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Clinical characteristics of 8p inversion duplication and other 8p rearrangements Jurenka, Stanislava B. 1998

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CLINICAL CHARACTERISTICS of 8p INVERSION DUPLICATION AND OTHER 8p REARRANGEMENTS by STANISLAVA B. JURENKA, M.D., FRCP(C) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES Department of Medical Genetics We accept this thesis as conforming to the required standard THTiXNWERSITY OF BRITISH COLUMBIA October 1998 © Stanislava B. Jurenka ln p resent ing this thesis in partial fu l f i lment of t h e requ i remen ts fo r an advanced degree at t h e Univers i ty o f Brit ish C o l u m b i a , I agree that the Library shall make it f reely available f o r re ference and study. I fu r ther agree that permiss ion f o r extens ive c o p y i n g o f th is thesis f o r scholar ly pu rposes may b e g ran ted by the head o f my d e p a r t m e n t o r by his o r her representat ives. It is u n d e r s t o o d that c o p y i n g or pub l i ca t ion o f this thesis fo r f inancial gain shall n o t be a l l o w e d w i t h o u t my w r i t t e n permiss ion . D e p a r t m e n t of The Univers i ty of Brit ish C o l u m b i a Vancouver , Canada Date Del 191U DE-6 (2/88) 11 ABSTRACT The main goal of the project reported in this thesis is to summarize the current knowledge of the 8p inversion duplication clinical features, to add observations of our own patients, and to correlate the resulting clinical definition with the new information on the molecular analysis of these cases. I present detailed clinical and cytogenetic findings of four patients with 8p inversion duplication, three of which were previously unreported. Two of these patients had an atypical inverted chromosome. In this atypical variant the center of symmetry was found to be more distal on the 8p arm of chromosome 8 so that the portion of 8p23.2 was duplicated in addition to more proximal bands. The report of our two patients is the first focused on this variant. The secondary goal of this thesis was to determine if these variants could be distinguished from the common form in order to add to our knowledge regarding the critical region responsible for the abnormal phenotype in this group of patients. Continued. Ill After a detailed clinical comparison of the two types of 8p inversion duplication, I found that in one of the patients with the atypical variant, the physical features were not clearly different from the common type, although the developmental deficit was less marked. In the second patient with the atypical variant the developmental delay and dysmorphism were minimal. In this case the duplication was short, terminating in 8p21.3 band. This case might point to the more exact location of the critical region on 8p. A cosmid 153G8, containing repetitive sequences hybridizing to 4p and to 8p, was used as FISH probe in four cases of 8p inversion duplication. In cases A. and B., 153G8 hybridized to the center of symmetry at 8p23.1. In cases C. and D. with the center of symmetry at 8p(23.2), two FISH signals flanked the center of symmetry. This finding provides the evidence that that repetitive sequences are present in the 8p23.1 region, and that in the atypical variant, the center of symmetry is distal to 8p23.1. iv TABLE OF CONTENTS Abstract ii List of tables vii List of figures VII'I* Acknowledgments ix CHAPTER 1: Introduction 1 1.1 Comparative analysis of clinical characteristics of reported cases 3 1.11 The natural history of 8p inversion duplication 7 1.12 Clinical characteristics of 8p inversion duplication compared with other 8p rearrangements 9 1.121 8p inversion duplication and tetrasomy 8 mosaic 9 1.122 8p inversion duplication and 8p terminal deletion 12 1.123 Atypical 8p inversion duplication-center of symmetry at 8p23.2 13 1.124 8p inversion duplication and 8p direct duplication-differential diagnosis 14 1.2 Cytogenetic and molecular characteristics of 8p inversion duplication 15 1.21 Models explaining the origin of 8p inversion duplication 17 1.3 Project goals 20 CHAPTER 2: Methods 22 2.1 Identification of patients 22 2.2 Fluorescence in situ hybridization with cosmid 153G8 22 2.21 Probe 153G8 description 22 2.22 FISH with the cosmid probe 153G8 - procedure 23 CHAPTER 3: Results 25 3.1 Patient reports 25 3.11 Patient A. 25 3.12 Patient B. 26 3.13 Patient C. 29 3.14 