1 HO 3-19 IV:2 HO 3-20 F a m i l y 3 165 JZr 1:1 11:1 Blood Blood o 111:1 HE 4-2 lll:2 HE 4-1 4 IV: 1 HE 4-4 Blood IV:2 HE 4-3 Blood F a m i l y 4 166 0 - r - 0 1:1 l:2 Blood I Blood o Q l l :2 T A 5 - 6 11:3 l l:4 : T A 5 - 5 Blood :4 111:1 T A 5 - 1 Blood l l l :2 111 3 T A 5 - 2 Blood IV: 1 T A 5 - 3 4 Blood IV:2 T A 5 - 4 F a m i l y 5 167 Q 1:1 O l:2 Blood Blood l l l :3 B l 6-2 4= l l :2 B l 6-3 # • LT ll:4 Blood Cells l l l :2 B l 6-1 l:5 ti F a m i l y 6 168 Q 111:1 l:2 Blood O IV: 1 F R 8-4 4 V:1 F R 8 - 1 Blood Cells Blood IV:2 F R 8-2 1 V : 2 F R 8 - 3 IV:3 U IV:6 IV:4 IV: 5 F a m i l y 8 169 D- 1:1 l:2 ll:2 11 :B BO 16-5 111:1 BO 16-2 V— •o lll:2 BO 16-3 CHr-6 ll:9 11:3 ll:6 ll:5 ll:7 Blood IV: 1 BO 16-1 6" IV:2 BO 16-4 F a m i l y 16 170 D- 1:3 o 1:4 11:1 KE 17-7 Blood 111:1 KE 17-1 Blood lll:2 KE 17-3 Blood 1:1 KE 17-5 ll:2 KE 17-2 111 3 KE 17-4 Blood -o l:2 KE 17-6 1 11:3 111:4 o 11:4 111:5 111:6 F a m i l y 17 171 • 1:1 l:2 I Blood Cells Blood 1 IV: 1 W H 18-1 IV:2 W H 18-4 IV: 3 W H 18-5 F a m i l y 18 172 8.6.2 Short tandem repeat (STR) Gels Family 1 EXT 1 Family Marker Member 1_2: 1 3: 1, 4 2, 4 1, 3 Family Marker Member 1_1: c, a 1_2: a, b 1_3: c, c Family 2 EXT 1 EXT 2 Family Marker Member 2_1: 3, 1 2_2: 1, 2 2_3: 3, 2 2_4:1, 3 2 5:lr 2 Family Marker Member 2_1: b, c 2_2: b, c 2_3: a, d 2_4:a, b 2_5:cr d 173 F a m i l y 3 E X T 1 Family Member Marker 3-1 2, 3 3-2 1, 1 3-3 1, 1 3-4 (1) 3-5 1, 1 3-6 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 1, 1 3-15 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 F a m i l y 3 E X T 2 F a m i l y M e m b e r M a r k e r 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 E X T 1 E X T 2 Family Marker Member 5 5_2 5_3 5 4 : 2, 2 1, 2 2, 1 2, 1 Family Marker Member 5_1: 5_2: 5_3: 5 4: a, d a, a d, a a, a Family 8 E X T 1 Family Marker Member 8_1: 8_2: 83: 8 4: 1, 1 1, 1 2, 1 1 , 2 E X T 2 Family Marker Member 8_l:b, a 8_2:a a 8_3: b a 8 4: b b 176 F a m i l y 16 F a m i l y 17 E X T 1 Family Marker Member 17_1: 3, 1 17_2: 4, 1 17_3: 1, 4 17_4:1, 1 17_5:1, 4 17_6:1, 2 E X T 2 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 177 F a m i l y 1 8 EXT 1 EXT 2 Family Marker Member mk . mk am 18 1: 2, 3 18 2: 1, 2 18_3: 2, 3 18 4:1, 2 Family Marker Member 18_1: a, c 18_2: a, d 18_3: c, c 18_4:a, c 178 8.6.3 Phenotype Data 8.6.3.1. Core Data 8.6.3.1.1 Lesion Quality Core Data S U B J E C T G E N D E R E X T Sense Severity Stage of M u t a t i o n T o t a l # lesions % small % medium B i g 6-01 male 42 35.7 26.2 B i g 6-02 male 27 22.2 33.3 B i g 6-03 female 28 35.7 46.4 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 Boe 16-05 Female 1 SS severe Late 20 25.0 35.0 F r i 8-01 Male 2 SS severe Late 33 13.8 39.0 F r i 8-02 Female 2 SS severe Late 18 27.7 16.6 G h u 1-01 Female 1 M S mi ld Late 28 29.6 33.3 G h u 1-03 Male 1 M S mi ld 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 H o i 3-01 Female 2 N S severe Ear ly 11 18.0 45.0 H o i 3-02 Female 2 N S severe Ear ly 13 7.7 23.1 H o i 3-04 Male 2 N S severe Early 13 7.7 23.1 H o i 3-08 Female 2 N S severe Ear ly 18 38.8 16.0 H o i 3-10 Female 2 N S severe Ear ly 9 22.2 22.2 H o i 3-15 Female 2 N S severe Ear ly 14 14.3 64.3 H o i 3-19 Male 2 N S severe Ear ly 39 43.5 30.7 H o i 3-22 Female 2 N S severe Ear ly 12 50.0 47.6 H o i 3-23 Male 2 N S severe Ear ly 28 18.0 21.4 K e r 17-01 Male 2 F S severe Ear ly 36 33.3 25.0 K e r 17-02 Female 2 F S severe Ear ly 24 27.7 47.0 K e r 17-05 Male 2 F S severe Ear ly 11 18.2 45.5 Nic 2-01 Male 2 N S severe Ear ly 27 37.0 25.9 Nic 2-02 Male 2 N S severe Ear ly 29 24.1 31.0 Nic 2-04 Male 2 N S severe Ear ly 20 55.0 35.0 T a b 5-01 Male 2 M S m i l d Ear ly 16 71.4 14.2 T a b 5-03 Female 2 M S m i l d Early 14 57.1 14.2 W h i 18-01 Male 1 N S severe Early 53 47.0 33.3 W h i 18-02 Male 1 N S severe Early 34 32.3 11.7 179 8.6.3.1.1 Lesion Quality Core Data (continued) S U B J E C T % large pelvic %pelvic flatbone % flatbone flare %flare Les ion R a n k 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 Boe 16-05 40.0 1 5.0 1 5.0 2 10.0 5 F r i 8-01 45.4 4 12.1 4 12.1 3 9.1 5 F r i 8-02 55.5 0 0.0 0 • 0.0 1 5.6 5 G h u 1-01 37.0 3 10.7 3 10.7 3 10.7 7 G h u 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 H e g 4-04 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 H o i 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 H o i 3-22 8.3 2 16.7 2 16.7 1 8.3 6 H o i 3-23 25.0 0 0.0 0 0.0 21 75.0 5 K e r 17-01 41.7 1 2.8 1 2.8 17 47.2 6 K e r 17-02 25.0 3 12.5 3 12.5 6 25.0 7 K e r 17-05 36.3 0 0.0 0 0.0 1 9.1 2 Nic 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 Nic 2-04 10.0 0 0.0 1 5.0 12 60.0 13 T a b 5-01 14.2 0 0.0 0 0.0 10 62.5 11 T a b 5-03 28.5 0 0.0 0 0.0 6 42.9 9 W h i 18-01 19.6 7 13.2 8 15.1 26 49.1 22 W h i 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 G e n d e r E X T Sense Severity Stage of M u t a t i o n # o f lesions C a r p a l S l i p R C a r p a l S l i p L B i g 6-01 male 42.0 8.0 10.0 B i g 6-02 male 27.0 too immature to see to immature to see B i g 6-03 female 28.0 2.0 3.0 Boe 16-01 female 1 SS severe Late 32.0 3.0 4.0 Boe 16-02 male 1 SS severe Late 36.0 8.0 5.0 Boe 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 G h u 1-01 female 1 M S m i l d Late 28.0 2.0 2.0 G h u 1-03 male 1 M S m i l d 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 N S severe Ear ly 11.0 2.0 6.0 H o i 3-02 female 2 N S severe Early 13.0 4.0 5.0 H o i 3-04 male 2 N S severe Ear ly 13.0 -8.0 -5.0 H o i 3-08 female 2 N S severe Ear ly 18.0 2.5 3.0 H o i 3-10 female 2 N S severe Ear ly 9.0 7.0 5.0 H o i 3-15 female 2 N S severe Early 14.0 -5.0 -5.0 H o i 3-19 male 2 N S severe Early 39.0 1.0 1.0 H o i 3-22 female 2 N S severe Early 12.0 3.0 6.0 H o i 3-23 male 2 N S severe Early 28.0 missing r wrist f i lm 9.0 K e r 17- 02 female 2 F S severe Ear ly 24.0 3.0 6.0 K e r 17-01 male 2 F S severe Ear ly 36.0 3.0 2.0 K e r 17-05 male 2 F S severe Ear ly 11.0 5.0 3.0 N i c 2-01 male 2 N S severe Ear ly 27.0 6.0 3.0 Nic 2-02 male 2 N S severe Ear ly 29.0 2.0 3.0 N i c 2-04 male 2 N S severe Ear ly 20.0 5.0 5.0 T a b 5-01 male 2 M S m i l d Ear ly 16.0 3.0 2.0 T a b 5-03 female 2 M S m i l d Ear ly 14.0 2.0 1.0 W h i 18-01 male 1 N S severe Ear ly 53.0 5.0 6.0 W h i 18-02 male 1 N S severe Early 34.0 r arm and forearm not fi lmed 8.0 181 8.6.3.1.2 Limb Alignment Core Data (continued) R a d R a d U l n R a d H e a d R a d H e a d Inclin Inclin Short U l n R a d B o w R a d Disclocation Disclocation Subject R L R Short L R Bow L R L B i g 6-01 39.0 31.0 -7.0 -10.0 9.0 12.0 N Y B i g 6-02 22.0 25.0 0.0 6.0 5.0 7.0 N N B i g 6-03 26.0 28.0 1.5 1.0 8.0 9.0 N N Boe 16- 01 27.0 36.0 3.0 2.0 9.0 20.0 N Y Boe 16- 02 28.0 32.0 0.0 8.0 7.0 11.0 Y N Boe 16- 05 27.0 33.0 0.0 11.0 9.0 9.5 N N F r i 8-01 27.0 28.0 4.0 1.0 9.0 8.0 N N F r i 8-02 28.0 21.0 2.0 4.0 8.0 8.0 N N G h u 1-01 20.0 22.0 1.0 1.0 6.0 10.5 N N G h u 1-03 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 missing missing r wrist r wrist missing r H o i 3-23 film 30.0 film 11.0 9.0 9.0 wrist film N K e r 17- 02 11.0 24.0 -11.0 -7.0 9.0 11.0 N N K e r 17- 01 30.0 34.0 -2.0 -9.0 7.0 12.0 N N K e r 17- 05 21.0 19.0 1.0 2.0 6.0 5.0 N N Nic 2-01 27.0 27.0 1.5 2.0 4.0 7.0 N N Nic 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 W h i 18- 01 29.0 35.0 -8.0 -2.0 11.0 9.0 N N r arm r arm and and forearm forearm r arm and r arm and W h i 18- not not forearm forearm not 02 filmed 22.0 filmed 5.0 not filmed 31.0 fi lmed Y 182 8.6.3.1.2 Limb Alignment Core Data (continued) Subject E l b J t R E l b J t L F e m A A R F e m A A L F e m N S A n g R F e m N S A n g L F e m M A R F e m M A 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 0.0 -4.0 Boe 16-01 17.0 3.0 0.0 -5.0 176.0 170.0 n/a n/a Boe 16-02 19.0 12.0 4.0 11.5 129.0 140.0 8.0 -10.0 Boe 16-05 -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 G h u 1-03 -22.0 -3.0 0.0 2.0 135.0 148.0 8.0 9.0 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 4.0 4.0 H o i 3-19 2.0 -11.0 -12.0 -14.0 146.0 139.0 n/a n/a 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 K e r 17- 02 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 Nic 2-01 -8.0 -30.0 -10.0 -5.0 149.0 142.0 0.0 4.0 Nic 2-02 -14.0 -18.0 0.0 -11.0 150.0 135.0 6.0 -6.0 Nic 2-04 9.0 -11.0 -6.0 -3.0 133.0 130.0 4.0 8.0 T a b 5-01 -13.0 -8.0 3.0 3.0 145.0 123.0 3.0 -2.0 T a b 5-03 -14.0 -11.0 -4.0 -2.0 140.0 155.0 1.0 3.0 W h i 18-01 2.0 0.0 0.0 -7.0 151.0 142.0 5.0 -7.0 W h i 18-02 r arm and forearm not filmed -7.0 -11.0 -12.0 141.0 143.0 -3.0 -3.0 183 8.6.3.1.2 Limb Alignment Core Data (continued) Subject Sharps R Sharps L F i b H t R F i b H t L A n k l e J t R A n k l e J t 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 Boe 16-01 43.0 40.0 50.0 58.0 3.0 3.0 Boe 16-02 36.5 40.0 53.0 56.0 32.0 21.0 Boe 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 F r i 8-02 36.0 33.0 28.0 49.0 -3.0 -5.0 G h u 1-01 n/a n/a 52.0 52.0 -6.0 1.0 G h u 1-03 37.0 33.0 53.0 67.0 -7.0 2.0 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 -12.0 -2.0 H o i 3-02 51.0 50.0 62.0 78.0 2.0 14.5 H o i 3-04 40.0 41.0 45.0 52.0 25.0 11.0 H o i 3-08 40.0 39.0 53.0 58.0 6.0 -11.0 H o i 3-10 39.0 39.0 37.5 23.0 -7.0 -5.0 H o i 3-15 50.0 45.0 62.0 48.0 0.0 2.0 H o i 3-19 36.0 42.0 28.5 37.0 n/a n/a 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 K e r 17- 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 Nic 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 13.0 T a b 5-01 46.0 41.0 58.0 30.0 long films didn't include distal ankle long films didn't include distal ankle T a b 5-03 n/a n/a 52.0 41.0 0.0 0.0 W h i 18- 01 40.0 47.0 64.0 64.0 -31.0 -34.0 W h i 18- 02 35.0 35.0 54.0 32.0 -21.0 -7.0 184 8.6.3.1.2 Limb Alignment Core Data (continued) Subject G e n d e r E X T Sense Severity % W t Bear R % W t B e a r L 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 Boe 16-01 female 1 SS severe 68.0 60.0 Boe 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 G h u 1-01 female 1 M S m i l d 20.0 70.0 G h u 1-03 male 1 M S m i l d 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 N S severe 19.0 79.0 H o i 3-02 female 2 N S severe 61.5 56.7 H o i 3-04 male 2 N S severe 67.0 63.0 H o i 3-08 female 2 N S severe 19.0 59.0 H o i 3-10 female 2 N S severe 45.0 24.0 H o i 3-15 female 2 N S severe 58.0 38.0 H o i 3-19 male 2 N S severe n/a n/a H o i 3-22 female 2 N S severe 32.0 15.0 H o i 3-23 male 2 N S severe 2.0 23.5 K e r 17- 02 female 2 F S severe 30.0 75.0 K e r 17-01 male 2 F S severe 11.0 37.0 K e r 17-05 male 2 F S severe 68.0 68.0 N i c 2-01 male 2 N S severe 54.0 62.0 N i c 2-02 male 2 N S severe 77.0 29.0 N i c 2-04 male 2 N S severe 68.0 78.0 T a b 5-01 male 2 M S m i l d 56.0 52.0 T a b 5-03 female 2 M S m i l d 61.0 65.0 W h i 18-01 male 1 N S severe 69.0 57.0 W h i 18-02 male 1 N S severe 54.0 49.0 185 8.6.3.1.3 Limb segments and percentile height core data S U B J E C T G E N D E R E X T Sense Severity Stage of M u t a t i o n Height (%ile) T o t A r m L e n g t h - L e f t Side T o t A r m L e n g t h - R i g h t Side T o t L e g L e n g t h - L e f t Side B i g 6-01 male 50 40 41 74 B i g 6-02 male 38 31 30 46 B i g 6-03 female 30 44 49.5 83 Boe 16-01 Female 1 SS Severe Late 3 43.0 42.0 75.0 Boe 16-02 Male 1 SS Severe Late 39 47.5 47.0 86.5 Boe16-05 Female 1 ss Severe Late 9 50.0 51.0 86.0 F r i 8-01 Male 2 ss Severe Late 25 47.0 44.0 77.5 F r i 8-02 Female 2 ss Severe Late 25 55.5 55.5 89.0 H e g 4-01 female 8 42.5 48.0 86.0 H e g 4-03 female 38 48.0 47.0 86.0 H e g 4-04 female 60 43.0 44.0 87.0 G h u 1-01 Female 1 M S M i l d Late 3 38.0 37.0 62.0 G h u 1-03 Male 1 M S M i l d Late 5 52.5 51.0 82.5 H o i 3-01 Female 2 N S Severe Ear ly 25 53.5 50.0 90.0 H o i 3-02 Female 2 N S Severe Ear ly 24 49.5 46.5 77.0 H o i 3-04 Male 2 N S Severe Ear ly 18 56.0 55.5 89.5 H o i 3-08 Female 2 N S Severe Ear ly 25 52.0 54.5 81.0 H o i 3-10 Female 2 N S Severe Ear ly 51 53.0 53.0 85.5 H o i 3-15 Female 2 N S Severe Ear ly 95 40.0 40.5 67.5 H o i 3-19 Male 2 N S Severe Ear ly 50 55.0 55.0 88.5 H o i 3-22 Female 2 N S Severe Early 90 56.0 57.0 97.0 H o i 3-23 Male 2 N S Severe Ear ly 60 53.5 53.5 92.0 K e r 17-01 Male 2 F S Severe Ear ly 18 50.5 52.5 86.5 K e r 17-02 Female 2 F S Severe Early 8 45.5 46.0 81.5 K e r 17-05 M a l e 2 FS Severe Ear ly 3 57.0 53.5 86.5 N i c 2-01 Male 2 N S Severe Ear ly 51 44.0 44.5 74.5 N i c 2-02 Male 2 N S Severe Early 77 52.5 50.5 94.0 N i c 2-04 Male 2 N S Severe Ear ly 85 59.0 58.5 95.0 T a b 5-01 Male 2 M S M i l d Early 15 54.0 54.5 88.0 T a b 5-03 Female 2 M S M i l d Early 63 36.5 38.5 61.5 W h i 18-01 Male 1 N S Severe Early 3 43.0 43.0 79.0 W h i 18-02 Male 1 N S Severe Early 3 41.0 43.0 81.0 186 8.6.3.1.3 Limb segments and percentile height core data (continued) S U B J E C T T o t L e g Length - Right Side U p p e r A r m R L o w e r A r m R U p p e r A r m L L o w e r A r m L U p p e r L e g R L o w e r L e g R U p p e r L e g L L o w e r L e g L 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 Boe 16-02 89.0 27.0 21.5 28.5 19.0 43.5 37.5 42.0 39.5 B o e 16-05 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 G h u 1-03 90.0 30.0 23.0 33.0 24.5 42.5 34.5 43.0 32.5 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 Nic 2-02 92.0 33.0 24.0 33.0 24.5 46.5 34.5 45.5 37.0 Nic 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 Corre la t ion M a t r i x # lesions carpa l slip r carpa l slip 1 r a d inclin r rad inclin 1 ulnar short r u lnar short 1 rad bow r 1 # lesions 1 0.433 0.738 0.361 0.665 -0.362 0.456 0.268 2 carpa l slip r 0.433 1 0.27 0.543 0.755 -0.495 -0.194 0.022 3 carpa l slip 1 0.738 0.27 1 0.346 -0.18 -0.297 0.192 -0.104 4 r a d inclin r 0.361 0.543 0.346 1 0.594 -0.14 0.022 0.34 5 r a d inclin 1 0.03 0.755 -0.18 0.594 1 -0.437 -0.323 0.089 6 ulnar short r -0.362 -0.495 -0.297 -0.14 -0.437 1 0.475 -0.268 7 ulnar short 1 0.456 -0.494 0.192 0.022 -0.323 