Patient D. 35 3.2 Fluorescence in situ hybridization with cosmid 153G8-results 36 CHAPTER 4: Discussion 38 4.1 Genotype-phenotype correlation in 8p rearrangements 38 4.2 Analysis of genotype-phenotype correlations in our patients 40 4.21 Patients A. and B. 40 4.22 Patient C. 40 4.23 Patient D. 42 4.3 Repetitive sequences (MS 153G8) involved in recombination mechanism of 8p inversion duplication 43 4.31 The FISH results in patients A. and B. 44 4.32 The FISH results in patients C.and D. 44 vf CHAPTER 5: Conclusions 44 5.1 Phenotype of 8p inversion duplication 44 5.2 Phenotype of 8p inversion duplication variant (center of symmetry at 8p23.2)45 5.3 Atypical variant of 8p inversion duplication with a short duplication 45 5.4 FISH analysis with cosmid probe 153G8 46 5.5 Areas for further research 46 Bibliography 47 V|| LIST OF TABLES: Table 1: Clinical symptoms of 8p inversion duplication Table 2: Literature data on 8p inversion duplication compared to our four patients Table 3: Clinical symptoms in 8p tetrasomy mosaicism (iso 8p) and 8p inversion duplication Table 4: Clinical symptoms of terminal deletion on 8p (23.1-ter) and 8p inversion duplication • •• VI f f LIST OF FIGURES: Figure 1: Ideogram of normal chromosome #8 and chromosomes with 8p inversion duplication. 2 Figure 2: Proposed mechanism of origin of 8p inversion duplication 21 Figure 3. Position of STS markers on 8p ideogram 18 Figure 4: Patients B and C at various ages 27 Figure 5: Recombinant chromosomes 8 and corresponding ideograms: patient B (center of symmetry at 8p23.1) and patient C (center of symmetry at 8p23.2) 28 Figure 6: Patient D. at various ages 32 Figure 7: Recombinant chromosome 8 and corresponding ideogram: patient D (center of symmetry at 8p23.2 and a short duplication) 33 Figure 8: Prophase of G banded chromosome 8 in patient D 34 Figure 9: Results of FISH with the MS probe 153G8 37 fx ACKNOWLEDGMENTS: This research was partially supported by a grant from the Roeher Institute. I would like to thank the families of our patients for their kind cooperation. To Dr Fred Dill for the example of scientific scepticism and clarity of thought. To Drs McGillivray, van Allen and Lewis, for sharing their clinical acumen. To Louise Hallmark, for her help in he lab. To Tanya Nelson for sharing her brilliance with me. To Tom, for giving me the original impulse. To Blanka, for English preceptorship and for unflagging support. Page 1 CHAPTER 1: INTRODUCTION. At birth, approximately 3% of all newborns are found to have some congenital anomaly. This number includes both minor and major anomalies. Not all the congenital anomalies have an identifiable genetic component, especially anomalies found in isolation. Systematic surveys of newborns show that about 0.5% have a chromosomal abnormality. Most of these abnormalities are numerical aberrations , i.e. trisomies and sex chromosome aneuploidies, whereas structural rearrangements are rare. In approximately 1:520 children with the rearrangement it is balanced, and in 1:1700 it is unbalanced [Jones, 1997]. Several of the multiple malformation / mental retardation syndromes are now known to be a discrete chromosomal anomaly, usually structural. The structural rearrangements are of an almost unlimited variety: translocations, inversions, ring chromosomes, isochromosomes, deletions and duplications. They can atise from errors in meiosis and in mitosis. Fluorescence in situ hybridization is particularly useful in characterizing these chromosomal anomalies. The structural rearrangements identified on the short arm of chromosome 8 are of several categories: there are isochromosomes 8p, mostly in the form of mosaic 8p tetrasomy, interstitial duplications and deletions, terminal deletions, translocations, and of a special interest to me, inverted duplications (Figure 1.). I decided to study the rearrangements on chromosome 8 from two reasons: First, during my training and in my practice I was fortunate to meet and examine the patients with 8p inversion duplication that I report here. Second, I have been allowed to benefit from the work done at mapping 8p by the research team of Dr. S.Wood at the University of British Columbia Department of Medical Genetics. Page 2 Normals Inv ilup (Spi Inv Uup (Kp) (;iiypiL"il) Figure 1. Chromosome 8. Normal chromosome (left) compared with two types of inversion duplication: Center of symmetry at 8p23.1 (center) and at 8p23.2 (right). Page 3 The following introduction is divided into two parts: The first includes the description of clinical aspects in 8p inverted duplication and other 8p rearrangements as reported in the literature. The second part summarizes the current information of the cytogenetic and molecular characteristics of 8p inverted duplication. 1.1 Comparative analysis of clinical presentation of reported cases. An inverted ("mirror") duplication on the short arm of chromosome #8 is found in association with multiple congenital anomalies and with mental retardation. 8p inversion duplication (8p invdup) is a rare condition. Since the first paper in 1977 [Taylor et al.], just over 50 individuals have been reported [Barber et al.,1994, Die-Smulders et al.,1995, Dill et al.,1987, Engelen et al.,1994,. Feldman et al.,1993, Fineman et al.,1979, Floridia etal.,1996, Fryns et al.,1985, Gorinati et al.,1991, Guo et al.,1995, Hongel et al.,1978, Hoo et al.,1995, Jensen et al.,1982, Mitchell et al.,1994, Nevin et al.,1990, Priest 1995, Ringer et al., 1995, Taylor et al., 1977, Walker et al.,1987 ]. Although all reports since 1977 contain valuable information, only the more recent papers are fully comparable , i.e. in these the authors use high resolution banding methods, and the cytogenetic diagnosis is supported by FISH or molecular testing (PCR of CA repeats, Mitchell et al.,1994). These criteria imposed a limit on the number of eligible cases: I reviewed in detail reports on forty-one patients published since 1985. Reports without a clear cytogenetic diagnosis or without clinical information were not included. Patient A., reported by Dill et al. in 1987, was also not included, as her follow up is given separately. Forty cases remained for analysis. Page 4 In twenty of these case reports, the authors presented the cytogenetic findings and the clinical picture of each case individually, although not all reports provided complete information. In the twenty remaining cases, the clinical descriptions were not the main focus of the paper and were not presented in an individual manner. I decided to list these description separately as "summary". (Table 1.) Comparison with our patients is presented in Table 2. The clinical picture of a child with 8p inversion duplication (8p invdup), based on the 20 detailed reports, includes severe to profound mental handicap (80%), muscle hypotonia in infancy (93%), prominent forehead (100%), upturned nose (64%), large mouth with everted lower lip (95%), CNS anomalies ranging from large lateral ventricles to agenesis of corpus callosum (80% of cases studied), large head circumference relative to the body size (54% of available measurements), and large, anteriorly rotated ears (55%). Sagging cheeks ( described as chubby in young infants) were reported recently [Die Smulders et al., 1996] . This feature is mentioned specifically in 30% of reports, but is seen more readily and more often in the available clinical photographs of children (thirteen out of fifteen). Abnormalities present in 25 to 50% of reviewed cases include a high palate, small receding chin, curly hair, thin at the temples, or sparse and straight hair, congenital heart disease (atrial septal defect, dextrocardia), long and hyperextensible fingers, or tapered and puffy fingers, and clinodactyly of the fifth finger. Non-specified orthopedic anomalies, or scoliosis/ contractures, are documented in older children. Occasional abnormalities, found in less than 25% of individuals with 8p inversion duplication, are hypertelorism, short palpebral fissures, a prominent chin (mostly in adults), a broad and short neck, a low posterior hair line, vascular naevi, cafe-au-lait Page 5 Table 1. Clinical symptoms of 8p invdup : Individual and summary reports. Individual Summary Total reports reports (N=20) (N=20) (N=40) Relative macrocephaly 13/16 0/8 13/24 (54%) Normal head circumference** 3/16 8/8 11/24 (46%) Prominent/large forehead: 20/20 10/20 30/40 (75%) Large mouth with downturned lower lip: 19/20 7/20 26/40 (65%) Upturned nose (children) 11/17 11/20 22/37 (59%) Large nose (adults) 2/2 Mental handicap-severe 16/20 18/20 34/40 (85%) Hypotonia in infancy 14/15 14/20 28/35 (80%) CNS abnormality* 8/10 10/20 18/30(60%) Large /misplaced ears 9/20 9/20 (45%) Curly hair 13/20 13/20 (65%) Hypotonia, residual 1/20 5/20 6/40(15%) Spasticity, later onset 3/20 7/20 10/40 (25%) * Agenesis