0.475 1 0.01 8 r a d bow r 0.268 0.22 -0.104 0.34 0.089 -0.268 0.01 1 9 r a d bow 1 0.749 0.427 0.349 0.463 0.312 -0.582 0.247 0.64 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 aa r -0.041 0.42 -0.475 0.226 0.297 0.275 0.479 0.355 13 fem aa 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 ma 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 21 fib ht 1 0.326 0.1 0.452 -0.159 -0.332 0.148 0.039 -0.434 22 ankle jt r -0.505 0.137 -0.564 -0.202 -0.141 861 0.384 -0.281 23 ankle jt 1 -0.552 -0.257 -0.496 -0.2 0.131 0.689 0.213 -0.398 24 % wt bear r 0.304 -0.027 -0.326 0.253 0.348 0.078 0.35 0.559 25 % wt bear 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 0.224 0.142 0.504 0.278 -0.114 0.249 0.158 -0.232 29 %prox -0.518 0.229 -0.79 -0.407 0.083 -0.07 -0.106 0.295 30 % pelvic 0.82 -0.277 0.724 0.51 0.083 -0.413 0.279 0.265 31 % d i a p h -0.244 0.288 -0.04 0.06 -0.115 0.217 -0.17 -0.219 32 %flat bones 0.002 -0.201 0.027 -0.053 0.278 -0.021 -0.229 -0.487 33 %complex 0.109 0.282 -0.05 0.435 0.346 -0.607 -0.149 0.656 34 %simple -0.446 0.087 -0.337 -0.535 -0.389 0.514 -0.211 -0.387 35 %flared 0.164 -0.327 0.118 0.104 0.29 -0.528 0.1 0.146 36 % not flared -0.084 0.28 -0.058 -0.045 -0.304 0.552 0.043 -0.096 37 % o f l -0.228 -0.249 -0.155 -0.195 -0.238 -0.244 -0.286 0.529 38 % o f 4 0.098 -0.233 -0.16 0.336 0.345 0.243 0.373 -0.324 39 avg # 1 0.028 0.738 0.361 0.03 -0.362 0.456 0.268 40 % left -0.087 0.433 -0.27 0.479 0.434 -0.325 -0.215 0.787 41 % right 0.087 0.104 0.27 -0.479 -0.434 0.325 0.042 -0.787 42 % h t -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 -0.439 188 8.6.4.2 Pearson Correlation Matrix (continued) 1 2 3 4 5 6 7 8 # lesions c a r p a l slip r carpa l slip 1 rad inclin r r a d inclin 1 ulnar short r u lnar short 1 rad bow r 46 ratio I a r m 0.72 -0.426 0.447 0.218 0.131 -0.288 -0.073 -0.027 47 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 upper -0.177 -0.143 -0.082 -0.655 -0.609 0.405 0.272 -0.404 53 1 leg lower -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 ratio 0.347 -0.402 0.601 0.354 -0.525 0.299 0.376 0.273 56 r leg upper -0.188 0.227 -0.121 -0.661 -0.414 0.279 0.262 -0.57 57 r leg lower -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 ratio 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 -0.339 189 8.6.4.2 Pearson Correlation Matrix (continued) 9 10 11 12 13 14 15 16 17 Corre la t ion M a t r i x r a d bow 1 e l b j t r elb jt 1 fem aa r fem aa 1 fem ns r fem ns 1 fem ma r fem ma 1 1 # lesions 0.749 0.473 0.465 -0.041 -0.397 0.256 0.364 0.217 -0.495 2 carpal slip r 0.427 0.104 0.078 0.42 -0.037 0.599 0.614 0.143 -0.008 3 carpa l slip 1 0.349 0.319 0.507 -0.475 -0.004 0.17 0.144 -0.379 -0.382 4 r a d inclin r 0.463 -0.055 -0.047 0.058 0.132 0.418 0.709 0.213 -0.285 5 r a d inclin 1 0.312 0.159 -0.397 0.226 -0.161 0.556 0.444 0.24 -0.021 6 ulnar short r -0.582 -0.656 -0.133 0.297 0.301 -0.532 0.079 0.388 0.124 7 ulnar short 1 0.247 0.093 0.324 0.275 -0.402 -0.03 0.352 0.593 0.345 8 r a d bow r 0.64 0.08 0.128 0.479 -0.028 0.271 0.312 0.355 0.342 9 rad bow I 1 0.452 0.415 0.355 -0.441 0.411 0.484 0.27 0.986 10 elb jt r 0.452 1 0.09 0.263 -0.704 0.421 -0.307 -0.127 0.67 11 elb jt 1 0.415 0.09 1 0.01 0.194 0.098 0.256 -0.186 0.875 12 fem aa r 0.263 0.01 0.058 1 -0.158 0.281 0.487 0.741 0.423 13 fem aa 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 1 0.53 0.234 16 fem ma r -0.55 -0.127 -0.186 0.741 -0.311 0.077 0.53 1 0.456 17 fem m a 1 -0.268 -0.563 -0.498 -0.489 0.44 -0.357 -0.28 -0.156 1 ' 18 sharps r 0.003 -0.254 0.085 0.253 0.636 0.011 -0.121 -0.184 0.246 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 ankle jt r -0.587 -0.626 -0.251 -0.165 0.333 -0.21 0.213 0.517 0.194 23 ankle jt 1 0.38 -0.498 -0.238 -0.105 -0.348 0.049 0.276 0.363 0.075 24 % wt bear r 0.106 0.101 -0.279 -0.103 0.158 0.253 0.402 0.902 -0.062 25 % wt bear 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 % d i a p h -0.375 -0.556 -0.162 0.406 -0.028 -0.599 -0.016 -0.251 0.245 32 %flat 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 % not 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 0.7 190 L6.4.2 Pearson Correlation Matrix (continued) 9 10 11 12 13 14 15 16 17 r a d bow 1 e l b j t r elb jt 1 fem aa r fem aa 1 fem ns r fem ns 1 fem ma r fem ma 1 40 % left -0.4 0.133 -0.203 -0.266 -0.017 0.575 0.297 0.21 0.9 41 % right -0.656 -0.133 0.203 -0.27 0.008 -0.575 -0.297 -0.21 0.65 42 % ht -0.389 0.188 -0.305 -0.137 -0.502 0.213 -0.401 -0.175 0.456 43 1 a r m 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 47 r a r m upper -0.517 0.254 -0.76 -0.052 -0.351 -0.274 -0.593 0.029 0.568 48 r a r m 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 A L D -0.334 -0.195 -0.084 -0.11 -0.465 -0.13 0.088 -0.065 0.56 52 1 leg upper -0.348 0.108 -0.309 -0.159 -0.596 -0.374 -0.448 0.033 0.67 53 1 leg lower -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 ratio -0.401 -0.229 -0.219 -0.185 -0.529 -0.075 0.266 -0.1 0.346 56 r leg upper -0.172 0.215 -0.366 -0.06 -0.619 -0.287 -0.519 -0.05 0.876 57 r leg lower -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 ratio -0.241 0.188 0.289 -0.009 0.123 0.124 -0.097 -0.356 0.113 60 L L D -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 20 21 22 23 24 25 26 Corre la t ion M a t r i x sharps r sharps 1 fib ht r fib ht 1 ankle j t r ankle jt 1 % wt bear r % wt bear 1 % ped 1 # lesions -0.557 -0.229 0.37 0.326 -0.505 -0.552 0.304 -0.087 0.352 2 carpa l slip r 0.073 -0.116 0.1 0.137 -0.257 -0.027 0.244 0.404 0.132 3 carpa 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 6 ulnar short r 0.012 -0.278 -0.275 0.148 0.861 0.689 0.078 -0.113 0.557 7 ulnar short 1 -0.537 -0.432 -0.02 0.039 0.384 0.213 0.35 -0.405 0.576 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 r a d 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 aa r 0.253 -0.011 -0.148 -0.438 0.453 0.507 0.615 -0.023 0.165 13 fem aa 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 ma r -0.184 0.156 -0.249 -0.211 0.517 0.363 0.902 0.36 0.666 17 fem ma 1 0.246 0.108 -0.098 0.168 0.194 0.075 -0.062 0.7 0.034 18 sharps r 1 0.821 0.3 -0.102 0.079 0.254 -0.098 0.138 0.408 19 sharps 1 0.821 1 0.685 0.062 -0.242 -0.165 -0.12 0.055 0.44 20 fibhtr 0.3 0.685 1 0.489 -0.343 -0.17 -0.123 -0.15 0.101 21 fib ht 1 -0.102 0.062 0.489 1 -0.113 0.896 -0.323 -0.027 0.214 22 ankle jt r 0.079 -0.242 -0.343 -0.113 1 0.895 0.226 0.082 0.368 23 ankle jt 1 0.254 -0.165 -0.32 -0.17 .896 1 0.109 -0.048 -0.13 24 % wt bear r -0.098 -0.12 -0.123 -0.323 0.226 0.109 1 0.483 0.618 25 % wt bear 1 0.138 0.055 -0.15 -0.027 0.082 -0.048 0.483 1 0.128 26 % ped 0.408 0.44 0.101 -0.214 -0.368 -0.13 -0.618 -0.128 1 27 % sess -0.131 0.02 0.272 0.303 -0.006 -0.185 0.456 0.189 0.797 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 % d i a p h -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 0.342 192 8.6.4.2 Pearson Correlation Matrix (continued) 18 19 20 21 22 23 24 25 26 Corre la t ion M a t r i x sharps r sharps 1 fib ht r fib ht 1 ankle j t r ankle jt 1 % wt bear r % wt bear 1 % ped 35 %flared 0.315 0.45 0.389 -0.306 -0.34 -0.037 -0.058 -0.433 0.562 36 % not 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 -0.455 -0.155 -0.113 -0.024 -0.038 0.379 38 % of 4 -0.528 -0.71 -0.694 7.95E- 05 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 -0.496 -0.498 -0.558 -0.071 0.149 -0.016 -0.031 0.118 -0.22 44 1 a r m lower 4.69E- 04 -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 47 r a r m 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 A L D -0.483 0.26 0.45 0.325 -0.082 -0.093 0.005 -0.162 0.349 52 1 leg upper 0.314 -0.345 -0.148 -0.031 0.355 0.107 -0.152 -0.428 0.258 53 1 leg lower -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 upper -0.074 -0.363 -0.204 -0.053 0.308 0.152 -0.206 -0.427 -0.14 57 r leg lower -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 ratio -0.044 -0.079 0.346 0.414 0.042 0.284 -0.435 -0.666 0.003 60 L L D -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 Corre la t ion M a t r i x % sess %distal % p r o x % pelvic % diaph % flatbones % complex % simple % flared 1 # lesions -0.495 -0.194 0.022 0.427 0.104 0.078 0.665 0.362 0.456 2 carpal slip r -0.297 0.192 -0.104 0.349 0.319 0.507 0.755 -0.495 -0.194 3 carpal slip 1 -0.14 0.022 0.34 0.463 0.055 -0.047 -0.18 -0.297 0.192 4 rad inclin r -0.437 -0.323 0.089 0.312 0.159 -0.397 0.594 -0.14 0.022 5 rad inclin I 1 0.475 -0.268 -0.582 0.656 -0.133 -0.89 -0.437 -0.323 6 ulnar short r 0.475 1 0.01 0.247 0.093 0.324 -0.437 0.765 0.475 7 ulnar short 1 -0.268 0.01 1 0.64 0.08 0.128 -0.323 0.475 0.346 8 rad bow r -0.582 0.247 0.64 1 0.452 0.415 0.089 -0.268 0.01 9 rad bow 1 -0.656 0.093 0.08 0.452 1 0.09 0.312 -0.582 0.247 10 elb jt r -0.133 0.324 0.128 0.415 0.09 1 0.159 -0.656 0.093 11 elb jt 1 0.275 0.479 0.355 0.263 0.01 0.058 -0.397 -0.133 0.324 12 fem aa r 0.301 -0.402 -0.028 -0.441 0.704 0.194 0.297 0.275 0.479 13 fem aa 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 ma r 0.124 -0.638 -0.12 -0.268 0.563 -0.498 0.24 0.388 0.593 17 fem ma I 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 19 sharps 1 -0.275 -0.02 0.085 -0.114 0.269 0.72 -0.17 -0.278 -0.432 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 ankle jt r 0.158 -0.232 0.224 0.142 0.504 0.278 -0.114 861 0.384 23 ankle jt 1 -0.106 0.295 -0.518 0.229 -0.79 -0.407 0.083 0.689 0.213 24 % wt bear r 0.279 0.265 0.82 -0.277 0.724 0.51 0.083 0.078 0.35 25 % wt bear 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 1 0.656 0.109 0.282 -0.05 0.435 0.346 0.267 0.146 28 % distal -0.211 1 -0 446 0.087 0.337 -0.535 -0.389 0.249 0.158 29 %prox 0.1 K 1 1« 1 -0.327 0.118 0.104 0.29 -0.07 -0.106 30 % pelvic 0.043 -0.096 -0.084 1 0.058 -0.045 -0.304 -0.413 0.279 31 % d i a p h -0.286 0.529 -0.228 -0.249 1 -0.195 -0.238 0.217 -0.17 32 %flat bones 0.373 -0.324 0.098 -0.233 -0.16 1 0.345 -0.021 -0.229 33 %complex 0.456 0.268 1 0.028 0.738 0.361 1 -0.607 -0.149 34 %simple -0.215 0.787 -0.087 0.433 -0.27 0.479 0.434 1 -0 211 35 %flared 0.042 -0.787 0.087 0.104 0.27 -0.479 -0.434 -0.528 1 36 % not flared -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 8.6.4.2 Pearson Correlation Matrix (continued) 27 28 29 30 31 32 33 34 35 Corre la t ion M a t r i x % sess %distal % p r o x % pelvic % diaph % flatbones % complex % simple % f lared 41 % right -0.027 -0.308 -0.23 0.181 0.296 -0.393 0.016 0.325 0.042 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 A L D 0.214 0.036 -0.163 -0.377 0.536 0.172 -0.126 0.265 0.522 52 1 leg upper -0.466 -0.514 -0.085 -0.362 0.456 0.268 0.073 0.405 0.272 53 1 leg lower 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 upper 0.178 0.137 -0.168 0.126 0.335 -0.432 -0.414 0.279 0.262 57 r leg lower -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 L L D 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 Corre la t ion M a t r i x % not flared % o f 1 % o f 4 avg # % left % right % ht 1 a r m upper 1 a r m lower 1 # lesions 0.268 1 0.433 0.738 0.361 0.665 -0.362 0.456 0.268 2 carpa l slip r 0.022 0.433 1 0.27 0.543 0.755 -0.495 -0.194 0.022 3 carpa l slip 1 0.104 0.738 0.27 1 0.346 -0.18 -0.297 0.192 -0.104 4 r a d inclin r 0.34 0.361 0.543 0.346 1 0.594 -0.14 0.022 0.34 5 r a d inclin I 0.089 0.03 0.755 -0.18 0.594 1 -0.437 -0.323 0.089 6 ulnar short r 0.268 0.362 -0.495 -0.297 -0.14 -0.437 1 0.475 -0.268 7 ulnar short 1 0.01 0.456 -0.494 0.192 0.022 -0.323 0.475 1 0.01 8 r a d bow r 0.634 0.268 0.22 -0.104 0.34 0.089 -0.268 0.01 1 9 r a d bow 1 0.64 0.749 0.427 0.349 0.463 0.312 -0.582 0.247 0.64 10 elb jt r 0.08 0.473 0.104 0.319 -0.55 0.159 -0.656 0.093 0.08 11 elb jt 1 0.128 0.465 0.78 0.507 -0.47 -0.397 -0.133 0.324 0.128 12 fem aa r 0.355 0.041 0.42 -0.475 0.226 0.297 0.275 0.479 0.355 13 fem aa 1 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 ankle jt r 0.281 0.505 0.137 -0.564 -0.202 -0.141 861 0.384 -0.281 23 ankle jt 1 0.398 0.552 -0.257 -0.496 -0.2 0.131 0.689 0.213 -0.398 24 % wt bear r 0.559 0.304 -0.027 -0.326 0.253 0.348 0.078 0.35 0.559 25 % wt bear 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 % d i a p h 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 Corre la t ion M a t r i x % not flared % o f 1 % o f 4 avg # % left % right % ht 1 a r m upper 1 a r m lower 33 %complex 0.656 0.109 0.282 -0.05 0.435 0.346 -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 36 % not flared 1 0.084 0.28 -0.058 -0.045 -0.304 0.552 0.043 -0.096 37 % of 1 0.529 1 -0.249 -0.155 -0.195 -0.238 -0.244 -0.286 0.529 38 % o f 4 0.324 0.098 1 -0.16 0.336 0.345 0.243 0.373 -0.324 39 avg # 0.268 1 0 028 1 0.361 0.03 -0.362 0.456 0.268 40 % left 0.787 0.087 0.433 -0.27 1 0.434 -0.325 -0.215 0.787 41 % right 0.787 0.087 0.104 0.27 -0.479 i 0.325 0.042 -0.787 42 % h t 0.475 -0.47 -0.104 -0.186 -0.353 -0.145 1 -0.075 -0.475 43 I a r m upper 0.432 -0.28 -0.336 -0.28 -0.376 -0.01 0.126 1 -0.432 44 1 a r m lower -0.37 0.744 -0.305 -0.607 -0.519 -0.078 0.274 -0.099 1 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 47 r a r m 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 A L D 0.302 0.242 0.162 0.415 0.268 -0.423 0.265 0.522 0.302 52 1 leg upper 0.404 0.177 -0.143 -0.082 -0.655 -0.609 0.405 0.272 -0.404 53 1 leg lower -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 ratio 0.273 0.347 -0.402 0.601 0.354 -0.525 0.299 0.376 0.273 56 r leg upper -0.57 0.188 0.227 -0.121 -0.661 -0.414 0.279 0.262 -0.57 57 r leg lower 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 ratio 0.679 0.128 -280 0.525 -0.024 -0.157 0.134 0.44 -0.679 60 L L D 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 46 47 48 49 50 51 52 53 Corre la t ion M a t r i x total a r m 1 ratio 1 a r m r a r m uppder r a r m lower total a r m r r a r m ratio A L D l l e g upper l l e g lower 1 # lesions 0.749 0.473 0.465 -0.041 -0.397 0.256 0.364 0.217 0.665 2 carpa l slip r 0.427 0.104 0.078 0.42 -0.037 0.599 0.614 0.143 0.755 3 carpa l slip 1 