of corpus callosum, large lateral ventricles, frontal atrophy ** No microcephaly noted in the remaining 16 case reports Page Table 2. Comparison of phenotypic features of cases of 8p inversion duplication reported in the literature and cases reported here. Total reports Detailed reports Our (N=40) (N=20) Relative macrocephaly 13/16(81%) 13/24 (54%) 4/4 Prominent forehead 30/40 (75%) 20/20 (100%o) 4/4 Large mouth, thick lower lip 24/40 (65%) 19/20/(95%) 2/4 Upturned nose (child) 22/37 (59%) 11/17(64%) 2/3 Mental handicap- severe 34/40 (85%) 16/20 (80%) 2/4 Mental handicap- unclear 6/40(15%) 4/20 (20%) -Hypotonia in infancy 28/35 (80%) 14/15 (93%) 3/4 Hypotonia in adult 2/3 Spasticity 10/17 (59%) 7/7 (100%) 1/4 Scoliosis 2/4 CNS abnormality 18/30(60%) 8/10 (80%) 2/4 Large/misplaced ears 17/24 (70%) 12/14 (85%) 2/4 Curly hair 13/15 13/15 3/4 Broad neck 8/30(26%) 4/10 (40%) 1/4 Short fingers 1/3 Cardiac anomaly 7/14 4/8 1/4 One case of anal atresia, gut malrotation and horseshoe kidney. One case of severe rib anomalies with an early death from ensuing respiratory failure. Our patient C is moderately and patient D mildly mentally delayed Page 7 pigmentations, and accessory nipple. Facial and skull asymmetry are documented. Organ malformations include renal anomalies, malrotation of the intestine, and diaphragmatic hernia. Skeletal anomalies include vertebral and rib malformations, as well as clubfoot. Failure to thrive in infancy, obesity in adulthood, hearing loss (not specified) , and seizures in early childhood are reported . No case of microcephaly was found. 1.11. The natural history of 8p inversion duplication. The clinical features of adults with 8p inversion duplication are not well defined, as a phenotypic description of only five adults and three older children has been reported [Die-Smulders et al.,1995, Dill et al.,1987, Guo et al,1995, and Feldman et al.,1993]. Severe mental handicap, macrocephaly relative to body size, a large mouth, and anomalies of muscle tone were present in all. Parental age at the time of the affected child's birth was recorded in thirteen cases. The youngest mother was 20 and the oldest 41 years of age, with an average age of 29 years, and standard deviation of 5.45 years. The youngest father was 22 years old and the oldest 46, with the average age 29.4 years and standard deviation of 7.31 years. Priest (1995) posed the question about prenatal loss of fetuses with 8p invdup. If such increased prenatal loss exists , how frequent it is remains unknown. Kalousek et al. (1993), did not detect any chromosome 8p rearrangements among chromosomal abnormalities in 3,912 cases of miscarriage. Floridia et al. [1996] estimated the incidence of 8p inversion duplication to be approximately 1:20,000 to 1:30,000, by comparing the numbers of 8p inversion duplication cases referred to genetic services with the incidence of Down syndrome in the catchment area. The question of prenatal loss in 8p inversion duplication is unresolved. Page 8 Individual data on birthweight are available in eight cases: The average birthweight was 2,995 gms, with a standard deviation of 263 gms. Included in the eight cases are two children born at 34 weeks and two at 37 weeks gestation, which makes the average weight of these children artificially low. (Average birthweight in North American full term newborns of both sexes is 3 200 gms.) Feldman et al. in 1993 found no instance of intrauterine growth retardation among his ten cases but does not quote their weight. Minelli et al. in 1993 reported birth weights under 10th centile in two of seven children, without giving gestational age or individual weights. These data are limited, but suggest that intrauterine growth retardation (IUGR) is uncommon in 8p inversion duplication. Almost all newborns with 8p inversion duplication are reported to have feeding difficulty due to muscle hypotonia, and their motor development is slow. In most cases the initial hypotonia changes as the child grows older: first, the truncal musculature remains weak, while the legs become progressively spastic. Later in childhood, contractures may appear, followed by secondary deformities and other orthopedic problems. The age when these changes first appear is not documented. In a minority of older individuals, muscle hypotonia persists and the spasticity does not appear. As a consequence, joint dislocations may occur. Ambulation is frequently limited or impossible. Scoliosis occurs frequently and is found both in patients who have vertebral anomalies and in those who do not. In men the facial features may coarsen and the nose become bulbous. Two children are reported to have seizures: one had a focal EEG abnormality and sporadic seizures until the age of six. No information is provided about the other child. Speech either does not develop or is limited to single words or short sentences. Behavior characteristics are not clearly defined. Several children are described as cheerful and interactive, and the same was reported in several adults. Page 9 Information on sexual development in reported adults described the adult men as sexually mature. In three adult women with 8p inversion duplication, normal menarche in two and premature puberty in one were observed. Body size is not specifically reported save in exceptional cases. Two adult women, one reported by Fryns et al. (1985), and our patient A., previously reported by Dill et al. (1987), are both significantly small (more than 3 SD below mean). An adult man reported by Die-Smulders et al. in 1995 is 205 cm tall. No signs of premature aging are reported. The oldest individual in the literature is a 44 years old man [Die-Smulders et al.,1995]. 1.12 Clinical characteristics of 8p inversion duplication compared with other 8p rearrangements: 1.121 8p inversion duplication and tetrasomy 8p mosaicism: Eight children with mosaic isochromosome (i.e.mosaic tetrasomy) have been reported [Newton et al.,1993, Robinow et al.,1989, Fisher et al., 1993, Schrander-Stumpel et al.,1994, Winters et al.,1995]. In the tetrasomy 8p mosaic, multiple copies of the whole short arm are present, including the areas which are either monosomic or deleted in 8p inversion duplication chromosomes. The phenotype of the children reported with 8p tetrasomy mosaic strongly resembles those with 8p inversion duplication. Their phenotype is remarkable for the same craniofacial dysmorphism as in the 8p inversion duplication: high square forehead, prominent mouth with a large lower lip, striking truncal hypotonia, and agenesis of corpus callosum. Seizures are not recorded in children with 8p tetrasomy mosaicism, and are rare in individuals with 8p inversion duplication (Table 3). Page 10 Table 3. Clinical symptoms in mosaic iso 8p and 8p invdup individuals: iso 8p 8pinvdup Prominent forehead 5/8(62%) 30/40 (75%) Upturned nose 5/8 (62%) 22/37 (59%) Large mouth 4/8 (50%) 24/40 (60%) Severe mental handicap 3/8 (38%) 34/40 (85%) Mild to moderate mental h. 5/8(62%) -CNS abnormality* 5/6 (83%) 18/30 (60%) Hypotonia in infancy 6/8 (75%) 28/35 (80%) Vertebral anomalies 6/8 (75%) 6/40(15%) Broad neck 1/8 (12%) 8/30 (26%) Curly hair 1/8 (12%) 13/15 (86%) Congenital heart disease 2/8 (25%) 7/14 (50%) Obesity 2/7 (28%) 1/40 (0.25%) Vertebral anomaly 1/12 (8%) 6/40 (15%) * CNS abnormalities : agenesis of corpus callosum, enlarged lateral ventricles T Table 4. Clinical symptoms of del 8p(23.1) (N=16) and 8p invdup (N=40) del 8p 8pinvdup Small skull 7/13 (53%) 0/24 (0%) Prominent forehead 4/16 (25%) 30/40 (75%) Upturned nose 6/16 (37%) 22/37 (59%) Large mouth 3/16(18%) 24/40 (60%) Broad neck 2/16 (12%) 8/30 (26%) Mild mental handicap 14/14 (100%) -Severe mental hand. - 34/40 (85%) Hypotonia in infancy - 28/35(80%) Congenital heart anomaly* 10/16 (62%) 7/14 (50%) CNS abnormalities** 3/5 (60%) 18/30 (60%) Seizures 6/16 (37%) 2/40 (5%) Normal, sex.development 2/2 2/3 Aggressive /hyperactive behaviour*** 10/16 (64%) ? *CHD in 8p del: AVSD, hypoplastic left heart, anomalies of great vessels. T-E fistula, horseshoe kidney, lx postaxial Polydactyly CHD in 8p invdup. dextrocardia 2x, ASD 2x, 3 unclear ** No details given *** Detailed description of behaviour in 3/5. No data on 8p invdup. Page 12 The degree of mental handicap varies considerably in 8p tetrasomy mosaicism, as opposed to severe to profound mental handicap found in most cases of 8p inversion duplication. The variability of developmental deficit reflects the different level of mosaicism , which varies both between cases and between individual body systems. 