0.349 0.319 0.507 -0.475 -0.004 0.17 0.144 -0.379 -0.18 4 r a d inclin r 0.463 0.055 -0.047 0.058 0.132 0.418 0.709 0.213 0.594 5 r a d inclin 1 0.312 0.159 -0.397 0.226 -0.161 0.556 0.444 0.24 -0.89 6 ulnar short r 0.582 0.656 -0.133 0.297 0.301 -0.532 0.079 0.388 -0.437 7 ulnar short 1 0.247 0.093 0.324 0.275 -0.402 -0.03 0.352 0.593 -0.323 8 r a d bow r 0.64 0.08 0.128 0.479 -0.028 0.271 0.312 0.355 0.089 9 rad bow 1 1 0.452 0.415 0.355 -0.441 0.411 0.484 0.27 0.312 10 elb jt r 0.452 1 0.09 0.263 -0.704 0.421 -0.307 -0.127 0.159 11 elb jt 1 0.415 0.09 1 0.01 0.194 0.098 0.256 -0.186 -0.397 12 fem aa r 0.263 0.01 0.058 1 -0.158 0.281 0.487 0.741 0.297 13 fem aa 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 ma r -0.55 0.127 -0.186 0.741 -0.311 0.077 0.53 1 0.24 17 fem ma 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 f ib ht r 0.114 0.269 0.72 0.615 0.208 0.063 -0.17 -0.249 -0.394 21 f ib ht 1 0.575 0.204 0.407 -0.023 0.235 -0.545 -0.09 -0.211 -0.332 22 ankle jt r 0.587 0.626 -0.251 -0.165 0.333 -0.21 0.213 0.517 -0.141 23 ankle jt 1 0.38 0.498 -0.238 -0.105 -0.348 0.049 0.276 0.363 0.131 24 % wt bear r 0.106 0.101 -0.279 -0.103 0.158 0.253 0.402 0.902 0.348 25 % wt bear 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 % p r o x 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 % d i a p h 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 0.278 198 8.6.4.2 Pearson Correlation Matrix (continued) 45 46 47 48 49 50 51 52 53 Corre la t ion M a t r i x total a r m 1 ratio 1 a r m r a r m uppder r a r m lower total a r m r r a r m ratio A L D H e g upper l l e g lower 35 %flared 0.379 0.605 0.47 -0.041 0.164 0.681 0.133 -0.148 0.29 36 % not 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 % o f 4 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 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 r a r m 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 1 -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 A L D 0.334 0.195 -0.084 -0.11 -0.465 -0 13 • -0 065 -0.126 52 1 leg upper 0.348 0.108 -0.309 -0.159 -0.596 -0.374 -0.448 0.073 53 1 leg lower 0.174 0.206 -0.359 -0.027 -0.742 -0.281 -0.513 0.068 1 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 upper 0.172 0.215 -0.366 -0.06 -0.619 -0.287 -0.519 -0.05 -0.414 57 r leg lower 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 L L D 0.214 0.041 0.456 -0.234 0.145 -0.064 0.009 0.132 -0.485 199 8.6.4.2 Pearson Correlation Matrix (continued) 54 55 56 57 58 59 60 Corre la t i on M a t r i x total l e g l H e g ratio r leg uppder r leg lower total l e g r r leg ratio L L D 1 # lesions 0.362 0.456 0.34 -0.754 0.445 0.125 0.297 2 carpa l slip r 0.495 0.194 0.647 -0.576 0.233 0.324 0.044 3 carpa l slip I 0.297 0.192 0.99 -0.75 0.34 0.859 0.26 4 r a d inclin r -0.14 0.022 0.322 -0.756 0.78 -0.94 -0.439 5 r a d inclin 1 0.437 0.323 0.538 0.34 0.98 0.23 -0.485 6 ulnar short r 0.765 0.475 0.283 0.76 0.5 0.35 0.308 7 ulnar short 1 0.475 0.346 0.73 0.23 0.55 -0.433 0.44 8 r a d bow r 0.268 0.01 0.93 0.123 0.456 -0.354 -0.339 9 r a d 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 aa r 0.275 0.479 883 0.83 0.04 0.94 -0.06 13 fem aa 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 ankle jt r 861 0.384 -0.734 -0.2 0.674 0.13 0.036 23 ankle jt 1 0.689 0.213 0.823 0.609 747 0.89 -0.163 24 % wt bear r 0.078 0.35 0.932 0.443 0.82 0.006 -0.377 25 % wt bear 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 % d i a p h 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 60 Corre la t ion M a t r i x total l e g l l l e g ratio r leg uppder r leg lower total leg r r leg ratio L L D 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 36 % not flared 0.552 0.043 0.566 0.54 0.009 0.345 -331 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 47 r a r m 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 A L D 0.265 0.522 -0.765 0.45 0.65 0.432 0.002 52 1 leg upper 0.405 0.272 0.789 0.576 0.7 0.35 0.922 53 1 leg lower 0.199 0.299 0.098 0.333 0.567 0.23 0.74 54 total leg 1 1 0.347 0.087 0.006 0.678 0.655 0.34 55 1 leg ratio 0 299 1 0.554 0.433 0.098 0.544 0.299 56 r leg upper 0.279 0 262 1 0.44 0.456 0.005 0.008 57 r leg lower 0.19 0.149 0.667 1 0.87 0.35 0.009 58 total leg r -0.03 0.158 0.453 0.333 1 0.234 0.343 59 r leg ratio 0.134 0.44 0.698 0.54 0.99 1 0.493 60 L L D 0.308 0.047 0.184 0.254 0.666 0.54 1 201 Appendix 8.7 Genotype - Phenotype Correlation Tables 8.7.1 Gene Table 8.7.1.1 Lesion Quality by Gene Variable 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 % R a n k 1 27.0 ± 10.1 31.6 ± 2 0 . 3 ( n = 1 7 ) 0.58 0.073 Lesion R a n k 2 6 . 4 1 2 . 9 3.9 ± 2 . 8 ( n = 17) 0.059 0.32 % R a n k 2 19.4 ± 7 . 5 22.6 ± 12.7 ( n = 17) 0.55 0.11 Lesion R a n k 3 5.0 ± 1.9 1.9+1.8 ( n = 17) <0.01 (0.0013) 0.94 % R a n k 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 ( n = 1 7 ) 0.019 0.50 % R a n k 4 37.7 ± 10.1 36.1 ± 1 8 . 9 ( n = 1 7 ) 0.83 0.053 Smal l (%) 28.4 ± 11.7 30.8 ± 1 7 . 9 ( n = 1 9 ) 0.74 0.061 M e d i u m (%) 30.6 ± 8.4 30.9 ± 13.9 ( n = 19) 0.96 0.050 L a r g e (%) 38.6 ± 15.7 36.5 ± 1 7 . 9 ( n - 1 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 No. 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 No. Distal 13.1 ± 5 . 0 8.1 ± 4 . 4 ( n = 19) 0.020 0.67 % Distal 40.2 ± 8.4 39.9 ± 14.5 ( n = 17) 0.97 0.051 No. P r o x i m a l 14.4 ± 5 . 2 9.4 ± 4 . 6 ( n = 19) 0.026 0.62 % P r o x i m a l 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 . 6 1 7 . 5 2.3 ± 5.2 (n = 17) 0.012 0.68 N o Diaphyseal 1.9 ± 1.7 1.3 ± 1.3 ( n = 19) 0.39 0.13 % Diaphyseal 6.5 ± 6 . 9 8.7 ± 12.0 (n = 17) 0.66 0.063 No. F lat 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. C o m p l e x 4.9 ± 5 . 9 2.7 ± 2 . 1 ( n = 19) 0.17 0.26 % C o m p l e x 12.4 ± 10.3 14.3 ± 9.3 (n = 17) 0.67 0.061 No. Simple 25.3 ± 5.4 17.3 ± 8.6 (n = 19) 0.32 0.58 % Simple 79.5 ± 10.6 84.1 ± 9 . 5 ( n = 17) 0.31 0.20 No. F l a r e d 14.1 ± 12.0 6 . 8 ± 5 . 9 ( n = 19) 0.047 0.52 % F l a r e d 38.6 ± 2 9 . 7 30.4 ± 2 4 . 2 ( n = 17) 0.48 0.099 No. Not F l a r e d 18.6 ± 7 . 8 12.9 ± 7.2 ( n = 17) 0.10 0.34 % Not F l a r e d 61.4 ± 2 9 . 7 69.6 ± 24.2 ( n = 17) 0.48 0.091 N o . Left 18.6 ± 6 . 7 10.1 ± 5 . 2 ( n = 19) <0.01 (0.0022) 0.92 % Left 56.6 ± 7 . 2 49.4 ± 10.09 ( n = 17) 0.13 0.39 N o . Right 14.3 ± 4 . 9 10.2 ± 4.9 ( n = 19) 0.076 0.42 % Right 43.8 ± 7 . 8 50.6 ± 10.9 ( n = 17) 0.15 0.34 202 Table 8.7.1.2. Limb Alignment by Gene Variable Normal Values EXT1 (n = 7) EXT 2 (n = 19) P-value Power 1. Carpal Slip Right 5 ± 2 m m 5.2 ± 2 . 8 2.2 ± 3 . 6 0.08 0.41 2. Carpal Slip Left 4.7 + 3.0 3.1 ± 3 . 5 0.29 0.17 3. Radial Inclination Right 21° ± 2 ° 2 8 . 0 ± 5 . 4 24.2 ± 5 . 0 0.13 0.31 4. Radial Inclination Left 30.6 ± 6.0 26.4 ± 4.8 0.08 0.41 5. Ulnar Shortening Right 0 ± 1 m m -1.2 ± 3 . 9 -1.7 ± 4 . 9 0.81 0.056 6. Ulnar Shortening Left 2.7 ± 5 . 8 -0.8 ± 4.9 0.14 0.30 7. Radial B o w Right 10° ± 5 ° 9.0 ± 2 . 3 7.6 ± 2 . 4 0.20 0.23 8. Radial B o w Left 14.2 + 8.4 7.7 + 2.4 <0.01 0.87 9. Radial Head Dislocation R 1 dislocation 1 dislocation 10. Radial Head Dislocation L 2 dislocations 1 dislocation 11. E lbow Joint Right 9° ± 3 ° -1.8 ± 18.5 -5.6 ± 12.9 0.58 0.082 12. E lbow Joint Left -3.9 ± 10.2 -8.5 ± 11.3 0.35 0.14 13. Femoral A . A . Right 7° ± 2 ° valgus -3.1 ± 7 . 8 -5.6 ± 9 . 1 0.53 0.093 14. Femoral A . A . Left -1.6 ± 8 . 3 -3.4 ± 9 . 0 0.64 0.073 15. Femoral N . S . Angle Right 135° ± 5 ° 143.1 ± 17.7 140.1 ± 8 . 0 0.55 0.088 16. Femoral N . S . Angle Left 146.4 ± 11.0 137.1 ± 9 . 1 0.04 0.56 17. Femoral M . A . Right 0 ° ± 5 ° varus 6.3 ± 5 . 4 -0.1 ± 6 . 0 0.03 0.59 18. Femoral M . A . Left -1.0 ± 7 . 0 1.1+5.0 0.42 0.12 19. Sharp's Right 35° ± 4 ° 38.5 ± 3 . 1 41.4 ± 5 . 7 0.29 0.23 20. Sharp's Left 38.5 ± 5 . 4 41.4 ± 4 . 9 0.31 0.16 21. Fibular Height Right 50 ± 10 52.0 ± 8.0 51.6 ± 11.7 0.94 0.051 22. Fibular Height Left 52.2+ 13.8 51.8 ± 14.4 0.95 0.052 23. Ank le Joint Angle Right 0 ° ± 5° -9.8 ± 13.6 -1.8 ± 10.1 0.14 0.062 24. Ank le Joint Angle Left -5.5 ± 14.4 -1.0 ± 10.6 0.42 0.052 25. % Weightbear Right 50 ± 10 61.0 ± 2 2 . 4 46.5 ± 22.2 0.18 0.36 26. % Weightbear Left 65.2 ± 11.9 51.4 ± 19.7 0.12 0.21 N u m b e r of parameters that fa l l beyond the no rma l range 1 5 / 2 4 4 / 2 4 203 Table 8.7.1.3. Segment Lengths and Percentile Height by Gene Variable E X T l (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 Upper L e g - Right 39.2 ± 4 . 5 44.0 ± 5.5 0.052 0.49 Lower 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 Lower 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 Upper A r m - Right 27.4 + 2.4 30.6 ± 3 . 8 0.052 0.49 Lower 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 Upper A r m - Left 27.5 ± 3 . 5 31.2 ± 4 . 4 0.059 0.47 Lower 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 V a r i a b l e M a l e s F e m a l e s P - v a l u e (n = 14) (n = 12) Les ion R a n k 1 9.1 ± 5 . 9 4 . 6 1 2 . 6 0.03 % R a n k 1 27.8 ± 16.8 32 .3119 .1 0.55 Les ion R a n k 2 5 . 3 1 3 . 4 3 . 8 1 2 . 4 0.24 % R a n k 2 18.8 ± 9 . 6 2 5 . 0 1 12.8 0.19 Les ion R a n k 3 3 . 7 1 2 . 5 1 .811 .7 0.04 % R a n k 3 12 .417 .0 1 0 . 3 1 7 . 6 0.49 Les ion R a n k 4 1 0 . 0 1 5 . 0 6 .9+4.5 0.13 % R a n k 4 3 6 . 5 1 1 6 . 3 3 6 . 7 1 1 7 . 9 0.97 Sma l l (%) 3 2 . 2 1 1 7 . 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 (%) 3 5 . 1 1 1 7 . 4 3 9 . 4 1 17.1 0.53 Average N u m b e r of Les ions 2 8 . 1 1 1 1 . 5 17 .21 7.2 0.01 No . Peduncu la ted 7 . 9 1 5 . 0 5 . 6 1 2 . 3 0.16 % Peduncu la ted 2 7 . 4 1 1 2 . 8 3 6 . 0 1 1 0 . 4 0.12 No . Sessile 19 .118 .5 11 .315 .4 0.01 % Sessile 68.0 + 14.6 5 8 . 2 1 6 . 7 0.08 No . D is ta l 11 .215 .4 7 . 4 1 3 . 9 0.05 % D is ta l 40.5 1 1 4 . 5 39 .5111 .1 0.86 No . P r o x i m a l 13 .415 .1 7 . 7 1 3 . 6 < 0.01 (0.0035) % P r o x i m a l 49 .3112 .1 4 1 . 4 1 1 9 . 5 0.30 No . Pe lv ic 1 .912.8 0 . 9 1 1 . 2 0.26 % Pe lv ic 5 . 2 1 7 . 6 3 . 5 1 5 . 7 0.55 N o D iaphysea l 1.1 1 1.1 1 .911 .6 0.13 % Diaphysea l 5 . 4 1 8 . 3 12 .9113 .7 0.16 No . F la t Bone 2 . 2 1 2 . 9 1 .211 .5 0.26 % F la t Bone 6 . 3 1 7 . 5 4 . 8 1 6 . 2 0.59 No . Comp lex 4 . 4 1 4 . 5 1 .911.4 0.07 % Comp lex 14 .517 .1 1 2 . 2 1 8 . 0 0.51 No . S imple 2 3 . 1 1 8 . 7 15 .216 .3 0.01 % S imple 8 3 . 9 1 8 . 7 8 3 . 3 1 8 . 0 0.88 N o . F l a r e d 12 .619 .1 4 . 3 1 4 . 9 0.01 % F l a r e d 45.0 1 2 5 . 2 18 .5117 .8 0.01 N o . No t F l a r e d 1 5 . 1 1 8 . 9 14 .016 .1 0.74 % Not F l a r e d 5 5 . 0 1 2 5 . 2 8 1 . 5 1 1 7 . 8 <0.01 (0.0079) N o . Lef t 1 5 . 2 1 7 . 2 9 . 0 1 4 . 3 0.02 % Lef t 5 2 . 1 1 10.6 5 1 . 0 1 1 0 . 0 0.83 N o . R igh t 1 3 . 4 1 5 . 6 8 . 8 1 3 . 5 0.02 % R igh t 4 7 . 9 1 1 0 . 6 49.3 110 .1 0.77 205 Table 8.7.2.2. Limb Alignment by Gender Var iable Normal Values Males (n = 14) Females (n = 12) P-value 1. Carpal Slip Right 5 ± 2mm 3.4 ± 4 . 3 ( n = 1 2 ) 2.5 ± 2 . 7 0.52 2. Carpal Sl ip Left 3.8 ± 3 . 6 3.3 ± 3 . 3 0.70 3. Radial Inclination Right 21° ± 2 ° 26.5 ± 4 . 6 ( n = 1 2 ) 23.8 ± 5 . 8 0.22 4. Radial Inclination Left 28.8 ± 5 . 7 26.1 ± 4 . 7 0.20 5. Ulnar Shortening Right 0 ± 1 m m -2.5 ± 4 . 6 ( n = 1 2 ) -0.58 ± 4 . 5 0.30 6. Ulnar Shortening Left 0.0 ± 5 . 6 0.33 ± 5 . 1 0.88 7. Radial B o w Right 10° ± 5 ° 8.1 ± 2 . 7 ( n = 1 3 ) 7.7 + 2.1 0.71 8. Radial B o w Left 10.0 ± 6 . 4 8.8 ± 4 . 1 0.58 9. Radial Head Dislocation R 1 dislocation 1 dislocation 10. Radial Head Dislocation L 1 dislocation 2 dislocations 11. E lbow Joint Right 9 ° ± 3° -3.8 ± 15.2 ( n = 1 3 ) -5.6 ± 13.4 0.77 12. E lbow Joint Left -7.9 ± 11.7 -6.5 ± 10.7 0.74 13. Femoral A . A . Right 7° ± 2 ° valgus 3.4 ± 4 . 3 ( n = 1 2 ) 2.5 ± 2 . 7 0.52 14. Femoral A . A . Left 3.8 ± 3 . 6 3.3 ± 3 . 3 0.70 15. Femoral N . S . Angle Right 1 3 5 ° ± 5 ° 140.9 ± 8.3 141.0 ± 14.1 0.97 16. Femoral N . S . Angle Left 138.0 + 8.1 141.4 ± 12.5 0.41 17. Femoral M . A . Right 0° ± 5° varus 2.2 + 6.7 ( n = 1 3 ) 0.55 ± 6 . 2 ( n = l l ) 0.53 18. Femoral M . A . Left -0.39 + 6.0 ( n = 1 3 ) 1.7 ± 4 . 7 ( n = l l ) 0.36 19. Sharp's Right 35° ± 4 ° 39.7 ± 3 . 9 ( n = 1 3 ) 42.3 ± 6.6 ( n = 1 0 ) 0.25 20. Sharp's Left 40.7 + 4.9 ( n = 1 3 ) 40.5 ± 5.3 ( n = 1 0 ) 0.93 21. Fibular Height Right 50 ± 10 54.5 ± 9.4 ( n = 1 3 ) 48.7 ± 11.7 0.18 22. Fibular Height Left 51.8 ± 12.8 ( n = 1 3 ) 51.9 ± 15.9 ( n = H ) 0.99 23. Ank le Joint Angle Right 0 ° ± 5° -4.2 ± 14.9 ( n = l l ) -3.7 ± 7 . 6 0.92 24. Ank le Joint Angle Left -2.0 ± 14.7 ( n = l l ) -2.3 ± 8.2 0.95 25. % Weightbear Right 50 ± 10 55.2 ± 24.9 ( n = 1 2 ) 45.1 ± 19.9 0.29 26. % Weightbear Left 54.2 ± 17.9 ( n = 1 2 ) 55.5 ± 2 0 . 4 ( n = 1 2 ) 0.87 Number of parameters that fall beyond the normal range 9 12 206 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 Lower 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 Lower L e g - Left 36.5 ± 4 . 5 33.6 ± 5 . 3 0.14 Total A r m Length - Right 50.4 ± 5.2 47.6 ± 6.9 0.25 Upper A r m - Right 31.0 ± 3 . 0 28.3 ± 4 . 1 0.072 Lower 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 Lower 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 V a r i a b l e M i s s e n s e N o n s e n s e S p l i c e S i t e F r a m e s h i f t p - v a l u e P o w e r (n=4) (n=14) (n=5) (n=3) Les ion R a n k 1 9.0 ± 1.6 7.6 ± 6 . 4 5.0 ± 0.0 5.0 ± 2 . 6 0.57 0.17 % R a n k 1 48.3 ± 2 1 . 6 30.0 ± 16.9 19.0 ± 5 . 5 21.3 ± 6 . 9 0.054 0.62 Les ion R a n k 2 4.0 ± 3 . 8 4.4 ± 2 . 6 6.0 ± 4 . 1 6.7 ± 3 . 1 0.53 0.18 % R a n k 2 16.0 ± 12.2 21.5 ± 11.8 23.8 ± 9 . 6 31.5 ± 13.1 0.38 0.24 Les ion R a n k 3 2.5 ± 1.9 2.6 ± 2 . 3 4.2 ± 2 . 6 2.3 ± 2 . 3 0.55 0.17 % R a n k 3 10.8 ± 5 . 9 10.1 ± 7 . 6 16.0 ± 7 . 1 9.0 ± 4 . 9 0.42 0.22 Les ion R a n k 4 5.5 ± 3 . 0 8.4 ± 5 . 1 12.6 ± 3 . 6 9.7 ± 8 . 1 0.22 0.36 % R a n k 4 24.8 ± 5 . 9 38.3 ± 20.2 41.2 ± 7 . 6 37.9 ± 14.0 0.47 0.20 Sma l l (%) 48.5 ± 19.3 29.7 ± 15.8 19.1 ± 6 . 7 26.4 ± 7 . 6 0.045 0.65 M e d i u m (%) 24.0 ±11 .3 30.7 ± 13.9 31 .4± 8.6 39.2 ± 12.3 0.49 0.19 L a r g e (%) 23.9 ± 10.8 37.3 ± 2 0 . 