1.122 8p inversion duplication and 8p terminal deletion: Since 1990, sixteen cases of small terminal deletions (8) p23.1-ter have been reported [Blennow et al., 1990, Claeys et al., 1997, Engelen et al., 1994, Hutchinson et al, 1992, Leistietal., 1977, Pettenati et al., 1992], The patients with the terminal deletion are very different from the individuals with 8p inversion duplication (Table 4). The face described in association with 8p terminal deletion is unremarkable or minimally dysmorphic. In 7/13 cases, microcephaly is noted, as opposed to a normal or large head size in 8p inversion duplication. Mental development is either within normal limits, or the delay is mild to moderate. No reported patient with 8p terminal deletion is severely mentally delayed, whereas in 8p inversion duplication the developmental handicap is severe or profound. . Specific behavioural phenotype, with aggressive and destructive outbursts, is reported by Clayes et al (1997) in four children with deletion (8)(p23.1-ter). The behavioural comments oin 8p inversion duplications, where available, report no problems, or comment on the cheerful disposition of the individual. Seizures are reported in five out of sixteen (31%) individuals with terminal 8p deletion. Three of these patients have a chronic recurrent seizure disorder, in two the seizures are episodic. Additional two children with 8p terminal deletion are reported as having abnormal EEG without overt seizures. Congenital heart defects (atriovetricular septal defects, outflow tract anomalies) are a prominent feature of 8p terminal deletion. Heart defects are found in ten out of sixteen cases (62%), with severity in some cases Page 13 increasing with the size of the deletion [Wu et al.,1996]. Heart defects in 8p inversion duplication less significant (dextrocardia, ASD. Table 4.) The frequency of seizure disorder and of congenital heart disease reported in terminal deletion of 8p could be due to a bias of ascertainment, i.e. children with seizure disorder or heart disease are more likely to be investigated (Table 4). Otherwise, the terminal deletion on 8p is considered to be a condition easily missed, both clinically and cytogenetically (Hutchinson et al.,1992, Wu et al.,1996). It is also compatible with maintenance of reproductive fitness. Pettenati et al.,1992, reports a family with an affected father and two affected children. 1.123 Atypical 8p inversion duplication - center of symmetry at 8p 23.2. From the forty patients with 8p inversion duplication, thirty seven have a duplication with the center of symmetry at 8p23.1. In only three cases the point of symmetry is at 8p(23.2). From these three, the eight-month old child reported by Hoo et al. as 8p invdup(21.2- 23.2 ) presented with failure to thrive and hypotonia as the main clinical features. The child had a triangular face and a cafe au lait macule on his back. The rest of the detailed assessment revealed no dysmorphic features. The infant is described as socially responsive and alert. Guo et al. in 1995 reports two children, one aged ten (case #4 ), with the duplicated segments 8p(l 1.2-23.2). The second child is 12 years old. This child has an unusual cytogenetic finding of 8p inverted duplication characterized as 8p(21.3-23.3), and an additional unidentified ring chromosome. Their clinical description shows a severe developmental and neurological deficit, i.e. spasticity and contractures. Page 14 The clinical information on these three cases shows no identifiable difference between the more typical 8p inversion duplication with the center of symmetry at 8p23.1, and the rare variant, where the center of symmetry is at 8p23.2. 1.124 8p direct and 8p inversion duplication - differential diagnosis. Six individuals have been reported with a direct duplication on 8p [Dhooge et al.,1994, Engelen et al., 1994]. Each of the two reports describes the findings in a woman and her two children. In the family reported by Dhooge et al., both the mother and the children are short and mildly dysmorphic, with a large head and an unusual triangular face. All three are mildly mentally handicapped. According to the authors, it was not possible to determine by the cytological examination, if the duplication is direct or inverted. The authors define the dark band 8p22 as clearly duplicated, but are not able to determine if the duplicated light band distal to 8p22 was 8p23 or 8p21 , and therefore the definitive diagnosis remains unresolved. The family reported by Engelen et al (1994)., presents much milder picture, with no discernible dysmorphism and no mental handicap. The authors define the duplication in their three patients as limited to the dark