1 48.5 ± 5 . 9 34.3 ± 8 . 5 0.19 0.37 Average N u m b e r 21.0 ± 7 . 0 22.9 ± 12.8 27.8 ± 8 . 2 23.7 ± 12.5 0.81 0.10 of Les ions No . Peduncu la ted 6.3 ± 2 . 1 6.7 ± 4 . 6 6.6 ± 2.7 8.3 ± 6.7 0.93 0.074 % Peduncu la ted 30.8 ± 8.9 30.4 ± 13.5 24.9 ± 9.4 35.5 ± 16.3 0.69 0.13 N o . Sessile 14.8 ± 5 . 7 14.6 ± 9 . 3 18.8 ± 7 . 2 15.3 ± 8 . 1 0.81 0.10 % Sessile 69.2 ± 8.9 63.6 ± 15.7 66.9 ± 11.9 64.5 ± 16.3 0.90 0.080 No . D is ta l 9.0 ± 4 . 7 9.6 ± 5 . 9 10.8 ± 3 . 0 7.3 ± 5.0 0.84 0.095 % D is ta l 42.3 ± 17.5 41.1 ± 12.6 39.8 ± 8 . 3 18.2 ± 0 . 0 0.39 0.23 No . P r o x i m a l 9.5 ± 3 . 0 10.1 ± 5 . 7 12.8 ± 6 . 1 11.7 ± 5.5 0.76 0.11 % P r o x i m a l 48.1 ± 19.5 44.9 ± 15.9 44.1 ± 10.9 50.6 ± 3 . 7 0.92 0.075 No . Pe lv ic 0.75 ± 1.5 1.5 ± 2.8 2.0 ± 1.6 1.3 ± 1.5 0.89 0.083 % Pe lv ic 2.7 ± 5 . 4 4.5 ± 7 . 9 6.3 ± 4 . 5 0.0 ± 0 . 0 0.79 0.10 N o D iaphysea l 1.5 ± 2 . 4 1.1 ± 1.3 2.0 ± 1.0 2.0 ± 1.0 0.61 0.15 % D iaphysea l 6.0 ± 8 . 4 7.2 ± 12.1 8.2 ± 5 . 3 11.4 ± 13.8 0.92 0.075 No . F la t Bone 1.3 ± 1.5 1.7 ± 2 . 9 2.4 ± 1.8 1.3 ± 1.5 0.89 0.081 % F la t Bone 4.6 ± 5 . 5 5.6 ± 7 . 9 7.6 ± 5 . 2 0.0 ± 0 . 0 0.78 0.11 No . C o m p l e x 2.8 ± 0.96 3.7 ± 4 . 8 3.0 ± 1.6 2.3 ± 1.5 0.92 0.075 % C o m p l e x 14.9 ± 9 . 4 14.3 ± 11.2 10.3 ± 3 . 5 12.6 ± 8 . 5 0.86 0.089 No . S imple 18.3 ± 7 . 5 18.2 ± 9 . 0 22.8 ± 6.5 21.3 ± 13.1 0.76 0.12 % S imple 85.1 ± 9 . 4 82.2 ± 11.3 82.6 ± 7 . 9 87.4 ± 8 . 5 0.85 0.091 No . F l a r e d 5.8 ± 3.1 9.6 ± 8 . 9 9.0 ± 11.7 8.0 ± 8 . 2 0.89 0.081 % F l a r e d 32.9 ± 24.3 36.5 ±25 .3 27.3 ± 3 1 . 4 27.1 ± 19.2 0.88 0.084 No . No t F l a r e d 15.3 ± 9 . 6 13.2 ± 7 . 1 18.8 ± 8 . 3 14.7 ± 4 . 5 0.57 0.18 % Not F l a r e d 67.1 ±24 .3 63.5 ±25 .3 72.7 ± 3 1 . 4 68.7 ± 19.8 0.92 0.075 No . Le f t 12.5 ± 2 . 6 12.1 ± 8 . 7 14.4 ± 2 . 9 9.7 ± 4 . 9 0.83 0.096 % Le f t 61.7 ± 10.2 49.1 ± 9 . 8 53.3 ± 6 . 9 40.8 ± 7.9 0.039 0.67 N o . R igh t 8.5 ± 4 . 7 10.7 ± 4 . 5 13.6 ± 5 . 6 14.0 ± 8 . 2 0.39 0.24 % R igh t 38.3 ± 10.2 50.9 ± 9 . 8 47.3 ± 7.4 59.2 ± 7 . 9 0.044 0.65 208 Table 8.7.3.2. imb Alignment by Mutation Type Variable Norma l Values Missense (n=4) Nonsense (n=14) Splice Site (n=5) Frameshift (n=3) P" value Power 1. Carpal Slip Right 5 ± 2 m m 4.0 ± 3 . 4 2.0 ±4 .4 3.8 ±2 .5 3.7 ± 1.2 0.71 0.13 2. Carpal Slip Left 3.3 ± 3 . 2 3.6 ± 4 . 2 3.6 ±2 .3 3 .7±2 .1 0.99 0.052 3. Radial Inclination Right 21° ±2° 26.5 ± 7.9 24.9 ±4.1 27.4 ±0.55 20.7 ±9 .5 0.36 0.25 4. Radial Inclination Left 27.0 ±5 .3 27.2 ±5.1 30.0 ±5 .8 25.7 ±7 .6 0.70 0.13 5. Ulnar Shortening Right 0 ± 1 mm -1.0 ±2 .3 -2.5 ±5 .1 1.8 ± 1.8 -4.0 ± 6.2 0.25 0.32 6. Ulnar Shortening Left -4.7 ± 5 . 9 0.14 ±4 .8 5.2 ± 4.2 -4.7 ± 5.9 0.033 0.69 7. Radial Bow Right 10° ± 5 ° 10.0 ±2 .8 7.2 ±2 .5 8.4 ± 0.89 7.3 ± 1.5 0.20 0.37 8. Radial Bow Left 9.5 ±0.91 8.9 ±6 .7 11.3 ±5 .0 9.3 ±3 .8 0.88 0.086 9. Radial Head Dislocation R 1 dislocation 0 1 dislocation 0 lO.Radial Head Dislocation L 1 dislocation 1 dislocation 1 dislocation 0 11. Elbow Joint Right 9° ± 3 ° -18.3 ±5 .6 -1.6 ± 13.9 1.0 ± 16.2 -9.3 ±10 .0 0.13 0.45 12. Elbow Joint Left -10.0 ±6 .3 -6.6 ± 12.7 -6.8 ± 13.6 -7.3 ± 4.5 0.97 0.063 13. Femoral A.A. Right 7° ± 2 ° valgus -4.5 ± 8.8 -5.6 ±9 .1 -0.9 ± 4.5 -8.7 ± 13.5 0.66 0.14 14. Femoral A.A. Left 2.3 ±3 .3 -4.4 ± 10.4 -1.8 ±7 .5 -4.7 ± 5.7 0.59 0.16 15. Femoral N.S. Angle Right 135° ± 5 ° 142.0 ±5 .7 142.4 ± 6 . 2 139.2 ± 22.9 135.7 ± 11.0 0.81 0.10 16. Femoral N.S. Angle Left 142.8 ± 13.8 138.6 ±6 .5 144.2 ± 16.8 132.3 ±6 .8 0.41 0.23 17. Femoral M.A. Right 0 ° ± 5 ° varus 4.5 ±3.1 -0.27 ± 5.7 8.1 ±4 .3 -4.0 ± 8.0 0.027 0.73 18. Femoral M.A. Left 3.3 ±4 .5 0.0 ±6 .0 -2.0 ± 5.4 3.0 ±3 .6 0.48 0.19 19. Sharp's Right 35° ± 4 ° 41.5 ±6 .4 40.6 ±5 .5 39.6 ± 3 . 4 42.7 ± 8.0 0.90 0.078 20. Sharp's Left 37.0 ±5 .7 40.8 ± 4 . 9 38.6 ±4 .6 44.3 ± 5.5 0.35 0.25 21. Fibular Height Right 50 ± 10 53.8 ± 2 . 9 51.3 ± 11.3 43.5 ± 12.7 62.2 ± 1.0 0.15 0.43 22. Fibular Height Left 47.5 ± 15.8 49.2 ± 13.9 52.5 ± 10.1 68.3 ± 7.6 0.17 0.39 23. Ankle Joint Angle Right 0°± 5° -4.3 ± 3 . 8 -3.2 ± 14.1 0.75 ± 2.9 -13.0 ± 6.1 0.48 0.19 24. Ankle Joint Angle Left 1.0 ± 1.0 -1.1 ± 14.4 -1.0 ± 4.1 -11.3 ± 5.9 0.55 0.17 25. % Weightbear Right 50 ± 10 55.5 ±26.8 48.1 ±23.2 61.8 ±8 .9 36.3 ±29 .0 0.50 0.19 26. % Weightbear Left 67.0 ± 12.0 48.7 ± 20.9 58.8 =b 11.1 60.0 ± 20.2 0.34 0.26 Number of parameters that fall beyond the normal range 13 11 12 12 209 Table 8.7.3.3. Segment Lengths and Percentile Height by Mutation Type Variable Missense (n=4) Nonsense (n=14) Splice Site (n=5) Frameshift (n=3) p-value 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 4 1 . 9 ± 3 . 7 42.3 ± 1.9 0.21 0.36 Lower 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 Lower 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 45.3 ± 5.4 50.3 ± 6 . 1 47.9 ± 5.4 50.7 ± 4 . 1 0.53 0.18 Upper A r m - Right 26.6 ± 4 . 5 30.9 ± 3 . 9 29.3 ± 3.5 30.5 ± 2 . 2 0.28 0.29 Lower 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 5 1 . 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 Lower 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 Mul tation Severity Variable Severe (n=22) Mild (n=4) P-value Power Lesion Rank 1 6.7 ± 5.5 (n=20) 9.0 ± 1.6 0.42 0.12 % Rank 1 26.7 ± 15.2 48.3 ±21.6 0.024 0.64 Lesion Rank 2 4.8 ± 2.9 4.0 ±3.8 0.66 0.070 % Rank 2 22.8 ± 11.2 16.0 ± 12.2 0.29 0.17 Lesion Rank 3 2.9 ± 2.4 2.5 ± 1.9 0.76 0.060 % Rank 3 11.5 ±7.6 10.8 ±5.9 0.85 0.054 Lesion Rank 4 9.2 ±5.1 5.5 ±3.0 0.18 0.25 % Rank 4 38.9 ± 17.2 24.8 ±5.9 0.12 0.32 Small (%) 26.8 ± 13.7 48.5 ± 19.3 0.011 0.76 Medium (%) 32.0 ± 12.5 24.0 ± 11.3 0.24 0.19 Large (%) 39.4 ± 17.0 23.9 ± 10.8 0.095 0.37 Average Number of Lesions 23.5 ± 11.8 21.0 ±7.0 0.69 0.067 No. Pedunculated 6.9 ± 4.4 6.3 ±2.1 0.77 0.059 % Pedunculated 29.8 ± 12.7 30.8 ±8.9 0.89 0.052 No. Sessile 15.6 ±8.6 14.8 ±5.7 0.85 0.054 % Sessile 63.9 ± 14.3 69.2 ± 8.9 0.49 0.10 No. Distal 9.5 ±5.2 9.0 ± 4.7 0.85 0.054 % Distal 39.6 ± 12.2 42.3 ± 17.5 0.71 0.064 No. Proximal 10.9 ±5.6 9.5 ±3.0 0.62 0.076 % Proximal 45.2 ± 14.3 48.1 ± 19.5 0.73 0.063 No. Pelvic 1.6 ±2.4 0.75 ± 1.5 0.50 0.098 % Pelvic 4.8 ±7.0 2.7 ±5.4 0.58 0.082 No Diaphyseal 1.5 ± 1.2 1.5 ±2.4 0.95 0.050 % Diaphyseal 8.4 ± 11.2 6.0 ±8.4 0.69 0.067 No. Flat Bone 1.8 ±2.5 1.3 ± 1.5 0.66 0.070 % Flat Bone 5.8 ±7.1 4.6 ±5.5 0.75 0.060 No. Complex 3.4 ±3.9 2.8 ± 0.96 0.76 0.060 % Complex 13.5 ±9.6 14.9 ±9.4 0.79 0.058 No. Simple 19.7 ±8.9 18.3 ±7.5 0.77 0.060 % Simple 82.3 ± 10.1 85.1 ±9.4 0.62 0.076 No. Flared 9.3 ± 9.0 5.8 ± 3.1 0.45 0.11 % Flared 32.8 ±26.4 32.9 ±24.3 0.99 0.050 No. Not Flared 14.5 ±7.5 15.3 ±9.6 0.85 0.054 % Not Flared 67.2 ± 26.4 67.1 ±24.3 0.99 0.050 No. Left 12.3 ±7.3 12.5 ±2.6 0.96 0.050 % Left 49.5 ± 9.4 61.7 ± 10.2 0.029 0.60 No. Right 11.8 ± 5.2 8.5 ±4.7 0.25 0.20 % Right 50.6 ±9.5 38.3 ± 10.2 0.028 0.61 211 Table 8.7.4.2. Limb Alignment by Mutation Severity Variable Normal Values Severe (n=22) Mild (n=4) P-value 1. Carpal Sl ip Right 5 ± 2mm 2.7 ± 3 . 7 4.0 ± 3 . 4 0.53 2. Carpal Sl ip Left 3.6 ± 3 . 5 3.3 ± 3 . 2 0.86 3. Radial Inclination Right 21° ± 2 ° 24.9 ± 4.9 26.5 ± 7.9 0.59 4. Radial Inclination Left 27.6 ± 5 . 5 27.0 ± 5 . 3 0.83 5. Ulnar Shortening Right 0 ± 1 m m -1.7 ± 4 . 9 -1.0 ± 2 . 3 0.79 6. Ulnar Shortening Left 0.6 ± 5 . 5 -2.5 ± 3.5 0.28 7. Radial B o w Right 10° ± 5 ° 7.5 ± 2 . 1 10.0 ± 2 . 8 0.05 8. Radial B o w Left 9.5 ± 5.9 9.5 ± 0 . 9 0.99 9. Radial Head Dislocation R 1 dislocation 1 dislocation 10.Radial Head Dislocation L 2 dislocations 1 dislocation 11. E lbow Joint Right 9° ± 3 ° -2.1 ± 13.8 -18.3 ± 5 . 6 0.03 12. E lbow Joint Left -6.8 ± 11.7 -10.0 ± 6 . 3 0.60 13. Femoral A . A . Right 7° ± 2 ° valgus -5.0 ± 8 . 9 -4.5 ± 8.8 0.92 14. Femoral A . A . Left -3.9 ± 9 . 1 2.3 ± 3 . 3 0.20 15. Femoral N . S . Angle Right 135° ± 5 ° 140.7 ± 11.9 142.0 ± 5 . 7 0.84 16. Femoral N . S . Angle Left 139.0 ± 9 . 8 142.8 ± 13.8 0.51 17. Femoral M . A . Right 0° ± 5° varus 0.9 ± 6 . 8 4.5 ± 3 . 1 0.31 18. Femoral M . A . Left 0.1 ± 5 . 6 3.3 ± 4 . 5 0.29 19. Sharp's Right 35° ± 4 ° 40.7 ± 5 . 4 41.5 ± 6 . 4 0.84 20. Sharp's Left 40.9 ± 4 . 9 37.0 ± 5 . 7 0.30 21. Fibular Height Right 50 ± 10 51.3 ± 11.7 53.8 ± 2 . 9 0.69 22. Fibular Height Left 52.7 ± 13.9 47.5 ± 15.8 0.51 23. Ank le Joint Angle Right 0 ° ± 5 ° -3.9 ± 12.2 -4.3 ± 3.8 0.95 24. Ank le Joint Angle Left -2.6 ± 12.3 1.0 ± 1.0 0.62 25. % Weightbear Right 5 0 ± 10 49.1 ± 2 2 . 4 55.5 ± 2 6 . 8 0.62 26. % Weightbear Left 52.5 ± 19.2 67.0 ± 12.0 0.16 N u m b e r o f parameters that fall beyond the normal range 7/24 5/24 8/24 212 Table 8.7.4.3. Segment Lengths and Percentile Height by Mutation Severity Variable Mild (n=4) Severe (n=22) P-value Power Total Leg Length- Right 74.3 ± 13.7 85.2 ±7.2 0.022 0.65 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 Mut tation Location Variable Early (n=19) Late (n=7) P-Value Power Lesion Rank 1 7.5 ± 6.0 5.9 ± 1.6 0.48 0.10 % Rank 1 33.6 ± 19.9 22.1 ± 7 . 6 0.16 0.28 Lesion Rank 2 3.9 ± 2 . 6 6.3 ± 3 . 5 0.085 0.39 % Rank 2 20.6 ± 12.4 24.3 ± 8.7 0.48 0.10 Lesion Rank 3 2.3 ± 2 . 2 4.1 ± 2 . 2 0.072 0.43 % Rank 3 9.6 ± 6 . 9 15.7 ± 6 . 2 0.058 0.47 Lesion Rank 4 7.5 ± 5 . 0 11.3 ± 3 . 7 0.084 0.39 % Rank 4 36.0 ± 19.2 (n=17) 37.9 ± 8.6 0.81 0.056 Small (%) 32.8 ± 17.8 22.9 ± 8.9 0.18 0.25 Medium (%) 30.3 ± 14.1 32.1 ± 7 . 1 0.75 0.061 Large (%) 35.1 ± 18.2 42.2 ± 13.2 0.36 0.14 Average Number of Lesions 2 1 . 2 ± 12.1 27.6 ± 6.7 0.21 0.22 No. Pedunculated 6.8 ± 4 . 6 6.9 ± 2.4 0.97 0.050 % Pedunculated 31.8 ± 13.1 25.6 ± 8.2 0.27 0.18 No. Sessile 14.2 ± 8 . 5 19.0 ± 5 . 9 0.19 0.24 % Sessile 63.3 ± 14.7 68.5 ± 10.4 0.41 0.12 No. Distal 8.8 ± 5 . 6 11.3 ± 2.8 0.27 0.18 % Distal 39.4 ± 14.3 41.7 ± 8 . 6 0.69 0.067 No. Proximal 10.3 ± 5 . 3 12.0 ± 5 . 3 0.47 0.11 % Proximal 47.2 ± 16.3 42.2 ± 10.8 0.46 0.11 No. Pelvic 1.3 ± 2 . 5 1.9 ± 1.6 0.59 0.080 % Pelvic 3.7 ± 7.4 6.1 ± 4 . 8 0.46 0.11 No Diaphyseal 1.2 ± 1.2 2.1 ± 1.7 0.13 0.31 % Diaphyseal 7.9 ± 12.1 8.4 ± 6.7 0.91 0.051 No. Flat Bone 1.5 ± 2 . 6 2.4 ± 1.5 0.37 0.14 % Flat Bone 4.6 ± 7 . 5 8.1 ± 4 . 4 0.27 0.18 No. Complex 3.4 ± 4 . 1 2.9 ± 1.3 0.73 0.063 % Complex 15.3 ± 10.7 10.0 ± 3 . 1 0.22 0.21 No. Simple 18.1 ± 9 . 2 23.3 ± 5 . 4 0.17 0.26 % Simple 81.9 ± 10.6 84.9 ± 7 . 7 0.50 0.097 No. Flared 9.3 ± 8 . 1 7.4 ± 9.9 0.63 0.074 % Flared 36.8 ± 2 4 . 8 23.2 ± 2 6 . 6 0.25 0.19 No. Not Flared 12.3 ± 6 . 7 20.1 ± 7 . 2 0.019 0.68 % Not Flared 63.2 ± 2 4 . 8 76.8 ± 26.6 0.25 0.19 No. Left 11.6 ± 7 . 7 14.4 ± 2.4 0.35 0.14 % Left 50.8 ± 11.9 53.4 ± 5 . 6 0.58 0.082 No. Right 10.6 ± 5 . 3 13.3 ± 4 . 6 0.25 0.19 % Right 49.2 ± 11.9 47.0 ± 6 . 0 0.65 0.072 214 Table 8.7.5.2. Limb Alignment by Mut ation Location Variable Normal Values Early (n=19) Late (n=7) P-Value 1. Carpal Slip Right 5 ± 2mm 2.4 ± 3 . 7 4.3 ± 3 . 0 0.25 2. Carpal Sl ip Left 3.4 ± 3 . 7 4.0 ± 2 . 6 0.68 3. Radial Inclination Right 21° ± 2 ° 24.1 ± 5.2 27.7 ± 5 . 0 0.13 4. Radial Inclination Left 26.8 ± 5 . 1 29.4 ± 5 . 9 0.28 5. Ulnar Shortening Right 0 ± 1 m m -2.6 ± 4.9 1.0 ± 2 . 3 0.08 6. Ulnar Shortening Left -0.9 ± 5.0 3.0 ± 5 . 5 0.10 7. Radial B o w Right 10° ± 5 ° 7.6 ± 2 . 5 8.6 ± 1.9 0.39 8. Radial B o w Left 9.0 ± 5 . 8 10.8 ± 4 . 2 0.47 9. Radial Head Dislocation R 1 dislocation 1 dislocation 10. Radial Head Dislocation L 1 dislocation 1 dislocation 11. E lbow Joint Right 9° ± 3 ° -4.2 ± 13.0 -5.9 ± 17.7 0.80 12. E lbow Joint Left -7.1 ± 11.0 -7.9 ± 12.0 0.87 13. Femoral A . A . Right 7° ± 2 ° valgus -5.6 ± 9 . 3 -3.1 ± 7 . 1 0.53 14. Femoral A . A . Left -3.9 ± 9 . 2 -0.1 ± 6 . 8 0.33 15. Femoral N . S . Angle Right 135° ± 5 ° 141.3 ± 7 . 0 139.9 ± 19.2 0.77 16. Femoral N . S . Angle Left 137.6 ± 8 . 3 144.9 ± 13.8 0.11 17. Femoral M . A . Right 0 ° ± 5 ° v a r u s -0.6 ± 5 . 8 7.8 ± 3 . 4 <0.01 18. Femoral M . A . Left 0.6 ± 5.4 0.7 ± 6.2 0.97 19. Sharp's Right 35° ± 4° 41.2 ± 5 . 7 39.1 ± 3 . 2 0.44 20. Sharp's Left 41.4 ± 4 . 8 37.5 ± 4 . 7 0.12 21. Fibular Height Right 50 ± 10 53.4 ± 10.5 46.5 ± 10.9 0.18 22. Fibular Height Left 50.9 ± 15.2 54.8 ± 9 . 9 0.56 23. Ank le Joint Angle Right 0 ° ± 5 ° -4.7 ± 13.0 -1.7 ± 4 . 4 0.59 24. Ank le Joint Angle Left -2.9 ± 13.3 -0.2 ± 3 . 4 0.63 25. % Weightbear Right 50 ± 10 47.3 ± 22.7 58.7 ± 2 2 . 2 0.30 26. % Weightbear Left 51.7 ± 19.7 64.3 ± 12.7 0.15 N u m b e r of parameters that fall beyond the no rma l range 11 11 215 Table 8.7.5.3. Segment Lengths and Percentile Height by Mutation Location Variable Early (n=19) Late (n=7) P-Value Power Total L e g Length-Right 84.7 ± 9.0 80.5 ± 9.2 0.31 0.16 Upper L e g - Right 43.6 ± 5 . 6 40.3 ± 5.4 0.19 0.24 Lower L e g - Right 34.7 ± 3 . 6 33.1 ± 4 . 3 0.34 0.15 Total L e g Length - Left 83.9 ± 9 . 2 79.8 ± 9 . 3 0.32 0.16 Upper L e g - Left 42.7 ± 5.2 40.1 ± 5 . 2 0.28 0.18 Lower L e g - Left 35.9 ± 4 . 9 33.3 ± 4 . 8 0.25 0.19 Total A r m Length - Right 50.0 ± 5 . 9 46.8 ± 6 . 3 0.24 0.20 Upper A r m - Right 30.2 ± 3 . 7 28.5 ± 3 . 8 0.31 0.16 Lower A r m - Right 23.1 ± 3 . 5 22.1 ± 2 . 7 0.48 0.10 Total A r m Length - Left 50.0 ± 6 . 5 47.6 ± 5.9 0.39 0.13 Upper A r m - Left 30.5 ± 4 . 5 29.3 ± 4.4 0.54 0.089 Lower A r m - Left 23.3 ± 3 . 9 21.5 ± 3 . 9 0.26 0.19 Percentile Height 40.2 ± 3 1 . 3 15.6 ± 14.1 0.058 0.47 216 8.7.6 Gene and Gender Table 8.7.6.1 Lesion Quality by Gene and Gender Variable E X T 1 Males (n=4) EXT 1 Females (n=3) EXT 2 Males (n=10) EXT 2 Females (n=9) P-value E X T P-value G e n d e r Les ion R a n k 1 11.8 + 7.3 5 .711 .2 7 .915 .3 4 . 3 1 2 . 9 0.18 0.032 % R a n k 1 30.8+11.9 2 2 . 0 1 5.2 33.1 122 .2 29 .9119 .3 0.59 0.56 Les ion R a n k 2 7 . 0 1 2 . 9 5 .713 .5 4 . 6 1 3 . 5 3.1 1 1.7 0.064 0.22 % R a n k 2 18.815.3 20 .3111 .1 18.8111.3 26 .8113 .6 0.55 0.20 Les ion R a n k 3 5.8 + 2.2 4 . 0 1 1 . 0 2 . 8 1 2 . 0 1.0 10 .93 0.0006 0.013 % R a n k 3 15.8 + 5.9 16 .317 .6 10.9 + 7.2 8 . 0 1 6 . 7 0.049 0.47 Les ion R a n k 4 12.8 + 3.9 11 .314 .9 8.8 + 5.0 5.3 13 .3 0.018 0.097 % R a n k 4 34 .8110 .0 41 .7110 .6 37 .2118 .9 34 .8120 .3 0.84 0.97 S m a l l (%) 32.3 113 .8 2 3 . 2 1 7 . 5 32 .2120 .0 29.3 116 .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 A v g # of lesions 37.3 111 .4 26 .716 .1 2 4 . 0 1 9 . 5 13 .613 .2 0.0011 0.0032 No. Pedunculated 9 . 0 1 3 . 2 8.3 13 .1 8 .1+5.6 4.9 1 0 . 9 0.27 0.15 % Pedunculated 24 .114 .1 3 0 . 5 1 4 . 9 28 .9113 .9 38 .8111 .6 0.24 0.097 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 64 .41 15.3 64 .114 .3 67.7 116 .0 5 5 . 2 1 5 . 7 0.75 0.15 N o . Distal 14 .815 .7 11 .014 .0 9 .814 .8 6.2 + 3.2 0.016 0.038 % Distal 40 .0110 .8 4 0 . 4 1 6 . 0 40.7 116 .5 39 .2112 .8 0.97 0.86 N o . P r o x i m a l 17 .315 .0 10 .713 .8 11 .814 .4 6 .713 .1 0.0095 0.0016 % P r o x i m a l 4 6 . 8 1 8 . 0 40.1110.1 48 .5113 .8 4 2 . 1 1 2 3 . 7 0.81 0.37 N o . Pelvic 4 . 5 1 3 . 7 1.0 1 1 . 0 0 .901 1.7 0 .561 1.1 0.0038 0.19 % Pelvic 11.3 + 9.8 7 . 3 1 3 . 0 2 . 5 1 4 . 9 2 . 1 1 5 . 9 0.015 0.51 N o Diaphyseal 2.0 10 .82 3 . 0 1 2 . 0 1 .211.2 1 .711.5 0.50 0.11 % Diaphyseal 2 . 4 1 1 . 6 11 .917 .8 6 .419 .1 13.4116.5 0.57 0.12 N o . F la t Bone 5.3 13 .2 2 . 7 1 1 . 5 1.0 1 1 . 6 0 .671 1.1 0.0004 0.15 % F la t Bone 13 .717 .4 9 . 4 1 3 . 9 2.0 1 4.9 3.0 16 .1 0.0026 0.52 217 Table 8.7.6.1 Lesion Quality by Gene and Gender (continued) Variable E X T 1 Males (n=4) E X T 1 Females (n=3) E X T 2 Males (n=10) E X T 2 Females (n=9) P-value E X T P-va lue G e n d e r N o . C o m p l e x 6.8 ± 7 . 7 2.3 ± 1.5 3.4 ± 2 . 5 2.1 ± 1 . 3 0.24 0.15 % C o m p l e x 15.6 ± 13.1 8.2 ± 3 . 9 14. 9 ± 7 . 9 14.1 ± 9 . 1 0.61 0.47 N o . S imple 26. ± 5.6 23.3 ± 5 . 5 23.1 ± 8 . 9 12.3 ± 4 . 2 0.039 0.0076 % S imple 73.6 ± 10.1 87.4 ± 4 . 7 85. 1 ± 7.9 81.2 ± 8 . 9 0.33 0.59 N o . F l a r e d 2 1 . 0 ± 11.5 5 .0+4.4 9.2 + 5.7 4 .1+5.3 0.019 0.0043 % F l a r e d 54.6 ± 29.4 17.3 ± 12.1 40.6 + 23.7 18.9 ± 2 0 . 3 0.43 0.0097 N o . No t F l a r e d 16.3 ± 9.8 21.7 ± 3 . 5 14.6 ± 9 . 1 11.1 ± 3 . 9 0.10 0.73 % No t F l a r e d 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 % Lef t 58.2 ± 8.9 54.5 ± 5 . 1 52.1 ± 1 1 . 9 49.3 ± 11.8 0.27 0.52 N o . R i g h t 15.5 ± 5.5 12.7 ± 4 . 5 13.2 ± 5 . 7 7.4 ± 2 . 4 0.11 0.022 % R i g h t 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 V a r i a b l e N o r m a l Values E X T l Males (n=4) E X T 1 Females (n=3) E X T 2 Males (n=10) E X T 2 Females (n=9) P -value E X T P -value G e n d e r 1. Carpal Sl ip Right 5 ± 2mm 7.3 ± 2 . 1 2.1 ± 4 . 1 3.0 ± 1.0 2.3 ± 3 . 1 0.076 0.4941 2. Carpal Sl ip Left 6.8 ± 1.5 2.6 ± 3 . 5 2.0 ± 2 . 0 3.7 ± 3 . 7 0.27 0.68 3. Radial Inclination Right 21° ± 2 ° 31.3 ± 4 . 9 24.9 ± 3.3 24.7 ± 4.0 23.6 ± 6 . 5 0.13 0.21 4. Radial Inclination Left 30.8 ± 5 . 9 28.0 ± 5 . 7 30.3 ± 7 . 4 24.7 ± 2 . 9 0.079 0.19 5. Ulnar Shortening Right 0 ± 1 m m -3.7 ± 4 . 0 -2.2 ± 4.9 1.3 ± 1.5 -1.2 ± 5 . 1 0.81 0.32 6. Ulnar Shortening Left 1.3 ± 6 . 4 -0.50 ± 5 . 5 4.7 ± 5 . 5 -1.1 ± 4 . 5 0.15 0.87 7. Radial B o w Right 10° ± 5 ° 10.0 ± 2 . 6 7.5 ± 2 . 6 8.0 ± 1.7 7.6 ± 2 . 2 0.21 0.71 8. Radial B o w Left 14.9 ± 10.8 8.1 ± 2 . 5 13.3 ± 5 . 8 7.3 ± 2 . 2 0.0061 0.53 9. Radial Head Dislocation R 1 dislocation 0 0 1 dislocation lO.Radial Head Dislocation L 1 dislocation 1 dislocation 0 1 dislocation 11. E lbow Joint Right 9° ± 3 ° -0.33 ± 20.6 -4.9 ± 14.4 -3.3 ± 2 0 . 5 -6.3 ± 11.8 0.59 0.77 12. E lbow Joint Left 0.50 ± 8 . 2 -11.4 ± 11.4 -9 .7+11.2 -5.4 ± 10.9 0.34 0.73 13. Femoral A . A . Right 7° ± 2 ° valgus -1.8 ± 6 . 4 -5.2 ± 9 . 9 -4.8 ± 10.6 -6.0 ± 8.8 0.54 0.68 14. Femoral A . A . Left -1.4 ± 10.4 -5.5 ± 7 . 8 -1.8 ± 6 . 8 -1.1 ± 10.1 0.65 0.40 15. Femoral N . S . Angle R 1 3 5 ° ± 5 ° 139 ± 9 . 4 141.6 ± 8 . 2 148.7 ± 2 7 . 0 138.4 ± 7 . 9 0.55 0.97 16. Femoral N . S . Angle L 143.3 ± 3 . 4 135.9 ± 8 . 6 150.7 ± 17.2 138.3 ± 9 . 9 0.040 0.38 17. Femoral M . A . Right 0 ° ± 5 ° varus 4.5 ± 5 . 2 1.2 ± 7 . 4 9.8 ± 5 . 3 -1.5 ± 4 . 4 0.033 0.49 18. Femoral M . A . Left -2.8 ± 8.3 0.68 ± 4 . 9 2.5 ± 0 . 7 1 1 .615.2 0.43 0.37 19. Sharp's Right 35° ± 4 ° 37.3 ± 2 . 5 40.3 ± 4 . 1 40.3 ± 3 . 9 42.8 ±7 .3 0.30 0.26 20. Sharp's Left 38.3 ± 7 . 6 41.4 ± 4 . 2 38.8 ± 1.8 40.9 ± 5 . 9 0.33 0.93 21. Fibular Height Right 50 ± 10 57.0 ± 6 . 1 53.8 ± 10.4 47.0 ± 7.0 49.2 ± 13.2 0.94 0.19 22. Fibular Height Left 54.3 ± 19.4 51.1 ± 11.5 50.0 ± 9.2 52.6 ± 18.3 0.96 0.99 23. Ank le Joint Angle Right 0 ° ± 5 ° -19.7 ± 12.1 1.6 ± 11.6 0.0± 5.2 -4.9 ± 8 . 1 0.099 0.90 24. Ank le Joint Angle Left -13.0 ± 18.7 2.1 ± 11.0 2.0 ± 1.0 -3.7 ± 9 . 1 0.40 0.95 25. % Weightbear Right 50 ± 10 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 Parameters outside of normal range 16 13 10 8 219 Table 8 .7 .6 .3 . Segment Lengths and Percentile Height by Gene and Gender Variable EXT 1 Males (n=4) E X T 1 Females (n=3) EXT 2 Males (n=10) EXT 2 Females (n=9) P-value EXT P-value Gender Total L e g Length- Right 83.4 ± 4.2 74.7 ± 10.8 87.6 ± 5 . 7 82.1 ± 11.5 0.082 0.18 Upper L e g - Right 40.8 ± 2.7 37.2 ± 6.3 44.5 ± 4.0 43.4 ± 7 . 1 0.47 0.058 Lower L e g - Right 3 4 . 0 1 2 . 7 30.2 ± 4.8 36.2 ± 2 . 9 33.7 ± 3 . 9 0.059 0.11 Total L e g Length - Left 82.3 ± 3.2 74.3 ± 12.0 87.2 ± 6.6 81.1 ± 11.2 0.080 0.18 Upper L e g - Left 40.4 + 2.6 36.8 ± 7 . 1 43.6 + 3.8 42.8 + 6.5 0.53 0.069 Lower L e g - Left 33.5 ± 4 . 1 31.0 ± 5 . 8 37.8 ± 4 . 1 34.5 ± 5 . 2 0.13 0.084 Total A r m Length - Right 46.0 ± 3 . 8 43.3 ± 7 . 1 52.2 ± 4.7 49.1 ± 6 . 6 0.22 0.028 Upper A r m - Right 28.3 ± 1.3 26.3 ± 3.5 32.1 ± 2 . 9 29.0 ± 4.2 0.058 0.043 Lower A r m - Right 20.9 ± 2 . 1 21.0 ± 3 . 3 24.7 ± 2.8 22.3 ± 3 . 6 0.21 0.069 Total A r m Length - L e f t 46.0 ± 5 . 1 43.7 ± 6 . 0 52.9 ± 4 . 6 49.1 ± 6 . 9 0.17 0.026 Upper A r m - Left 28.6 ± 3 . 1 26.0 ± 4 . 0 32.3 ± 3 . 2 29.9 ± 5.3 0.17 0.058 Lower A r m - Left 19.6 ± 3 . 9 20.3 ± 2.4 25.0 ± 2 . 8 22.7 ± 3 . 6 0.30 0.011 Percentile Height 12.5 ± 17.7 5.0 ± 3 . 5 40.2 + 28.3 45.1 + 31.4 0.79 0.011 220 8.7.7 Gene and Mutation Type Table 8.7.7.1 Lesion Quality by Gene and Mutation Type Variable E X T 1 E X T 2 P-value Power E X T 1 E X T 2 P - Power Missense Missense Nonsense Nonsense value (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 % R a n k 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 % R a n k 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 % R a n k 3 15.0 ± 5 . 7 6.5 ± 0 . 7 1 0.17 0.24 11.0 ± 2.8 9.9 ± 8.2 0.87 0.053 Lesion R a n k 4 8.0 ± 1.4 3.0 ± 0 . 0 0.038 0.72 15.0 ± 1.4 7.3 ± 4 . 6 0.041 0.55 % R a n k 4 29.5 ± 3 . 5 20.0 ± 1.4 0.072 0.48 36.5 ± 38 .6±21 .5 0.89 0.052 14.8 Smal l (%) 32.8 ± 4 . 5 64.3 ± 0.057 0.57 39.7 ± 28.0 ± 16.3 0.36 0.14 10.1 10.4 M e d i u m (%) 33 .8± 14.2 ± 0 . 0 0.0007 1.0 22.5 ± 32.1 ± 13.9 0.39 0.13 0.71 15.3 L a r g e (%) 26.5 ± 21 .4± 0.72 0.058 37.8 ± 37.2 ± 20.5 0.97 0.050 14.8 10.1 25.7 Average 27.0 ± 1.4 15.0 ± 1.4 0.013 0.97 43.5 ± 19.4 ± 9 . 3 0.007 0.86 N u m b e r of 13.4 1 Lesions N o . 7.5 ± 2 . 1 5.0 ± 1.4 0.29 0.14 11.5 ± 2.1 5.9 ± 4 . 5 0.12 0.33 Pedunculated % 27.6 ± 6 . 4 33.9 ± 0.59 0.068 26.9 ±3.5 31 .0± 14.5 0.71 0.064 Pedunculated 12.6 N o . Sessile 19.5 ± 10.0 ± 2 . 8 0.044 0.66 27.5 ± 12.4 ± 6 . 2 0.027 0.64 0.71 17.7 % Sessile 72.3 ± 6.4 66.1 ± 0.59 0.068 59.8 ± 64.2 ± 15.6 0.73 0.062 12.6 22.2 N o . 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 % Distal 46.6 ± 37.9 ± 0.72 0.058 37.9 ± 7 . 8 41.6 ± 13.4 0.72 0.063 10.3 27.1 N o . P r o x i m a l 10.0 ± 2 . 8 9.0 ± 4 . 2 0.81 0.054 18.0 ± 5 . 7 8.8 ± 4 . 7 0.027 0.64 % P r o x i m a l 37.4 ± 58.9 ± 0.36 0.11 41.3 ± 45.6 ± 17.3 0.74 0.061 12.4 22.7 0.24 N o . 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 N o Diaphyseal 2.5 ± 3 . 5 0.50 ± 0.51 0.078 1.5 ± 0 . 7 1 1.1 ± 1.4 0.69 0.066 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 N o . F lat 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 % Fla t 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 N o . C o m p l e x 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 % C o m p l e x 9.3 ± 3 . 1 20.5 ± 0.31 0.13 18.5 ± 13.6 ± 10.1 0.59 0.079 11.4 21.9 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 Missense (n=2) E X T 2 Missense (n=2) P-value Power E X T 1 Nonsense (n=2) E X T 2 Nonsense (n=12) P - value Power % Simple 90.7 ±3.1 79.5 ± 11.4 0.31 0.13 68.3 ±3.2 84.5 ± 10.5 0.057 0.48 No. F l a r e d 3.5 ±0.71 8.0 ±2.8 0.16 0.25 25.5 ± 0.71 7.0 ± 6.4 0.001 9 0.96 % F l a r e d 13.0 ±3.3 52.7 ± 13.9 0.059 0.55 61.3 ± 17.3 32.3 ±24.5 0.14 0.29 No. Not F l a r e d 23.5 ±2.1 7.0 ± 1.4 0.011 0.98 18.0 ± 12.7 12.4 ±6.3 0.32 0.15 % Not F l a r e d 86.9 ±3.3 47.3 ± 13.9 0.059 0.55 38.7 ± 17.3 67.7 ±24.5 0.14 0.29 No. Left 14.5 ± 0.71 10.5 ±2.1 0.13 0.31 27.5 ±4.9 9.6 ±6.1 0.002 1 0.96 % Left 53.7 ± 0.19 69.6 ±7.6 0.097 0.38 64.5 ± 8.6 46.5 ± 7.5 0.009 3 0.82 No. Right 12.5 ± 0.71 4.5 ±0.71 0.0077 0.99 16.0 ±8.5 9.8 ±3.4 0.072 0.43 % Right 46.3 ± 0.19 30.4 ±7.6 0.097 0.38 35.5 ±8.6 53.5 ±7.5 0.009 3 0.82 222 Table 8.7.7.1 Lesion Quality by Gene and Mutation Type (continued) Variable E X T l E X T 2 P-value P o w e r E X T 2 Splice Site Splice Site F S (n=3) (n=2) (n=3) Les ion 5.0 ± 0 . 0 5.0 ± 0 . 0 2.0 ± 0 . 0 R a n k l % R a n k 1 18.3 ± 5 . 9 20.0 ± 7 . 1 0.79 0.055 18.0 ± 0 . 0 Les ion 5.7 ± 3 . 5 6.5 ± 6.4 0.86 0.052 4.0 ± 0 . 0 R a n k 2 % R a n k 2 18.0 ± 7 . 6 32.5 ± 0 . 7 1 0.082 0.43 36.0 ± 0.0 Les ion 5.7 ± 2 . 1 2.0 ± 1.4 0.12 0.32 1.0 ± 0 . 0 R a n k 3 % R a n k 3 20.0 ± 6 . 2 10.0 ± 1.4 0.12 0.32 9.0 ± 0 . 0 Les ion 13.0 ± 4 . 6 12.0 ± 2 . 8 0.81 0.054 4.0 ± 0 . 0 R a n k 4 % R a n k 4 44.0 ± 7 . 8 37.0 ± 7 . 1 0.39 0.11 36.0 ± 0 . 0 S m a l l (%) 17.9 ± 6 . 1 20.8 ± 9 . 8 0.71 0.060 26.4 ± 7.6 M e d i u m (%) 33.9 ± 0 . 9 8 27.8 ± 15.8 0.52 0.082 39.2 ± 12.3 L a r g e (%) 47.2 ± 6 . 3 50.5 ± 7 . 1 0.62 0.068 34.3 ± 8.5 Average 29.3 ± 8.3 25.5 ± 10.6 0.68 0.063 11.0 ± 0 . 0 N u m b e r of Lesions N o . 7.7 ± 3 . 1 5.0 ± 1.4 0.35 0.12 8.3 ± 6 . 7 Peduncula t ed % 26.3 ± 7.5 22.7 ± 14.9 0.74 0.058 45.5 ± 0 . 0 Peduncula t ed N o . Sessile 18.0 ± 4 . 6 20.0 ± 12.7 0.81 0.054 15.3 ± 8 . 1 % Sessile 61.8 ± 2 . 9 74.5 ± 18.9 0.30 0.14 54.5 ± 0.0 N o . Dis ta l 11.0 ± 4 . 0 10.5 ± 2 . 1 0.89 0.052 7.3 ± 5 . 0 % Dis t a l 37.5 ± 8 . 4 43.2 ± 9 . 6 0.53 0.080 18.2 ± 0 . 0 N o . 15.0 ± 6 . 0 9.5 ± 6.4 0.39 0.11 11.7 ± 5.5 P r o x i m a l % P r o x i m a l 50.1 ± 7 . 2 35.1 ± 10.4 0.15 0.28 54.5 ± 0 . 0 N o . Pe lv ic 2.0 ± 1.0 2.0 ± 2 . 8 0.050 1.3 ± 1.5 % Pe lv ic 6.5 ± 1.7 6.1 ± 8 . 6 0.93 0.051 0.0 ± 0 . 0 N o 1.7 ± 1 . 2 2.5 ± 0 . 7 1 0.44 0.097 2.0 ± 1.0 Diaphysea l % 6.9 ± 6 . 9 10.1 ± 1.4 0.59 0.072 27.3 ± 0.0 Diaphysea l N o . F la t 2.7 ± 1.5 2.0 ± 2 . 8 0.75 0.058 1.3 ± 1.5 Bone % F la t 8.6 ± 3 . 8 6.1 ± 8 . 6 0.66 0.064 0.0 ± 0.0 Bone N o . 3.3 ± 2 . 1 2.5 ± 0 . 7 1 0.64 0.066 2.3 ± 1.5 C o m p l e x % C o m p l e x 10.5 ± 4 . 8 10.1 ± 1.4 0.93 0.051 18.2 ± 0 . 0 223 Table 8.7.7.1 Lesion Quality by Gene and Mutation Type (continued) Variable E X T 1 Spl ice Site (n=3) E X T 2 Spl ice Site (n=2) P-value P o w e r E X T 2 F S (n=3) No. S imple 23.0 ± 5 . 3 22.5 ± 10.6 0.95 0.050 21.3 ± 13.1 % S imple 79.6 ± 8 . 8 87.1 ± 5 . 4 0.37 0.12 81.8 ± 0 . 0 No . F l a r e d 13.7 ± 13.9 2.0 ± 1.4 0.34 0.13 8.0 ± 8 . 0 % F l a r e d 40.6 ± 36.2 7.3 ± 2 . 5 0.31 0.14 9.1 ± 0 . 0 N o . No t F l a r e d 15.7 ± 7 . 8 23.5 ± 9 . 2 0.38 0.12 10.0 ± 0 . 0 % Not F l a r e d 59.4 ± 36.2 92.7 ± 2.5 0.31 0.14 90.9 ± 0 . 0 N o . Lef t 15.3 ± 3 . 1 13.0 ± 2 . 8 0.45 0.094 9.7 ± 4 . 9 % Lef t 53.3 ± 5 . 8 53.3 ± 11.1 0.99 0.050 36.4 ± 0 . 0 No . R igh t 14.3 ± 5 . 5 12.5 ± 7 . 8 0.77 0.056 14.0 ± 8 . 2 % R igh t 47.7 ± 6.9 46 .7± 11.1 0.91 0.051 63.6 ± 0 . 0 224 Table 8.7.7.2. Limb Alignment by Gene and Mutation Type V a r i a b l e N o r m a l Values E X T l M S (n=2) E X T 2 M S (n=2) P - value Powe r E X T 1 N S (n=2) E X T 2 N S (n=12) P - value Power 1. Carpal Sl ip Right 5 ± 2mm 5.5 ± 4 . 9 2.5 ± 0.71 0.49 0.083 5.0 1.8 ± 4 . 5 0.51 0.093 2. Carpal Sl ip Left 5.0 ± 4 . 2 1.5 ± 0.71 0.37 0.11 7.0 ± 1.4 3.0 ± 4 . 2 0.22 0.21 3. Radial Inclination Right 21° ± 2 ° 28.5 ± 12.0 24.5 ± 4.9 0.71 0.059 29.0 24.5 ± 4.0 0.32 0.15 4. Radial Inclination Left 28.0 ± 8.5 26.0 ± 2.8 0.78 0.055 28.5 ± 9.2 27.0 ± 4.7 0.71 0.064 5. Ulnar Shortening Right 0 ± 1 m m -1.0 ± 2.8 - 1 . 0 ± 2 . 8 0.050 -8.0 -2.0 ± 5.0 0.28 0.17 6. Ulnar Shortening Left -2.5 ± 4.9 -2.5 ± 3.5 0 1.5 ± 4 . 9 -0.83 ± 4 . 9 0.68 0.067 7. Radial B o w Right 10° ± 5 ° 9.0 ± 4 . 2 1 1 . 0 ± 1.4 0.59 0.068 11.0 6.9 ± 2 . 4 0.12 0.32 8. Radial B o w Left 9.5 ± 1.4 9.5 ± 0.71 0.050 20.0 ± 15.6 7.0 ± 2 . 1 0.0048 0.89 9. Radial Head Dislocation R 0 1 dislocatio n 0 0 10. Radial Head Dislocation L 0 1 dislocatio n 1 dislocati on 0 11 E lbow Joint Right 9° ± 3 ° -23.0 ± 1.4 -13.5 ± 0.71 0.014 0.97 2.0 -1.9 ± 14.5 0.80 0.056 12. E lbow Joint Left -10.5 ± 10.6 -9.5 ± 2 . 1 0.91 0.051 -3.5 ± 4.9 -7.2 ± 13.7 0.72 0.063 13. Femoral A . A . Right 7° ± 2 ° valgus -8.5 ± 12.0 -0.50 ± 4.9 0.48 0.084 -5.5 ± 7.8 -5.7 ± 9 . 7 0.98 0.050 14. Femoral A . A . Left 4.0 ± 2 . 8 0.50 ± 3.5 0.39 0.10 -9.5 ± 3.5 -3.6 ± 11.0 0.48 0.10 15. Femoral N . S . Angle Right 1 3 5 ° ± 5 ° 141.5 ± 9.2 142.5 ± 3.5 0.90 0.051 146.0 ± 7.1 141.8 ± 6 . 2 0.39 0.13 16. Femoral N . S . Angle Left 146.5 ± 2.1 139.0 ± 22.6 0.69 0.060 142.5 ± 0.71 137.9 ± 6 . 8 0.37 0.13 17. Femoral M . A . Right 0 ° ± 5 ° varus 7.0 ± 1.4 2.0 ± 1.4 0.072 0.48 1.0 ± 5 . 7 -0.50 ± 5 . 9 0.75 0.060 18. Femoral M . A . Left 6.0 ± 4 . 2 0.50 ± 3.5 0.29 0.14 -5.0 ± 2.8 0.91 ± 6 . 0 0.21 0.22 19. Sharp's Right 35° ± 4° 37.0 (n=l) 46 (n=l) 37.5 ± 3.5 41.1 ± 5 . 7 0.42 0.12 225 Table 8.7.7.2. Limb Alignment by Gene and Mutation Type (continued) V a r i a b l e N o r m a l V a l u e s E X T l M S (n=2) E X T 2 M S (n=2) P - value Power E X T l N S (n=2) E X T 2 N S (n=12) P - value Power 20. Sharp's Left 33.0 (n=l) 41.0 (n=l) 41 .0± 8.5 40.8 ± 4.6 0.96 0.050 21. Fibular Height Right 50 ± 10 52.5 ± 0.71 55.0 ± 4.2 0.49 0.081 59.0 ± 7.1 49.9 ± 11.5 0.31 0.16 22. Fibular Height Left 59.5 ± 10.6 35.5 ± 7.8 0.12 0.31 48.0 ± 22.6 49.5 ± 13.5 0.90 0.052 23. Ank le Joint Ang le Right 0 ° ± 5° -6.5 ± 0.71 0.0 0.084 0.44 -26.0 ± 7.1 1.0 ± 10.5 0.005 5 0.89 24. Ank le Joint Ang le Left 1.5 ± 0.71 0.0 0.33 0.11 -20.5 ± 19.1 2.4 ± 11.1 0.03 0.61 25. % Weightbear Right 50 ± 10 52.5 ± 45.9 58.5 ± 3.5 0.87 0.052 61.5 ± 10.6 45.7 ±24 .