band 8p22 and the more distal 8p23, and do not consider the band 8p21 duplicated in these cases. They suggest that duplication of 8p21 is the critical factor determining the severity of the phenotype. Diagnosis of a direct duplication in Engelen's patients is supported by FISH studies using cosmid probes from the (8)(p23-ter) region. In all three cases, the probes gave a double signal, one at the approximate breakpoint site and one in the subtelomeric region, suggesting a true direct duplication. The authors conclude that in order to Page 15 differentiate between a direct and inverted duplication where the cytogenetic picture is ambiguous, multicolored FISH can be used to show the sequence of loci within the duplication, i.e if the FISH signals are repeated in a direct or in a mirror pattern.. 1.2 Cytogenetic and molecular characteristics of 8p inversion duplication. The descriptions of an elongated 8p arm in the earlier reports are based on cytogenetic resolution of only 300 bands. Fineman et al. in 1979, in his paper on partial trisomies 8, reports a severely affected child with an unbalanced translocation between chromosomes 8 and 10, resulting in a duplication of 8p(21>ter). The band resolution in 8p duplication reported by Mattei et al. in 1980 is not sufficient to distinguish between inverted or tandem duplication. The more recent papers on 8p duplication still occasionally discuss the diagnostic uncertainty in distinguishing direct from inverted duplication. The diagnostic methods used to make a more accurate diagnosis include high resolution banding (550 bands or higher), FISH (probes like D8S7, defensin I gene , centromere, telomere), gene dosage measurements (Factor VII, glutathione reductase), andPCRof(CA) repeats. For the following review, I select reports of forty patients, where there are sufficient data for reasonable cytogenetic and clinical comparison. In 8p inversion duplication, the rearranged short arm of chromosome 8 has a discernible center of symmetry region. The duplicated bands extend from this region in opposite directions in a mirror like fashion. The large dark band (G band) 8p22 makes the mirror arrangement quite conspicuous. This band is duplicated in all cases of inversion duplication reported to date. The light band (R-band) 8p23.1 lies between the two p22 and is broader than on a normal 8p chromosome, suggesting that it is partially duplicated. The inverted duplicated segment may be long and involve almost the whole 8p arm, or be Page 16 as short as to terminate in the light 8p21.3 band, just distal to the dark band 8p22. In all 8p inversion duplication cases reported since 1977, at least a part of the band 8p21 was duplicated. From the cases analyzed, seven have a duplication 8p(ll.2-23.1), 8p(12-22) is seen in eight, 8p(12 -23.1) in nine. In thirteen the duplication varies between 8p(l 1.2-23) to 8p(21.1-23.1). In rare instances (three out of 40 reported cases), the center of symmetry has a different appearance than described above. Instead of a broad light band in the middle of the rearranged arm (duplicated 8p23.1), there is an additional dark band in the center, between the two light bands 8p23.1. The interpretation is that this band represents 8p23.2 in the center of the duplication [Guo et al.,1995 , Hoo et al., 1995]. (Figurel.) In two of these cases, the duplication is characterized as 8p(l 1.2-23.2), and 8p(21.2-23.2). Guo's patient #5, whose duplication is diagnosed as 8p(21.3-23.3), has also an additional (supernumerary) ring chromosome, not defined. The common segment duplicated in 39/40 cases (Guo's patient #5 is the exception) is 8p(21.1-22). In addition to the duplication of the 8p bands, a deletion of the terminal sequences ( D8S7) in 8p inversion duplication is reported on the molecular level by Dill et al. (1987), thus defining the rearrangement as duplication deficiency. Subsequently, many authors confirm the deletion [Gorinati et al.,1991, Minelli et al.,1993, Mitchell et al.,1994, Barber et al.,1994, Engelen et al.,1994, Guo et al.,1994, Priest, 1995, Floridia et al.,1996]. Mitchell et al., in 1994, reports the Defensin I gene locus deleted in his patient, as well as the D8S7. All cases of 8p inversion duplication with the center of symmetry at 8p23.1 are deleted at the D8S201 and D8S349 loci [Floridia et al.,1996]. The STS marker D8S349 is the more proximal, located in the 8p23 region, as is D8S201 and D8S7. Page 17 In 17 informative cases [Minelli et al., 1993, Floridia et al., 1996, and our patient B), the deleted alleles are shown to be of maternal origin. The molecular analysis also demonstrate an unduplicated central segment between the two "mirror" sections. This unduplicated region at 8p23.1, called the single copy region, is reported by Floridia et al., 1996. 