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 beyond the normal range 111 14 15 9 226 Table 8.7.7.2. Limb Alignment by Gene and Mutation Type (continued) Variable Normal E X T l E X T 2 P-value Power E X T 2 Values Splice Site (n=3) Splice Site (n=2) FS (n=3) 1. Carpal 5 ± 2mm 5.0 ± 2 . 6 2.0 ± 0 . 0 0.23 0.19 3.7 ± 1.2 Slip Right 2. Carpal 3.0 ± 2 . 6 4.5 ± 2 . 1 0.56 0.076 3.7 ± 2 . 1 Sl ip Left 3. Radial 21° ± 2 ° 27.3 ± 0.58 27.5 ± 0 . 7 1 0.79 0.055 20.7 ± 9 . 5 Inclination Right 4. Radial 3 3 . 7 ± 2 . 1 24.5 ± 4.9 0.057 0.54 25.7 ± 7 . 6 Inclination Left 5. Ulnar 0 ± 1 m m 1.0 ± 1.7 3.0 ± 1.4 0.27 0.16 -4.0 ± 6.2 Shortening Right 6. Ulnar 7.0 ± 4 . 6 2 . 5 ± 2 . 1 0.29 0.14 -4.7 ± 5.9 Shortening Left 7. Radial 10° ± 5 ° 8.3 ± 1.2 8.5 ± 0 . 7 1 0.87 0.052 7.3 ± 1.5 B o w Right 8. Radial 13.5 ± 5 . 7 8.0 ± 0 . 0 0.28 0.15 9.3 ± 3 . 8 B o w Left 9. Radial 1 dislocation 0 Head Dislocation R 10. Radial 1 dislocation 0 Head Dislocation L 11 E lbow 9° ± 3 ° 11.0 ± 12.2 -14.0 ± 2 . 8 0.073 0.46 -9.3 ± 10.0 Joint Right 12. E lbow 0.33 ± 13.2 -17.5 ± 2 . 1 0.17 0.24 -7.3 ± 4.5 Joint Left 13. Femoral 7 ° ± 2° 2.2 ± 2 . 0 -5.5 ± 0 . 7 1 0.016 0.89 -8.7 13.5 A . A . Right valgus 14. Femoral 0.0 ± 9 . 9 -4.5 ± 0 . 7 1 0.59 0.82 -4.7 ± 5.7 A . A . Left 15. Femoral 135° ± 5 ° 142.3 ± 29.4 134.5 ± 17.7 0.76 0.057 135.7 ± 11.0 N . S . Angle Right 16. Femoral 149.0 ± 18.2 137.0 ± 16.9 0.51 0.083 132.3 ± 6 . 8 N . S . Angle Left 17. Femoral 0 ° ± 5° 10.8 ± 3 . 9 5.5 ± 3 . 5 0.29 0.14 -4.0 ± 8.0 M . A . Right varus 18. Femoral -4.0 ± 8 . 5 0.0 ± 0 . 0 0.57 0.070 3.0 ± 3 . 6 M . A . Left 19. Sharp's 35° ± 4° 40.3 ± 3 . 9 39.0 ± 4 . 2 0.79 0.054 42.7 ± 8 . 0 Right 20. Sharp's 38.8 ± 1.8 38.5 ± 7 . 8 0.97 0.050 44.3 ± 5 . 5 Left 227 Table 8.7.7.2. Limb Alignment by Gene and Mutation Type (continued) V a r i a b l e N o r m a l V a l u e s E X T l Spl ice Site (n=3) E X T 2 Spl ice Site (n=2) P-va lue P o w e r E X T 2 F S (n=3) 21. Fibular Height Right 50 ± 10 44.5 ± 7.8 42.5 ± 20.5 0.91 0.051 62.2 ± 1.0 22. Fibular Height Left 49.0 ± 12.7 56.0 ± 9 . 9 0.60 0.067 68.3 ± 7 . 6 23. Ank le Joint Angle Right 0 ° ± 5 ° 3.0 ± 0 . 0 -1.5 ± 2 . 1 0.096 0.39 -13.0 ± 6 . 1 24. Ank le Joint Angle Left 2.5 ± 0 . 7 1 -4.5 ± 0 . 7 1 0.010 0.99 -11.3 ± 5 . 9 25. % Weightbear Right 50 ± 10 69.0 ± 1.4 54.5 ± 4 . 9 0.058 0.56 36.3 ± 29.0 26. % Weightbear Left 67.0 ± 9 . 9 50.5 ± 0 . 7 1 0.14 0.28 60.0 ± 20.2 Parameters beyond the no rma l range 14 9 12 228 Table 8.7.7.3. Segment Lengths and Percentile Height by Gene and Mutation Type V a r i a b l e E X T l M S (n=2) E X T 2 M S (n=2) P - value Power E X T 1 N S (n=2) E X T 2 N S (n=12) P - value Power Total Leg Length- Right 73.5 ± 14.1 75.0 ± 19.1 0.94 0.050 80.5 ±2.1 86.7 ± 8.4 0.34 0.15 Upper Leg - Right 36.3 ±8.8 38.3 ± 10.3 0.85 0.052 38.5 ±0.71 45.7 ±5.0 0.075 0.42 Lower Leg - Right 30.0 ±6.4 33.0 ±9.2 0.74 0.057 32.0 ± 1.4 35.5 ±3.2 0.17 0.25 Total Leg Length - Left 72.3 ± 14.5 74.8 ± 18.7 0.90 0.051 80.0 ± 1.4 85.9 ±9.1 0.39 0.13 Upper Leg - Left 36.0 ±9.9 38.5 ±8.5 0.81 0.053 38.3 ± 1.1 44.6 ±5.0 0.11 0.35 Lower Leg - Left 29.0 ± 4.9 32.0 ±8.5 0.71 0.059 31.0± 1.4 37.2 ±4.9 0.12 0.33 Total Arm Length - Right 44.0 ± 9.9 46.5 ± 11.3 0.84 0.053 43.0 ±0.0 51.6 ±5.4 0.051 0.51 Upper Arm -Right 26.5 ± 4.9 26.8 ± 6.0 0.97 0.050 28.0 ±0.0 31.1 ±3.8 0.29 0.17 Lower Arm -Right 20.3 ± 3.9 21.8 ±6.0 0.79 0.054 19.5 ±2.1 23.9±3.1 0.084 0.40 Total Arm Length - Left 45.3 ± 10.3 45.3 ± 12.4 0.050 42.0 ± 1.4 52.0 ±5.3 0.025 0.66 Upper Arm -Left 27.5 ±7.8 25.3 ± 6.0 0.78 0.055 26.5 ±0.71 31.8 ± 4.3 0.12 0.32 Lower Arm -Left 22.0 ±3.5 22.8 ±7.4 0.91 0.051 17.5 ±3.5 24.5 ±2.7 0.0065 0.87 Percentile Height 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 Table 8.7.7.3. Segment Lengths and Percentile Height by Gene and Mutation Type (continued) Variable E X T 1 Splice Site (n=3) E X T 2 Splice Site (n=2) P-value Power E X T 2 F S (n=3) Total L e g Length-Right 83.2 ± 6 . 9 83.5 ± 8 . 5 0.96 0.050 85.7 ± 4 . 3 Upper L e g - Right 41.7 ± 2 . 0 42.3 ± 6.7 0.89 0.051 42.3 ± 1.9 Lower L e g - Right 34.2 ± 3 . 8 34.5 ± 3 . 5 0.93 0.051 34.7 ± 2 . 0 Total L e g Length - Left 82.5 ± 6.5 83.3 ± 8 . 1 0.92 0.051 84.8 ± 2.9 Upper L e g - Left 41.2 ± 2 . 4 42.8 ± 6.0 0.69 0.062 41.0 ± 0 . 9 Lower L e g - Left 35.7 ± 4 . 6 34.0 ± 4.2 0.71 0.060 36.7 ± 1.2 Total A r m Length - Right 46.7 ± 4.5 49.8 ± 8 . 1 0.61 0.069 50.7 ± 4 . 1 Upper A r m - Right 27.7 ± 2 . 1 3 1 . 8 ± 4 . 6 0.25 0.17 30.5 ± 2 . 2 Lower A r m - Right 22.3 ± 1.4 23.5 ± 3 . 5 0.63 0.068 23.5 ± 4 . 4 Total A r m Length - Left 46.8 ± 3 . 5 51.3 ± 6 . 0 0.36 0.12 5 1 . 0 ± 5 . 8 Upper A r m - Left 28.2 ± 2 . 0 32.8 ± 3 . 9 0.17 0.24 31.5 ± 1.8 Lower A r m - Left 20.2 ± 2.5 23.0 ± 2 . 8 0.32 0.14 23.0 ± 4 . 6 Percentile Height 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 Variable E X T l Severe (n=5) EXT 2 Severe (n=17) P - value P o w er E X T l M i l d (n=2) EXT 2 M i l d (n=2) P - value P o w e r Les ion R a n k 1 9.6 ± 7 . 4 5.7 ± 4 . 4 0.16 0.28 8.0 ± 1.4 10.0 ± 1.4 0.29 0.14 % R a n k 1 25.8 ± 11.6 26.5 ± 15.7 0.93 0.05 1 30.0 ± 7 . 1 66.5 ± 3.5 0.022 0.88 Les ion R a n k 2 6.2 ± 3 . 3 4.7 ± 2 . 9 0.34 0.15 7.0 ± 2 . 8 1.0 ± 0 . 0 0.096 0.39 % R a n k 2 17.0 ± 6 . 0 25.2 ± 12.1 0.16 0.27 25.5 ± 9.2 6.5 ± 0 . 7 1 0.10 0.37 Les ion R a n k 3 5.4 ± 2 . 1 2.2 ± 1.9 0.0043 0.88 4.0 ± 1.4 1.0 ± 0 . 0 0.096 0.39 % R a n k 3 16.4 ± 6 . 8 9.8 ± 7 . 0 0.079 0.41 15.0 ± 5 . 7 6.5 ± 0 . 7 1 0.17 0.24 Les ion R a n k 4 13.8 ± 3 . 5 8.2 ± 5 . 1 0.035 0.57 8.0 ± 1.4 3.0 ± 0 . 0 0.038 0.72 % R a n k 4 4 1 . 0 ± 10.1 38.3 ± 18.6 0.76 0.06 0 29.5 ± 3 . 5 20.0 ± 1.4 0.072 0.48 S m a l l (%) 26.6 ± 13.7 26.9 ± 14.2 0.97 0.05 0 32.8 ± 4 . 5 64.3 ± 10.1 0.057 0.57 M e d i u m (%) 29.3 ± 9.9 32.8 ± 13.4 0.59 0.08 0 33.8 ±0.71 14.0 ±0.0 0.0007 1.0 L a r g e (%) 43.4 ± 14.5 38.3 ± 17.9 0.57 0.08 5 26.5 ± 14.8 21.4 ± 10.1 0.72 0.058 Average N u m b e r o f Lesions 35.0 ± 11.8 20.9 ± 9.6 0.012 0.75 27.0 ± 1.4 15.0 ± 1.4 0.014 0.97 N o . Peduncula ted 9.2 ± 3 . 2 6.2 ± 4 . 5 0.19 0.24 7.5 ± 2 . 1 5.0 ± 1.4 0.29 0.14 % Peduncula ted 26.6 ± 5 . 6 30.8 ± 14.3 0.53 0.09 2 27.6 ± 6.4 33.9 ± 12.6 0.59 0.068 N o . Sessile 21.8 ± 10.8 13.8 ± 7 . 2 0.065 0.45 19.5 ± 0 . 7 1 10.0 ± 2 . 8 0.044 0.66 % Sessile 6 1 . 0 ± 11.3 65.5 ± 15.3 0.55 0.08 7 72.4 ± 6.4 66.1 ± 12.6 0.59 0.068 N o . Dis ta l 13.4 ± 6 . 1 8.4 ± 4 . 5 0.057 0.47 12.5 ± 2 . 1 5.5 ± 3 . 5 0.14 0.28 % Dis t a l 37.6 ± 7 . 1 39.8 ± 13.7 0.75 0.06 1 46.6 ± 10.3 37.9 ± 2 7 . 1 0.72 0.058 N o . P r o x i m a l 16.2 ± 5 . 4 9.4 ± 4 . 7 0.013 0.75 10.0 ± 2 . 8 9.0 ± 4 . 2 0.81 0.054 % P r o x i m a l 46.6 ± 6 . 9 45.2 ± 15.3 0.85 0.05 4 37.4 ± 12.4 58.9 ± 2 2 . 7 0.36 0.11 N o . Pelv ic 4.2 ±3.1 0.82 ±1.5 0.0024 0.93 1.5 ± 2.1 0.0 ± 0 . 0 0.42 0.095 % Pe lv ic 11.3 ± 7.5 3.2 ± 5 . 7 0.017 0.70 5.4 ± 7 . 6 0.0 ± 0 . 0 0.42 0.095 N o Diaphysea l 1.6 ± 0 . 8 9 1.4 ± 1.3 0.77 0.05 9 2.5 ± 3 . 5 0.50 ± 0 . 7 1 0.51 0.078 % Diaphysea l 5.5 ± 5 . 3 8.7 ± 11.9 0.58 0.08 3 8.9 ± 12.6 3.1 ± 4 . 4 0.60 0.067 N o . F la t Bone 4.8 ±3.1 0.94 ±1.4 0.0007 0.98 2.5 ± 0 . 7 1 0.0 ± 0 . 0 0.038 0.72 % F la t Bone 12.9 ± 7 . 1 3.9 ± 5 . 7 0.0081 0.81 9.2 ± 2 . 1 0.0 ± 0 . 0 0.026 0.85 N o . C o m p l e x 5.8 ± 7 . 1 2.6 ± 2 . 2 0.11 0.34 2.5 ± 0 . 7 1 3.0 ± 1.4 0.69 0.059 % C o m p l e x 13.7 ± 12.3 13.0 ± 8 . 9 0.89 0.05 2 9.3 ± 3 . 1 20.5 ± 11.4 0.31 0.13 N o . S imple 25.6 ± 6 . 5 17.9 ± 8 . 9 0.090 0.38 24.5 ± 2 . 1 12.0 ± 2 . 8 0.038 0.72 % Simple 75.1 ± 8 . 9 85.4 ± 9.4 0.042 0.54 90.7 ± 3 . 1 79.5 ± 11.4 0.31 0.13 231 Table 8.7.8.1 Lesion Quality by Gene and Severity (continued) Variable E X T 1 Severe (n=5) E X T 2 Severe (n=17) P - value Pow er E X T l M i l d (n=2) E X T 2 M i l d (n=2) P - value Power No. F l a r e d 18.4 ± 11.8 6.6 ± 6.3 0.0068 0.83 3.5 ± 0 . 7 1 8.0 ± 2 . 8 0.16 0.25 % F l a r e d 48.9 ± 29.3 28.5 ± 2 2 . 9 0.11 0.33 13.0 ± 3.3 52.7 ± 13.9 0.059 0.55 No. Not F l a r e d 16.6 ± 8 . 5 14.1 ± 6 . 9 0.51 0.09 6 23.5 ± 2 . 1 7.0 ± 1.4 0.011 0.98 % Not F l a r e d 51.1 ± 29.3 70.8 ± 2 3 . 0 0.13 0.31 86.9 ± 3 . 3 47.3 ± 13.9 0.059 0.55 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.S 1.2. Limb A ignmenl t by Gene and Severity V a r i a b l e N o r m a l Values E X T 1 Severe (n=5) E X T 2 Severe (n=17) P - value Power E X T l M i l d (n=2) E X T 2 M i l d (n=2) P - value Power 1. Carpal S l i p R 5 ± 2mm 5.0 ± 2.2 2.2 ± 3 . 8 0.17 0.26 5.5 ± 4 . 9 2.5 ± 0.71 0.49 0.083 2. Carpal S l i p L 4.6 ± 2.9 3.3 ± 3 . 7 0.48 0.10 5.0 ± 4 . 2 1.5 ± 0.71 0.37 0.11 3. Radial Inclination R 21° ± 2 ° 27.8 ± 0.96 24.2 ± 5 . 2 0.19 0.23 28.5 ± 12.0 24.5 ± 4.9 0.71 0.059 4. Radial Inclination L 31.6 ± 5.6 26.5 ± 4.9 0.063 0.45 28.0 ± 8 . 5 26.0 ± 2.8 0.78 0.055 5. Ulnar Shortening R 0 ± 1 m m -1.3 ± 4.7 -1.8 ± 5.1 0.85 0.054 - 1 . 0 ± 2 . 8 -1.0 ± 2.8 0.050 6. Ulnar Shortening L 4.8 ± 5.1 -0.59 ± 5 . 1 0.050 0.50 -2.5 ± 4 . 9 -2.5 ± 3.5 0.050 7. Radial B o w R 10° ± 5 ° 9.0 ± 1.6 7.1 ± 2 . 1 0.12 0.33 9.0 ± 4 . 2 11.0 ± 1.4 0.59 0.068 8. Radial B o w Left 16.1 ± 9.5 7.5 ± 2 . 4 0.0020 0.94 9.5 ± 1.4 9.5 ± 0.71 0.050 9. Radial Head Dislocation R 1 0 0 1 10. Radial Head Dislocation L 2 0 0 1 11. E lbow Joint R 9 ° ± 3° 8.8 ± 10.9 -4.6 ± 13.3 0.079 0.41 -23.0 ± 1.4 -13.5 ± 0.71 0.014 0.97 12. E lbow Joint L - 1 . 2 ± 9.9 -8.4 ± 11.9 0.24 0.20 -10.5 ± 10.6 -9.5 ± 2.1 0.91 0.051 13. Femoral A . A . R 7° ± 2 ° valgus -0.90 ± 5.9 -6.2 ± 9.4 0.25 0.19 -8.5 ± 12.0 -0.50 ± 4.9 0.48 0.084 14. Femoral A . A . L -3.8 ± 8.9 -3.9 ± 9.4 0.99 0.050 4.0 ± 2 . 8 0.50 ± 3.5 0.39 0.10 15. Femoral N . S . Angle R 135° ± 5 ° 143.8 ± 21.2 139.8 ± 8 . 4 0.53 0.092 141.5 ± 9.2 142.5 ± 3 . 5 0.89 0.051 16. Femoral N . S . Angle L 146.4 ± 13.4 136.8 ± 7 . 8 0.053 0.49 146.5 ± 2.1 139.0 ± 2 2 . 6 0.69 0.060 17. Femoral M . A . R 0° ± 5° varus 5.9 ± 6.9 -0.46 ± 6.4 0.099 0.36 7.0 ± 1.4 2.0 ± 1.4 0.072 0.48 18. Femoral M . A . L -4.5 ± 5.2 1.2 ± 5 . 2 0.066 0.45 6.0 ± 4 . 2 0.50 ± 3.5 0.29 0.14 19. Sharp's Right 35° ± 4° 38.9 ± 3.4 41.1 ± 5 . 7 0.47 0.11 37.0 (n = 1) 46.0 (n=l) 20. Sharp's Left 39.9 ±5 .2 41.1 ± 5 . 0 0.65 0.071 33.0 (n=l) 41 (n=l) 233 Table 8.7.8 .2. Limb A ignmenl t by Gene and Severity (continued) V a r i a b l e N o r m a l Values E X T l Severe (n=5) E X T 2 Severe (n=17) P - value Power E X T 1 M i l d (n=2) E X T 2 M i l d (n=2) P - value Power 21. Fibular Height R 50 ± 1 0 51.8 ± 10.3 51.2 ± 12.3 0.94 0.051 52.5 ± 0.71 55.0 ± 4.2 0.49 0.081 22. Fibular Height L 48.5 ± 15.0 53.8 ± 13.9 0.51 0.095 59.5 ± 10.6 35.5 ± 7.8 0.12 0.31 23. Ank le Joint Angle R 0 ° ± 5 ° -11.5 ± 17.2 -1.9 ± 10.4 0.17 0.26 -6.5 ± 0.71 0.0 (n=l) 24. Ank le Joint Angle L -9.0 ± 17.3 -1.0 ± 10.9 0.26 0.19 1.5 ± 0 . 7 1 0.0 (n=l) 25. % Weightbear R 50 ± 10 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 26. % Weightbear L 60.0 ± 10.4 50.5 ± 20.6 0.39 0.13 75.5 ± 7 . 8 58.5 ± 9.2 0.18 0.22 N u m b e r of parameters that fall beyond the n o r m a l range 15 14 11 7 234 Table 8.7.8.3. Segment Lengths and Percentile Height by Gene and Severity Variable E X T l Severe (n=5) E X T 2 Severe (n=17) P - va lue P o w e r E X T 1 M i l d (n=2) E X T 2 M i l d (n=2) P - va lue P o w e r Total L e g Length-Right 82.1 ± 5 . 2 86.1 ± 7 . 5 0.28 0.18 73.5 ± 14.1 75.0 ± 19.1 0.94 0.050 Upper L e g - Right 40.4 ± 2.3 44.7± 4.8 0.071 0.43 36.3 ± 8 . 8 38.3 ± 10.3 0.85 0.052 Lower L e g - Right 33.3 ± 3 . 0 35.2 ± 2 . 9 0.22 0.21 30.0 ± 6 . 4 33.0 ± 9 . 2 0.74 0.057 Total L e g Length - Left 81.5 ± 4 . 8 85.4 ± 7.9 0.31 0.16 72.3 ± 14.5 74.8 ± 18.7 0.89 0.051 Upper L e g - Left 40.0 ± 2 . 4 43.8 ± 4 . 7 0.10 0.36 36.0 ± 9 . 9 38.5 ± 8 . 5 0.81 0.053 Lower L e g - Left 33.8 ± 4 . 2 36.7 ± 4 . 4 0.21 0.23 29.0 ±4.9 32.0 ± 8 . 5 0.71 0.059 Total A r m Length - Right 45.2 ± 3 . 8 51.2 ± 5 . 2 0.026 0.63 44.0 ± 9.9 46.5 ± 11.3 0.84 0.053 Upper A r m - Right 27.8 ± 1.5 31.1 ± 3.5 0.057 0.48 26.5 ± 4 . 9 26.8 ± 6 . 0 0.97 0.050 Lower A r m - Right 2 1 . 2 ± 2 . 1 23.8 ± 3 . 1 0.11 0.35 20.3 ± 3.9 21.8 ± 6 . 0 0.79 0.054 Total A r m Length - Left 44.9 ± 3 . 7 5 1 . 7 ± 5 . 1 0.012 0.76 45.3 ± 10.3 45.3 ± 12.4 0.050 Upper A r m - Left 27.5 ± 1.7 31.9 ± 3.8 0.023 0.65 27.5 ± 7 . 8 25.3 ± 6 . 0 0.78 0.055 Lower A r m - Left 19.1 ± 2 . 9 24.1 ± 2 . 9 0.0034 0.90 22.0 ± 3 . 5 22.8 ± 7.4 0.91 0.051 Percentile Height 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 8.7.9 Gender and Severity Table 8.7.9.1 Lesion Quality by Gender and Severity Variable Males Severe (n=12) Females Severe (n=10) P - value Power Male s M i l d (n=2) Females M i l d (n=2) P-value Power Les ion R a n k 1 8.7 ± 6 . 2 4.2 ± 2.3 0.044 0.52 10.0 ± 1.4 8.0 ± 1.4 0.29 0.14 % R a n k 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 2 5.8 ± 3 . 3 4.2 ± 2 . 6 0.24 0.19 3.0 ± 2 . 8 5.0 ± 5 . 7 0.69 0.059 % R a n k 2 19.7 ± 9 . 2 27.8 ± 12.7 0.099 0.36 12.5 ± 9.2 19.5 ± 17.7 0.67 0.061 Lesion R a n k 3 3.9 ± 2 . 4 1.7 ± 1.7 0.025 0.64 3.0 ± 2 . 8 2.0 ± 1.4 0.69 0.059 % R a n k 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 4 11.6 ± 5.1 7.0 ± 4 . 6 0.040 0.54 5.0 ± 2 . 8 6.0 ± 4 . 2 0.81 0.054 % R a n k 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 Smal 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 M e d i u m (%) 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 N u m b e r of Lesions 29.9 ± 11.6 17.1 ± 6 . 9 0.0061 0.84 21.0 ± 7.1 21.0 ± 9 . 9 0.050 N o . Pedunculated 8.3 ± 5 . 3 5.2 ± 2 . 2 0.097 0.34 5.0 ± 1.4 7.5 ± 2 . 1 0.29 0.14 % Pedunculated 30.0 ± 14.4 33.2 ± 12.6 0.64 0.073 24.0 ± 1.4 37.5 ± 7 . 6 0.13 0.29 No. 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 60 .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 % Distal 39.7 ± 14.2 38.8 ± 10.6 0.88 0.053 36.3 ± 24.8 48.2 ± 12.6 0.61 0.067 No. P r o x i m a l 13.6 ± 5 . 5 7.8 ± 3 . 9 0.011 0.76 12.0 ± 0 . 0 7.0 ± 1.4 0.038 0.72 % P r o x i m a l 46.5 ± 8.5 43.9 ±20 .3 0.73 0.062 60.6 ± 20.4 35 .7± 10.1 0.26 0.16 N o . Pelvic 2.3 ± 2 . 9 0 .80± 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 N o Diaphyseal 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 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. F lat 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. C o m p l e x 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 % C o m p l e x 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 18.0± 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. F l a r e d 14.3 ± 9 . 3 3.2 ± 3 . 2 0.0018 0.94 7.0 ± 4 . 2 4.5 ± 2 . 1 0.53 0.075 % F l a r e d 46.1 ± 24.2 17.4 ± 16.8 0.0049 0.87 38.9 ±33 .3 26.8 ± 2 2 . 7 0.71 0.058 No. Not F l a r e d 15.6 ± 8 . 7 13.6 ± 4 . 9 0.53 0.091 14.0 ± 11.3 16.5 ± 12.0 0.85 0.052 236 Table 8.7.9.1 Lesion Quality by Gender and Severity (continued) Variable Males Severe (n=12) Females Severe (n=10) P - value Power Males M i l d (n=2) Females M i l d (n=2) P - value Power % Not F l a r e d 53.9 ± 24.2 81.3 ± 17.9 0.0075 0.82 61.1 ±33 .3 73.2 ± 2 2 . 7 0.71 0.058 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 V a r i a b l e N o r m a l Values Males Severe (n=12) Females Severe (n=10) P - value Power Males M i l d (n=2) Females M i l d (n=2) P - value Power 1. Carpal S l i p R 5 ± 2mm 2.9 ± 4 . 4 2.6 ± 3 . 0 0.84 0.054 6.0 ± 4.2 2.0 ± 0 . 0 0.31 0.13 2. Carpal S l i p L 3.6 ± 3 . 6 3.6 ± 3 . 6 0.99 0.050 5.0 ± 4 . 2 1.5 ± 0 . 7 1 0.37 0.11 3. Radia l Inclination R 21° ± 2 ° 25.3 ± 3 . 4 24.5 ± 6.2 0.72 0.063 32.5 ± 6.4 20.5 ± 0 . 7 1 0.12 0.33 4. Radia l Inclination L 28.4 ± 5 . 9 26.7 ± 4 . 9 0.48 .10 31.0 ± 4 . 2 23.0 ± 1.4 0.13 0.31 5. Ulnar Shortening R 0 ± 1 mm -2.5 ± 5 . 1 -0.90 ± 4.9 0.49 0.098 -3.0 ± 0 . 0 1.0 ± 0 . 0 6. Ulnar Shortening L 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. Radia l B o w R 10° ± 5 ° 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. Radia l B o w Left 10.3 ± 6 . 9 8.6 ± 4 . 5 0.52 0.094 8.8 ±0 .35 10.3 ±0 .35 0.051 0.61 9. Radia l Head Dislocat ion R 1 dislocation 0 0 1 dislocation 10. Radia l Head Dislocation L 1 dislocation 1 dislocation 0 1 dislocation 11. E lbow Joint R 9° ± 3 ° -1.4 ± 15.1 -2.9 ± 12.9 0.81 0.056 -17.5 ± 6.4 -19.0 ± 7 . 1 0.84 0.052 12. E lbow Joint L -8.4 ± 12.6 -4.9 ± 10.9 0.49 0.099 -5.5 ± 3 . 5 -14.5 ± 4 . 9 0.17 0.23 13. Femoral A . A . R 7° ± 2 ° valgus 1.6 ± 7.1 -0.11 ± 6 . 6 0.58 0.082 5.5 ± 3 . 5 3.5 ± 3 . 5 0.63 0.065 14. Femoral A . A . L -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 15. Femoral N.S. Ang le R 135° ± 5 ° 141.0± 8.8 140.4 ± 15.4 0.91 0.051 140.0 ± 7.1 144.0 ± 5.7 0.59 0.068 16. Femoral N .S . Ang le L 138.4 ± 6 . 9 139.7 ± 12.9 0.77 0.059 135.0 ± 17.7 150.0 ± 7 . 1 0.39 0.10 17. Femoral M . A . R 0 ° ± 5 ° varus 1.6 ± 7.1 -0.11 ± 6 . 6 0.58 0.082 5.5 ± 3 . 5 3.5 ± 3 . 5 0.63 0.065 18. Femoral M . A . L -1.1 ± 5 . 8 1.4 ± 5 . 2 0.32 0.15 3.5 ± 7 . 8 3.5 ± 0 . 0 0.94 0.050 19. Sharp's Right 35° ± 4° 39.3 ± 3 . 7 42.3 ± 6.6 0.21 0.22 Data not available Data not available 20. Sharp's Left 41.3 ± 4 . 8 40.5 ± 5.3 0.69 0.066 Data not available Data not available 21. Fibular Height R 50 ± 10 54.4 ± 10.3 48.0 ± 12.8 0.22 0.21 55.5 ± 3 . 5 52.0 ± 0 . 0 0.29 0.14 22. Fibular Height L 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) V a r i a b l e N o r m a l Values Males Severe (n=12) Females Severe (n=10) P - value Power Males M i l d (n=2) Females M i l d (n=2) P - value Power 23. Ank le Joint Ang le R 0 ° ± 5° -3.9 ± 15.7 -3.8 ± 8 . 3 0.99 0.050 -7.0 ± 4.3 -7.0 (n=l) 0.58 0.065 24. Ank le Joint Ang le L -2.4 ± 15.5 -2.9 ± 8.9 0.94 0.051 0.50 ± 0 . 7 1 2.0 (n=l) 0.33 0.11 25. % Weightbear R 50 ± 10 52.1 ± 25.5 46.1 ± 19.7 0.56 0.085 70.5 ± 20.5 40.5 ± 28.9 0.35 0.11 26. % Weightbear L 51.8 ± 17.5 53.1 ± 2 1 . 7 0.88 0.052 66.5 ± 20.5 67.5 ± 3 . 5 0.95 0.050 N u m b e r of parameters that fall beyond the normal range 10 9 11 12 239 Table 8.7.9.3. Variable Males Severe (n=12) Females Severe (n=10) P-value Power Males M i l d (n=2) Females M i l d (n=2) P - value Power Total L e g Length-Right 86.5 ± 5 . 9 83.8 ± 8 . 6 0.39 0.13 86.0 ± 3.5 62.5 ± 1.4 0.013 0.98 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 Lower L e g - Right 35.3 ± 2 . 9 34.2 ± 3 . 1 0.39 0.13 37.0 ± 3.5 26.0 ± 0 . 7 1 0.049 0.62 Total L e g Length - Left 85.9 ± 6 . 6 82.9 ± 8 . 5 0.37 0.13 85.3 ± 3.9 61.8 ± 0 . 3 5 0.014 0.97 Upper L e g - Left 42.5 ± 4.0 43.5 ± 5.2 0.62 0.076 43.8 ± 1.1 30.8 ± 2 . 5 0.021 0.90 Lower 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 3 1 . 0 ± 3 . 3 29.5 ± 3.5 0.28 0.17 30.5 ± 0.71 22.8 ± 0.35 0.0052 1.0 Lower 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 Lower 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 Males Missense (n=2) Females Missense (n=2) P-value P o w e r Ma le s Nonsense (n=8) E X T 2 Nonsense (n=6) P-value P o w e r Les ion R a n k 1 10.0 ± 1.4 8.0 ± 1.4 0.29 0.14 10.8 ± 6 . 7 3.3 ± 2 . 5 0.025 0.65 % R a n k 1 52.0 ± 24.0 44.5 ± 27.6 0.79 0.054 33.7 ± 17.7 25.2 ± 16.1 0.37 0.13 Les ion R a n k 2 3.0 ± 2 . 8 5.0 ± 5 . 7 0.69 0.059 4.9 ± 3 . 2 3.7 ± 1.6 0.41 0.12 % R a n k 2 12.5 ± 9 . 2 19.5 ± 17.7 0.67 0.061 15.7 ± 6 . 9 29.2 ± 12.9 0.026 0.64 Les ion R a n k 3 3.0 ± 2 . 8 2.0 ± 1.4 0.69 0.059 3.8 ± 2 . 3 1.0 ± 1.1 0.020 0.69 % R a n k 3 12.5 ± 9 . 2 9.0 ± 2 . 8 0.66 0.062 11.9 ± 7 . 4 7.7 ± 7 . 8 0.32 0.16 Les ion R a n k 4 5.0 ± 2 . 8 6.0 ± 4 . 2 0.81 0.054 11.0 ± 4.8 4.8 ± 3 . 1 0.018 0.72 % R a n k 4 23.0 ± 5 . 7 26.5 ± 7 . 8 0.66 0.062 38.9 ± 19.6 37.6 ± 2 2 . 9 0.91 0.051 S m a l l (%) 53.7 ± 2 5 . 0 43.4 ± 19.4 0.69 0.060 33.1 ± 15.8 25.2 ± 16.0 0.38 0.13 M e d i u m (%) 24.3 ± 14.2 23.8 ± 13.5 0.97 0.050 26.5 ± 7 . 7 36.4 ± 18.8 0.20 0.23 L a r g e (%) 15.1 ± 1.3 32.8 ± 6 . 0 0.056 0.58 35.9 ± 19.8 39.1 ± 2 2 . 2 0.78 0.058 Average 21.0 ± 7.1 21.0 ± 9 . 9 — 0.050 30.4 ± 12.1 12.8 ± 3.1 0.0050 0.89 N u m b e r of Lesions N o . 5.0 ± 1.4 7.5 ± 2 . 1 0.29 0.14 8.5 ± 5 . 6 4.3 ± 0.82 0.098 0.37 Pedunculated % 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 16.0 ± 5 . 7 13.5 ± 7 . 8 0.75 0.056 19.8 ± 9 . 3 7.7 ± 2 . 4 0.0093 0.82 % Sessile 75.9 ± 1.4 62.5 ± 7.6 0.13 0.29 64.8 ± 16.8 53.2 ± 6 . 1 0.23 0.19 N o . Dis ta l 8.5 ± 7 . 8 9.5 ± 2 . 1 0.88 0.051 13.4 ± 4 . 9 4.5 ± 2 . 1 0.0013 0.98 % Dis ta l 36.3 ± 24.8 48.2 ± 12.6 0.61 0.067 46.1 ± 12.1 34.4 ± 10.7 0.084 0.39 N o . P r o x i m a l 12.0 ± 0 . 7.0 ± 1.4 0.038 0.72 13.1 ± 5 . 4 6.2 ± 2 . 9 0.015 0.74 % P r o x i m a l 60.6 ± 20.4 35.7 ± 10.1 0.26 0.16 43.2 ± 8 . 7 45.5 ± 29.2 0.87 0.052 N o . Pe lv ic 0.0 ± 0.0 1.5 ± 2.1 0.42 0.095 2.4 ± 3 . 5 0.33 ± 0.82 0.19 0.24 % Pe lv ic 0.0 ± 0.0 5.4 ± 7 . 6 0.42 0.095 5.9 ± 8 . 9 2.8 ± 6 . 8 0.49 0.098 N o Diaphysea l 0.50 ± 0 . 7 1 2.5 ± 3 . 5 0.51 0.078 0.75 ± 0.89 1.7 ± 1.6 0.20 0.23 % Diaphysea l 3.1 ± 4 . 4 8.9 ± 12.6 0.60 0.067 1.7 ± 2 . 4 17.4 ± 19.3 0.11 0.34 N o . F la t Bone 1.0 ± 1.4 1.5 ± 2 . 1 0.81 0.054 2.6 ± 3 . 6 0.50 ± 0 . 8 4 0.18 0.24 % F la t Bone 3.8 ± 5 . 4 5.4 ± 7 . 6 0.84 0.052 6.7 ± 8 . 9 4.1 ± 6 . 9 0.55 0.086 N o . C o m p l e x 2.5 ± 0 . 7 1 3.0 ± 1.4 0.69 0.059 5.8 ± 5 . 5 1.0 ± 0 . 6 3 0.061 0.47 % C o m p l e x 12.0 ± 0 . 6 8 17.9 ± 15.2 0.64 0.064 15.9 ± 9 . 8 11.3 ± 7 . 4 0.46 0.10 N o . S imple 18.5 ± 6 . 4 1 8 . 0 ± 11.3 0.96 0.050 23.5 ± 8 . 5 11.2 ± 2 . 5 0.0051 0.89 % S imple 87.9 ± 0 . 6 8 82.1 ± 15.2 0.64 0.064 84.1 ± 9 . 8 83.1 ± 9 . 7 0.89 0.052 N o . F l a r e d 7.0 ± 4.2 4.5 ± 2 . 1 0.53 0.075 15.3 ± 7 . 7 2.2 ± 2 . 3 0.0019 0.96 % F l a r e d 38.9 ± 3 3 . 3 26.8 ± 22.7 0.71 0.058 50.9 ± 18.2 17.1 ± 2 0 . 5 0.0067 0.86 N o . No t F l a r e d 14.0 ± 12.0 16.5 ± 12.0 0.85 0.052 15.1 ± 8 . 6 10.7 ± 3 . 6 0.26 0.19 % Not F l a r ed 6 1 . 6 ± 3 3 . 3 73.2 ± 2 2 . 7 0.71 0.058 49.0 ± 18.2 82.9 ± 2 0 . 5 0.0067 0.86 N o . Left 1 3 . 0 ± 1.4 12.0 ± 4 . 2 0.78 0.055 17.1 ± 8 . 5 5.5 ± 1.9 0.0068 0.86 % Left 64.4 ± 14.9 58.9 ± 7 . 6 0.69 0.060 52.0 ± 6 . 8 42.6 ± 7 . 1 0.081 0.41 N o . R i g h t 8.0 ± 5 . 7 9.0 ± 5 . 7 0.88 0.051 13.3 ± 4 . 5 7.3 ± 1.2 0.0087 0.83 % R i g h t 35.6 ± 14.9 41.1 ± 7 . 6 0.69 0.060 47.9 ± 6 . 8 57.4 ±7 .1 0.081 0.41 241 Table 8.7.10.1 Lesion Quality by Gender and Mutation Type (continued) Variable Males Females P - P o w e r Ma le s F S Females P-va lue P o w e r Splice Site Splice Site value (n=2) F S (n=2) (n=3) (n=l) Les ion R a n k 1 5.0 ± 0 . 0 5.0 ± 0 . 0 — — 4.0 ± 2 . 8 7.0 0.55 0.069 % R a n k 1 14.5 ± 0 . 7 1 22.0 ± 5 . 2 0.15 0.27 17.4 ± 0 . 9 2 29.2 0.060 0.59 Les ion R a n k 2 10.0 ± 1.4 3.3 ± 2 . 3 0.038 0.67 5.0 ± 1.4 10.0 0.21 0.18 % R a n k 2 29.0 ± 5 . 7 20.3 ± 11.1 0.39 0.11 26.4 ± 13.7 41.7 0.53 0.071 Les ion R a n k 3 5.5 ± 3 . 5 3.3 ± 2 . 1 0.44 0.098 3.0 ± 2 . 8 1.0 0.67 0.058 % R a n k 3 15.5 ± 9 . 2 16.3 ± 7 . 6 0.92 0.051 11.5 ± 3 . 5 4.2 0.34 0.11 Les ion R a n k 4 14.0 ± 0 . 0 11.7 ± 4 . 7 0.56 0.076 11.5 ± 10.6 6.0 0.75 0.055 % R a n k 4 40.5 ± 2 . 1 41.7 ± 10.6 0.89 0.051 44. ± 11.9 25.0 0.41 0.090 S m a l l (%) 13.8 ± 0 . 0 22.6 ± 6 . 7 0.18 0.23 25.8 ± 10.7 27.7 0.91 0.051 M e d i u m (%) 36.2 ± 4 . 0 28.3 ± 10.2 0.39 0.11 35.3 ± 14.5 47.0 0.63 0.061 L a r g e (%) 47.7 ± 3 . 3 49.0 ± 8.0 0.85 0.053 39.0 ± 3 . 8 25.0 0.21 0.19 Average N u m b e r 34.5 ± 2 . 1 23.3 ± 7 . 6 0.15 0.27 23.5 ± 17.7 24.0 0.99 0.050 of Lesions N o . Pedunculated 5.5 ± 2 . 1 7.3 ± 3.2 0.54 0.079 10.5 ± 7 . 8 4.0 0.62 0.062 % Pedunculated 15.8 ± 5 . 2 30.9 ± 5 . 1 0.049 0.59 44.9 ± 0.75 16.7 0.021 0.99 N o . Sessile 25.5 ± 4 . 9 14.3 ± 4 . 2 0.071 0.47 13.0 ± 9 . 9 20.0 0.67 0.058 % Sessile 74.5 ± 18.9 61.8 ± 2 . 9 0.30 0.14 55.1 ± 0 . 7 5 83.3 0.021 0.99 N o . Dis ta l 11.5 ± 0 . 7 1 10.3 ± 4 . 2 0.73 0.058 5.0 ± 4 . 2 12.0 0.41 0.091 % Dis t a l 33.5 ± 4 . 1 43.9 ± 7 . 9 0.19 0.22 20.2 ± 2.9 50.0 0.074 0.49 N o . P r o x i m a l 17.5 ± 4 . 9 9.7 ± 5 . 0 0.19 0.23 11.5 ± 7 . 8 12.0 0.97 0.050 % P r o x i m a l 50.4 ± 11.2 39.9 ± 10.5 0.36 0.12 50.9 ± 5 . 2 50.0 0.91 0.050 N o . Pe lv ic 3.5 ± 0 . 7 1 1.0 ± 1.0 0.058 0.53 0.50 ± 0 . 7 1 3.0 0.21 0.18 % Pe lv ic 10.2 ± 2.7 3.8 ± 3 . 3 0.11 0.35 1.4 ± 1.9 12.5 0.14 0.28 N o Diaphysea l 2.0 ± 1.4 2.0 ± 1.0 — 0.050 2.0 ± 1.4 2.0 - 0.050 % Diaphysea l 5.9 ± 4 . 5 9.7 ± 6 . 1 0.51 0.084 15.0 ± 17.3 4.0 0.69 0.057 N o . F la t Bone 3.5 ± 0 . 7 1 1.7 ± 2.1 0.33 0.13 0.50 ± 0 . 7 1 3.0 0.21 0.18 % F la t Bone 10.2 ± 2 . 7 5.8 ± 6 . 3 0.44 0.098 1.4 ± 1.9 12.5 0.14 0.28 N o . C o m p l e x 4.0 ± 1.4 2.3 ± 1.5 0.31 0.14 1.5 ± 0 . 7 1 4.0 0.21 0.18 % C o m p l e x 11.5 ± 3 . 4 9.5 ± 3 . 9 0.61 0.069 10.5 ± 10.9 16.7 0.72 0.055 N o . S imple 27.5 ± 3 . 5 19.7 ± 6 . 4 0.23 0.19 22.0 ± 18.4 20.0 0.94 0.050 % S imple 80.2 ± 15.2 84.2 ± 0.84 0.65 0.065 89.5 ± 10.9 83.3 0.72 0.055 N o . F l a r e d 16.0 ± 18.4 4.3 ± 4 . 9 0.34 0.13 9.0 ± 11.3 6.0 0.86 0.051 % F l a r e d 44.8 ± 50.5 15.6 ± 13.7 0.38 0.11 28.2 ± 26.9 25.0 0.94 0.050 N o . No t F l a r e d 18.5 ± 16.3 19.0 ± 2 . 6 0.96 0.050 14.5 ± 6.4 15.0 0.96 0.050 % No t F l a r e d 55.2 ± 5 0 . 5 84.4 ± 13.7 0.38 0.11 71.9 ± 2 6 . 9 62.5 0.82 0.052 N o . Left 16.5 ± 2 . 1 13.0 ± 2 . 6 0.22 0.19 8.5 ± 6.4 12.0 0.73 0.055 % Left 47.7 ± 3 . 2 57.0 ± 6 . 1 0.15 0.27 36.2 ± 0 . 1 9 50.0 0.011 1.0 N o . R i g h t 18.0 ± 0 . 0 10.7 ± 5 . 5 0.17 0.24 15.0 ± 11.3 12.0 0.86 0.051 % R i g h t 52.3 ± 3 . 2 44.0 ± 7.9 0.27 0.16 63.8 ± 0 . 1 9 50.0 0.011 1 242 Table 8.7. 0.2. Limb Alignment by Gender and Mutation Type V a r i a b l e N o r m a l Values Males Missense (n=2) Females Missense (n=2) P - value Power Males Nonsense (n=8) E X T 2 Nonsense (n=6) P - value Power 1. Carpal Slip Right 5 ± 2mm 6.0 ± 4 . 2 2.0 ± 0 . 0 0.31 0.13 1.8 ± 5 . 2 2.3 ± 3 . 9 0.88 0.052 2. Carpal Sl ip Left 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 3. Radial Inclination Right 21° ± 2 ° 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 4. Radial Inclination Left 3 1 . 0 ± 4 . 2 23.0 ± 1.4 0.13 0.31 28.5 ± 6 . 0 25.5 ± 3 . 1 0.29 0.17 5. Ulnar Shortening Right 0 ± 1 m m -3.0 ± 0 . 0 1.0 ± 0 . 0 ~ -4.6 ± 5.3 -0.50 ± 4.3 0.18 0.25 6. Ulnar Shortening Left -5.5 ± 0.71 0.50 ± 0 . 7 1 0.014 0.97 1.1 ± 5 . 1 -1.2 ± 4 . 4 0.39 0.12 7. Radial B o w Right 10° ± 5 ° 12.0 ± 0 . 0 8.0 ± 2 . 8 0.18 0.22 7.4 ± 2 . 8 6.9 ± 2 . 4 0.73 0.062 8. Radial B o w Left 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 9. Radial Head Dislocation R 0 1 dislocation 0 0 10. Radial Head Dislocation L 0 1 dislocation 1 dislocation 0 11 E lbow Joint Right 9° ± 3 ° -17.5 ± 6.4 -19.0 ± 7 . 1 0.84 0.052 1.1 ± 14.9 -4.8 ± 13.3 0.46 0.10 12. E lbow Joint Left -5.5 ± 3 . 5 -14.5 ± 4 . 9 0.17 0.23 -9.3 ± 13.1 -3.1 ± 12.5 0.39 0.13 13. Femoral A . A . Right 7° ± 2 ° valgus 1.5 ± 2 . 1 -10.5 ± 9 . 2 0.21 0.19 -5.8 ± 10.9 -5.5 ± 8 . 3 0.95 0.050 14. Femoral A . A . Left 2.5 ± 0 . 7 1 2.0 ± 5 . 7 0.91 0.051 -7.4 ± 7.9 -0.50 ± 12.6 0.24 0.20 15. Femoral N . S . Angle Right 1 3 5 ° ± 5° 140.0 ± 7.1 144.0 ± 5 . 7 0.59 0.068 144.6 ± 6 . 3 139.3 ± 5.0 0.12 0.33 16. Femoral N . S . Angle Left 135.5 ± 17.6 150.0 ± 7 . 1 0.39 0.10 139.4 ± 5 . 3 137.5 ± 8.2 0.61 0.076 17. Femoral M . A . Right 0 ° ± 5 ° varus 5.5 ± 3 . 5 3.5 ± 3 . 5 0.63 0.065 1.4 ± 6 . 3 -2.3 ± 4.8 0.27 0.18 18. Femoral M . A . Left 3.5 ± 7 . 8 3.0 ± 0 . 0 0.94 0.050 -0.71 ± 6 . 2 0.83 ± 6.2 0.66 0.069 19. Sharp's Right 35° ± 4° Data not available Data not available 39.1 ± 3 . 8 42.5 ± 7.2 0.28 0.18 20. Sharp's Left Data not available Data not available 40.6 ± 4 . 8 41.2 ± 5 . 4 0.83 0.055 21. Fibular Height Right 50 ± 10 55.5 ± 3 . 5 52.0 ± 0.0 0.29 0.14 51.9 ± 11.1 50.3 ± 12.5 0.80 0.056 22. Fibular Height Left 48.5 ± 26.2 46.5 ± 7.8 0.93 0.050 48.3 ± 10.3 50.8 ± 19.9 0.76 0.059 23. Ank le Joint Angle Right 0 ° ± 5 ° -7.0 (n=l) -3.0 ± 4 . 2 0.58 0.065 -2.9 ± 18.8 -3.5 ± 7.2 0.94 0.051 24. Ank le Joint Angle Left 2.0 (n=l) 0.50 ± 0 . 7 1 0.33 0.11 -0.57 ± 18.5 -1.8 ± 9 . 2 0.89 0.052 243 Table 8.7. 0.2. Limb Alignment by Gender and Mutation Type (continued) V a r i a b l e N o r m a l Values 50 ± 10 Males Missense (n=2) Females Missense (n=2) P - value Power Males Nonsense (n=8) E X T 2 Nonsense (n=6) P - value Power 25. % Weightbear Right 70.5 ± 20.5 40.5 ± 28.9 0.35 0.11 55.9 ± 25.2 39.1 ± 18.7 0.21 0.22 26. % Weightbear Left 66.5 ± 20.5 67.5 ± 3 . 5 0.95 0.050 51.6 ± 19.5 45.3 ± 23.9 0.61 0.076 Parameters beyond the normal range 244 Table 8.7.1 0.2. Lim b Alignment by Gender and Mutal ion Type (continued) Variable Normal Values Males Splice Site (n=2) Females Splice Site (n=3) P-value P o w e r M a l e s F S (n=2) Females F S (n=l) P - value P o w e r 1. Carpal Sl ip Right 5 ± 2mm 5.0 ± 4 . 2 3.0 ± 1.0 0.46 0.093 4.0 ± 1.4 3.0 0.67 0.058 2. Carpal Sl ip Left 4.0 ± 1.4 3.3 ± 3 . 1 0.79 0.055 2.5 ± 0 . 7 1 6.0 0.15 0.25 3. Radial Inclination Right 21° ± 2 ° 27.5 ± 0 . 7 1 27.3 ± 0 . 5 8 0.79 0.055 25.5 ± 6.4 11.0 0.31 0.12 4. Radial Inclination Left 30.0 ± 2 . 8 30.0 ± 7 . 9 - 0.050 26.5 ± 10.6 24.0 0.88 0.051 5. Ulnar Shortening Right 0 ± 1 m m 2.0 ± 2 . 8 1.7 ± 1.5 0.87 0.052 -0.50 ± 2 . 1 -11.0 0.15 0.25 6. Ulnar Shortening Left 4.5 ± 4 . 9 5.7 ± 4 . 7 0.81 0.054 -3.5 ± 7 . 8 -7.0 0.78 0.053 7. Radial B o w Right 10° ± 5 ° 8.0 ± 1.4 8.7 ± 0 . 5 8 0.49 0.086 6.5 ± 0 . 7 1 9.0 0.21 0.18 8. Radial B o w Left 9.5 ± 2 . 1 12.5 ± 6 . 5 0.59 0.072 8.5 ± 4.9 11.0 0.75 0.054 9. Radial Head Dislocation R 1 dislocation 0 0 10. Radial Head Dislocation L 0 1 dislocation 0 11 E lbow Joint Right 9° ± 3 ° 3.5 ± 2 1 . 9 -0.67 ± 16.6 0.82 0.054 -15.0 ± 2 . 8 2.0 0.13 0.30 12. E lbow Joint Left -3.5 ± 2 1 . 9 -9.0 ± 10.4 0.72 0.059 -9.5 ± 3 . 5 -3.0 0.37 0.099 13. Femoral A . A . Right 7° ± 2 ° valgus -0.50 ± 6 . 4 -1.2 ± 4 . 5 0.89 0.051 -8.5 ± 19.1 -9.0 0.99 0.050 14. Femoral A . A . Left 3.8 ± 10.9 -5.5 ± 0 . 8 7 0.21 0.20 -7.0 ± 5 . 7 0.0 0.49 0.075 15. Femoral N . S . Angle Right 1 3 5 ° ± 5 ° 138.0 ± 12.7 140.0 ± 3 1 . 2 0.94 0.050 129.5 ± 3 . 5 148.0 0.15 0.26 16. Femoral N . S . Angle Left 144.5 ± 6.4 144.0 ± 2 3 . 3 0.98 0.050 128.5 ± 2 . 1 140.0 0.14 0.27 17. Femoral M . A . Right 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) Variable Normal Values Males Splice Site (n=2) Females Splice Site (n=3) P-value Power Males F S (n=2) Females F S (n=l) P - value Power 18. Femoral M . A . Left -5.0 ± 7 . 1 1.0 ± 1.4 0.36 0.11 1.5 ± 3 . 5 6.0 0.49 0.076 19. Sharp's Right 35° ± 4° 39.3 ± 3 . 9 38.8 ± 3 . 7 0.91 0.051 38.5 ± 4 . 9 51.0 0.29 0.13 20. Sharp's Left 42.0 ± 2.8 36.8 ± 3 . 5 0.19 0.22 43.0 ± 7 . 1 47.0 0.72 0.055 21. Fibular Height Right 50 ± 10 55.0 ± 2 . 8 39.0 ± 11.0 0.15 0.27 62.8 ± 0 . 3 5 61.0 0.15 0.25 22. Fibular Height Left 59.5 ± 4 . 9 49.0 ± 9.0 0.24 0.18 63.9 ± 1.3 77.0 0.075 0.49 23. Ank le Joint Angle Right 0 ° ± 5 ° 16.0 ± 2 2 . 6 1.0 ± 3 . 5 0.31 0.14 -9.5 ± 0 . 7 1 -20.0 0.052 0.66 24. Ank le Joint Angle Left 8.5 ± 17.7 0.0 ± 4.4 0.45 0.095 -8.0 ± 1.4 -18.0 0.11 0.35 25. % Weightbear Right 50 ± 10 63.5 ± 17.7 65.3 ± 6.4 0.87 0.052 39.5 ± 40.3 30.0 0.88 0.051 26. % Weightbear Left 45.5 ± 7.8 61.3 ± 12.1 0.21 0.20 52.5 ± 2 1 . 9 75.0 0.56 0.067 Parameters beyond the normal range 246 Table 8.7.10.3. Segment Lengths and Percentile Height by Gender and Mutation Type Variable Males Missense (n=2) Females Missense (n=2) P-value P o w e r M a l e s Nonsense (n=8) E X T 2 Nonsense (n=6) P-va lue P o w e r Total L e g Length-Right 86.0 ± 3.5 62.5 ± 1.4 0.013 0.98 86.9 ± 6 . 4 84.4 ± 10.4 0.59 0.078 Upper L e g - Right 44.0 ± 2.1 30.5 ± 0.71 0.013 0.97 44.2 ± 4 . 7 45.3 ± 6.4 0.73 0.062 Lower L e g - Right 37.0 ± 3.5 26.0 ± 0.71 0.049 0.62 35.3 ± 3 . 4 34.5 ± 3 . 3 0.66 0.069 Total L e g Length - L e f t 85.3 ± 3.9 61.8 ± 0.35 0.014 0.97 86.7 ± 7 . 6 83.0 ± 10.3 0.45 0.11 Upper L e g - Left 43.8 ± 1.1 30.8 ± 2.5 0.021 0.90 43.3 ± 4.7 44.3 ± 6 . 1 0.71 0.064 Lower L e g - Left 35.3 ± 3.9 25.8 ± 0.35 0.075 0.47 37.1 ± 5 . 3 35.3 ± 5 . 2 0.53 0.089 Total A r m Length - Right 52.8 ± 2.5 37.8 ± 1.1 0.016 0.95 50.4 ± 6 . 2 50.3 ± 6.0 0.96 0.050 Upper A r m - Right 30.5 ± 0.71 22.8 ± 0.35 0.0052 1.0 31 .7± 3.6 29.3 ± 3 . 5 0.23 0.20 Lower A r m - Right 24.5 ± 2.1 17.5 ± 0.0 0.04 0.67 23.3 ± 3 . 7 23.2 ± 3 . 1 0.94 0.051 Total A r m Length - Left 53.3 ± 1.1 37.3 ± 1.1 0.0044 1.0 50.5 ± 6 . 8 50.7 ± 5 . 6 0.96 0.050 Upper A r m - Left 31.3 ± 2.5 21.5 ± 0.71 0.033 0.77 31.5 ± 4 . 5 30.4 ± 4.7 0.67 0.068 Lower A r m - Left 26.3 ± 2.5 18.5 ± 1.4 0.062 0.54 23.1 ± 4 . 4 24.0 ± 2 . 7 0.68 0.067 Percentile Height 10.0 ± 7.1 33.0 ± 42.4 0.53 0.076 43.4 ± 3 1 . 9 51.7 ± 33.3 0.65 0.071 247 Table 8.7.10.3. Segment Lengths and Percentile Height by Gender and Mutation Type (continued) Variable Males Splice Site (n=2) Females Splice Site (n=3) P-value Power Males F S (n=2) Females F S (n=l) P-value Powe r Total L e g Length- Right 83.3 ± 8 . 1 83.3 ± 7 . 1 0.99 0.050 88.0 ± 2 . 1 81.0 0.23 0.17 Upper L e g - Right 40.5 ± 4.2 42.8 ± 3 . 8 0.57 0.075 42.8 ± 2 . 5 41.5 0.75 0.054 Lower L e g - Right 34.8 ± 3 . 9 34.0 ± 3 . 6 0.84 0.053 35.8 ± 1.1 32.5 0.24 0.16 Total L e g Length - L e f t 82.0 ± 6 . 4 83.3 ± 7 . 4 0.85 0.053 86.5 ± 0.0 81.5 O . 0 0 0 1 1.0 Upper L e g - Left 40.3 ± 2.5 42.8 ± 4 . 3 0.51 0.084 41.5 ± 0 . 0 40.0 <0.0001 - Lower L e g - Left 35.3 ± 6 . 0 34.8 ± 3 . 8 0.93 0.051 37.0 ± 1.4 36.0 0.67 0.058 Total A r m Length - Right 45.5 ± 2 . 1 49.5 ± 6.9 0.50 0.085 53.0 ± 0 . 7 1 46.0 0.078 0.47 Upper A r m Right 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 - L e f t 47.3 ± 0.35 49.5 ± 6.3 0.66 0.064 53.8 ± 4 . 6 45.5 0.38 0.097 Upper A r m - Left 29.3 ± 1.1 30.5 ± 4 . 8 0.75 0.057 32.3 ± 1.8 30.0 0.49 0.076 Lower A r m - Left 20.0 ± 1.4 22.2 ± 3 . 3 0.46 0.092 25.5 ± 2 . 1 18.0 0.21 0.18 Percentile Height 32.0 ± 9 . 9 12.3 ± 11.4 0.14 0.28 10.5 ± 10.6 8.0 0.88 0.051 248 8.7.11 Gene and Mutation Location Table 8.7.11.1 Lesion Quality by Gene and Mutation Location Variable E X T 1 E a r l y (n=2) E X T 2 E a r l y (n=17) P - value Power E X T 1 Late (n=2) E X T 2 L a t e (n=17) P - value Power Les ion R a n k 1 16.5 ± 7 . 8 6.3 ± 4 . 9 0.019 0.69 6.2 ± 1.8 5.0 ± 0 . 0 0.41 0.11 % R a n k 1 37.0 ± 7 . 1 3 3 . 2 ± 2 1 . 1 0.81 0.056 23.0 ± 8.4 20.0 ± 7 . 1 0.69 0.065 Lesion R a n k 2 7.0 ± 4 . 2 3.5 ± 2 . 2 0.076 0.42 6.2 ± 2.9 6.5 ± 6.4 0.93 0.051 % R a n k 2 15.5 ± 4 . 9 21.2 ± 12.9 0.55 0.086 21 .0± 8.2 32.5 ± 0 . 7 1 0.12 0.33 Lesion R a n k 3 5.0 ± 2 . 8 1.9 ± 1.9 0.058 0.48 5.0 ± 1.9 2.0 ± 1.4 0.10 0.36 % R a n k 3 11.0 ± 2 . 8 9.5 ± 7 . 4 0.79 0.058 18.0 ± 5.9 10.0 ±1.4 0.13 0.30 Lesion R a n k 4 15.0 ± 1.4 6.5 ± 4 . 4 0.018 0.70 11.0 ± 4.3 12.0 ± 2 . 8 0.78 0.057 % R a n k 4 36.5 ± 14.8 35.9 ± 2 0 . 2 0.97 0.050 38.2 ± 9.8 37.0 ± 7 . 1 0.88 0.052 Smal l (%) 39.7 + 10.4 31.9 ±18.5 0.58 0.082 23.9 ± 9.5 20.8 ± 9 . 8 0.71 0.062 M e d i u m (%) 22.5 ±15.3 31.2 ±14.2 0.42 0.12 33.8 ± 0.78 27.8 ± 15.8 0.36 0.13 L a r g e (%) 37.8 ± 25.7 34.8 ± 18.2 0.84 0.054 38.9 ± 14.2 50.5 ± 7 . 1 0.34 0.14 Average N u m b e r of Lesions 43.5 ± 13.4 18.3 ± 8 . 6 0.0021 0.95 28.4 ± 6.1 25.5 ± 10.6 0.65 0.068 N o . Pedunculated 11.5 + 2.1 6.2 ± 4 . 5 0.13 0.31 7.6 ± 2.4 5.0 ± 1.4 0.22 0.19 % Pedunculated 26.9 ±3.5 32.2 ±13.8 0.61 0.077 26.8 ± 6.3 22.7 ± 14.9 0.60 0.074 N o . Sessile 27.5 ± 17.7 12.6 ± 6 . 1 0.015 0.73 18.6 ± 3.4 20.0 ± 12.7 0.81 0.055 % Sessile 59.8 ± 22.2 64.5 ± 14.5 0.68 0.067 66.1 ± 6.9 74.5 ± 18.9 0.38 0.12 No. Distal 17.0 ± 8 . 5 7.8 ±4.6 0.023 0.66 11.6 ± 3.1 10.5 ± 2 . 1 0.68 0.065 % Distal 37.9 ± 7 . 8 39.5 ± 15.2 0.88 0.052 41.1 ± 9.3 43.2 ± 9 . 6 0.80 0.055 No. P r o x i m a l 18.0 ± 5 . 7 9.4 ± 4 . 6 0.024 0.65 13.0 ± 5.2 9.5 ± 6 . 4 0.48 0.095 % P r o x i m a l 41.3 ± 0.24 48.0 ± 16.1 0.58 0.083 44.9 ± 10.6 35.1 ± 10.4 0.31 0.15 No. Pelvic 7.5 ± 0 . 7 1 0.59 ± 1.2 <0.001 1.00 1.8± 1.3 2.0 ± 2 . 8 0.89 0.052 % Pelvic 18.4 ± 7 . 3 1.8 ± 4 . 9 <0.001 0.99 6.1 ± 4.0 6.1 ± 8 . 6 0.99 0.050 249 Table 8.7.11.1 Lesion Quality by Gene and Mutation Location (continued) Variable E X T l E a r l y (n=2) E X T 2 E a r l y (n=17) P - value Power E X T 1 L a t e (n=2) E X T 2 L a t e (n=17) P - value Power N o Diaphyseal 1.5 ± 0 . 7 1 1.2 ± 1.3 0.74 0 . 0 6 2 2.0 ± 2.0 2.5 ± 0 . 7 1 0 . 7 6 0 . 0 5 8 % Diaphyseal 3.4 ± 0 . 5 9 7.9 ± 12.1 0.61 0 . 0 7 6 7.8 ± 8.1 10.1 ± 1.4 0.71 0 . 0 6 2 N o . F la t Bone 8.0 ± 0 . 0 0.71 ± 1.2 <0.001 1.00 2.6 ± 1.1 2.0 ± 2 . 8 0 . 6 8 0 . 0 6 5 % Fla t Bone 19.3 ± 5 . 9 2.6 ± 5 . 1 O . 0 0 1 0 . 9 9 8.8 ± 2.9 6.1 ± 8 . 6 0 . 5 0 0 . 0 9 0 N o . C o m p l e x 9.5 ± 12.0 2.7 ± 2 . 2 0 . 0 2 3 0 . 6 6 3 .0 ± 1.6 2.5 ± 0 . 7 1 0 . 6 9 0 . 0 6 3 % C o m p l e x 18.5 ± 2 1 . 9 14.2 ± 9 . 6 0.61 0 . 0 7 8 10.0 ± 3.8 10.1 ± 1.4 0 . 9 8 0 . 0 5 0 N o . Simple 2 9 . 5 ± 7 . 8 16.7 ± 8 . 5 0 . 0 5 9 0 . 4 7 2 3 . 6 ± 3.9 2 2 . 5 ± 10.6 0 . 8 3 0 . 0 5 4 % Simple 6 8 . 3 ± 3 . 2 84.5 ± 9.9 0 . 0 3 8 0 . 5 6 8 4 . 0 ± 8.8 87.1 ± 5 . 4 0 . 6 7 0 . 0 6 5 N o . F l a r e d 2 5 . 5 ± 0.71 7.4 ± 6 . 1 O . 0 0 1 0 . 9 8 9.6 ± 11.3 2.0 ± 1.4 0.41 0.11 % F l a r e d 61.3 ± 17.3 33 .5 ± 2 4 . 1 0 . 1 4 0 . 2 9 2 9 . 6 ± 2 9 . 8 7.3 ± 2 . 5 0 . 3 6 0 .13 N o . Not F l a r e d 1 8 . 0 ± 12.7 11.5 ± 5.9 0.21 0 . 2 2 1 8 . 8 ± 7.1 2 3 . 5 ± 9 . 2 0 . 4 9 0 . 0 9 3 % Not F l a r e d 3 8 . 7 ± 17.3 66.5 ± 2 4 . 1 0 . 1 4 0 . 2 9 7 0 . 4 ± 2 9 . 8 9 2 . 7 ± 2 . 5 0 . 3 6 0 .13 N o . Left 27.5 ± 4.9 9.7 ± 5 . 4 O . 0 0 1 0 . 9 9 15.0 ± 2.2 13.0 ± 2 . 8 0 . 3 6 0 .13 % Left 64.5 ± 8.6 4 8 . 2 ± 10.9 0 . 0 6 0 0 . 4 6 53.5 ± 4.1 53 .2 ± 11.1 0 . 9 7 0 . 0 5 0 N o . Right 16.0 ± 8 . 5 9.9 ± 4 . 8 0 .13 0.31 13.6 ± 4 . 0 12.5 ± 7 . 8 0 . 8 0 0 . 0 5 5 % Right 35.5 ± 8 . 6 51.8 ± 10.9 0 . 0 6 0 0 . 4 6 47.1 ± 4 .9 4 6 . 7 ± 11.1 0 . 9 4 0 . 0 5 0 2 5 0 Table 8.7. 1.2. Limb Alignment by Gene and Mutation Location V a r i a b l e N o r m a l Values E X T l E a r l y (n=2) E X T 2 E a r l y (n=17) P - value Power E X T l L a t e (n=2) E X T 2 L a t e (n=17) P - value Power 1. Carpal S l i p R 5 ± 2mm 5.0 2.2 ± 3 . 8 0.49 0.099 5.2 ± 3 . 1 2.0 ± 0.0 0.23 0.19 2. Carpal S l i p L 7.0 ± 1.4 2.9 ± 3 . 6 0.15 0.29 3.8 ± 3 . 0 4.5 ± 2.1 0.78 0.057 3. Radial Inclination R 21° ± 2 ° 29.0 23.8 ± 5 . 2 0.94 0.14 27.8 ± 6 . 1 27.5 ± 0.71 0.95 0.050 4. Radial Inclination L 28.5 ± 9.2 26.6 ± 4 . 8 0.64 0.073 3 1 . 4 ± 5 . 5 24.5 ± 4.9 0.18 0.24 5. Ulnar Shortening R 0 ± 1 m m -8.0 -2.3 ± 4.8 0.27 0.18 0.20 ± 2.2 3.0 ±1 .4 0.16 0.26 6. Ulnar Shortening L 1.5 ± 4.9 -1.2 ± 5 . 0 0.49 0.10 3.2 ± 6 . 6 2.5 ± 2.1 0.89 0.052 7. Radial B o w R 10° ± 5 ° 11.0 7.4 ± 2 . 5 0.18 0.25 8.6 ± 2 . 3 8.5 ± 0.71 0.96 0.050 8. Radial B o w Left 20.0 ± 15.6 7.7 ± 2 . 5 0.0019 0.95 11.9 ± 4 . 6 8.0 ± 0.0 0.31 0.15 9. Radial Head Dislocation R 0 1 1 1 10. Radial Head Dislocation L 1 1 1 1 11. E lbow Joint R 9° ± 3 ° 2.0 -4.6 ± 13.3 0.64 0.073 -2.6 ± 20.5 -14.0 ± 2.8 0.49 0.092 12. E lbow Joint L -3.5 ± 4.9 -7.5 ± 11.5 0.64 0.072 -4.0 ± 12.3 -17.5 ±2 .1 0.20 0.22 13. Femoral A . A . R 7° ± 2 ° valgus -5.5 ± 7.8 -5.6 ± 9 . 7 0.99 0.050 -2.1 ± 8 . 5 -5.5 ± 0.71 0.62 0.072 14. Femoral A . A . L -9.5 ± 3.5 -3.3 ± 9 . 5 0.38 0.13 1.6 ± 7 . 5 -4.5 ± 0.71 0.33 0.14 15. Femoral N . S . Angle R 135° ± 5 ° 146.0 ± 7.1 140.8 ± 6 . 9 0.33 0.15 142.0 ± 21.3 134.5 ± 17.7 0.68 0.065 16. Femoral N . S . Angle L 142.5 ± 0.71 137.1 ± 8 . 6 0.39 0.13 148.0 ± 13.0 137.0 ± 16.9 0.39 0.12 17. Femoral M . A . R 0° ± 5° varus 1.0 ± 5.7 -0.84 ± 5.9 0.69 0.067 8.9 ± 3 . 2 5.5 ± 3.5 0.30 0.15 18. Femoral M . A . L -5.0 ± 2.8 1.3 ± 5 . 3 0.12 0.32 1.0 ± 7 . 9 0.0 ± 0.0 0.88 0.052 19. Sharp's Right 35° ± 4 ° 37.5 ± 3.5 4 1 . 7 ± 5 . 9 0.35 0.14 38.5 ± 3 . 0 39.0 ± 4.2 0.87 0.052 20. Sharp's Left 4 1 . 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) V a r i a b l e N o r m a l Values E X T l E a r l y (n=2) E X T 2 E a r l y (n=17) P - value P o w e r E X T 1 L a t e (n=2) E X T 2 La te (n=17) P - value P o w e r 21. Fibular Height R 50 ± 10 59.0 ± 7.1 52.7 ± 10.8 0.44 0.11 49.4 ± 5.9 42.5 ± 20.5 0.47 0.096 22. Fibular Height L 48.0 ± 22.6 51.2 ± 15.1 0.79 0.058 54.6 ± 9 . 8 56.0 ± 9.9 0.87 0.052 23. Ank le Joint Angle R 0 ° ± 5 ° -26.0 ± 7.1 -1.9 ± 10.8 0.0085 0.82 5.0 ± 15.8 -1.5 ± 2.1 0.61 0.073 24. Ank le Joint Angle L -20.5 ± 19.1 -0.50 ± 11.2 0.041 0.55 5.8 ± 8 . 5 -4.5 ± 0.71 0.17 0.25 25. % Weightbear R 50 ± 10 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 26. % Weightbear L 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 N u m b e r o f parameters that fal l beyond the n o r m a l range 252 Table 8.7.11.3. Segment Lengths and Percentile Height by Gene and Mutation Location Variable E X T 1 E a r l y (n=2) E X T 2 E a r l y (n=17) P - value Power E X T 1 Late (n=2) E X T 2 L a t e (n=17) P - value Power Total L e g Length-Right 80.5 ± 2 . 1 85.1 ± 9 . 4 0.51 0.096 79.3 ± 10.1 83.5 ± 8 . 5 0.63 0.070 Upper L e g - Right 38.5 ± 0 . 7 1 44.2 ± 5.6 0.18 0.25 39.5 ± 5 . 5 42.3 ± 6.7 0.59 0.075 Lower L e g Right 32.0 ± 1.4 35.0 ± 3 . 7 0.28 0.18 32.5 ± 4 . 8 34.5 ± 3 . 5 0.62 0.071 Total L e g Length - Left 80.0 ± 1.4 84.4 ± 9.7 0.54 0.090 78.4 ± 10.3 83.3 ± 8 . 1 0.58 0.076 Upper L e g - Left 38.3 ± 1.1 43.3 ±5 .2 0.20 0.23 39.1 ± 5 . 9 42.8 ± 6 . 0 0.49 0.091 Lower L e g - Left 31.0 ± 1.4 36.5 ± 4 . 9 0.15 0.29 33.0 ± 5 . 5 34.0 ± 4 . 2 0.83 0.054 Total A r m Length - Right 43.0 ± 0 . 0 50.8 ± 5 . 7 0.077 0.41 45.6 ± 6 . 1 49.8 ± 8 . 1 0.48 0.094 Upper A r m - Right 28.0 ± 0 . 0 30.5 ± 3 . 8 0.39 0.13 27.2 ± 2 . 9 31.8 ± 4 . 6 0.16 0.26 Lower A r m - Right 19.5 ± 2 . 1 23.6 ± 3 . 4 0.13 0.32 2 1 . 5 ± 2 . 5 23.5 ± 3 . 5 0.42 0.11 Total A r m Length - Left 42.0 ± 1.4 5 1 . 0 ± 6 . 2 0.059 0.47 46.2 ± 5 . 8 51.3 ± 6 . 0 0.35 0.13 Upper A r m Left 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 Lower A r m - Left 17.5 ± 3 . 5 24.0 ± 3 . 4 0.021 0.67 20.9 ± 2 . 7 23.0 ± 2 . 8 0.39 0.12 Percentile Height 3.0 ± 0 . 0 44.6 ± 3 0 . 1 0.074 0.42 11.8 ± 15.4 25.0 ± 0 . 0 0.30 0.15 253

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