1.21 Models explaining the origin of 8p inversion duplication In most cases reported, the 8p inversion duplication arose de novo [first noted by Jensen et al., 1982], except in one case reported by Feldman et al (1993), the mother has a balanced pericentric inversion. Maternal origin of 8p inversion duplication has been demonstrated by using polymorphic DNA markers in 17 informative cases.. Sex specific genetic maps indicate that the genetic distance in this region is much larger in women, indicating that the region 8p23.1 is a site of frequent recombination. There is a molecular evidence that the initial event in formation of 8p inversion duplication takes place in meiosis 1 as an interchromosomal event [Floridia et al.,1996]. It is likely that the characteristics of female meiosis predispose to a more frequent recombination in this area. Several models have been constructed to explain the origin of the inversion duplication and the accompanying deletion. One model proposes an inversion in the parental germ line, with a subsequent formation of an inversion loop, followed by a crossover with U type exchange in the loop. This mechanism would preserve the telomere [Gorinati et al.,1991; Feldman et al.,1993; Engelen et al., 1994; Mitchell et al., 1994, Hoo etal.,1995). The objections against this mechanism are that it does not explain why the center of symmetry is almost always at 8p23.1, and it would require more than one primary event occuring in the same cell: the parental germline inversion, and the the U type exchange in the inversion loop. Page 18 The model consistent with our data presumes a single primary error in meiosis I. The aberrant chromosome arises through a mispairing and a recombination on the short arm of the homologous chromosomes, end to end, or by a misalignment of the segments of the chromosome. The end result would be like a U-type exchange between sister chromatids, but in this case it is an exchange between homologous chromosomes. Recombination at the mispaired sequences would produce a dicentric chromosome in which the terminal sequences including the telomeres of the original chromosomes are deleted. A random break of the dicentric chromosome at the next cell division produces an inversion duplication of a variable size. [Dill et al.,1987, Henderson et al.,1992, Barber et al.,1994, Floridia et al.,1996].(Figure 2.) This hypothesis requires only one primary event: the mispairing on 8p. The secondary events follow the formation of the dicentric chromosome. This hypothesis is supported by findings of a dicentric variant of 8p inversion duplication. A molecular analysis of 16 cases by Floridia et al. (1996) demonstrates the dicentric configuration in several cases. In 1997, Piantanida et al. reports a case of dicentric isochromosome 8, with nullisomy for the regions distal to 8p 23.1. These reports point out that the constant site of the center of symmetry is an important feature in 8p rearrangements. In this model, the regions distal to 8p23.1 including the telomeres of the original chromosomes are lost in the recombination. The telomere on the rearranged chromosome is subsequently reconstituted. Reconstitution of a telomere in terminal deletion is reported on chromosome 16 [Flint et al., 1994]. This model presumes that the mispairing is facilitated by the presence of repetitive sequences, tandem or of a varied orientation, on the short arm of chromosome 8 [Floridia et al., 1996]. Such repetitive sequences would then appear in the middle of the inverted duplication. Analysis of a novel repetitive sequence (megasatellite, MS) located at 8p is Page 19 in progress in Dr. Wood's laboratory at UBC (T.Nelson). This megasatellite is a candidate for a repetitive DNA sequence involved in the formation of 8p inverted duplication. Figure 2. Proposed model explaining the origin of 8p inversion duplication: Mispairing and recombination of homologous chromosomes end-to-end, with formation of a dicentric chromosome and subsequent random break. Page 20 1.3 Project goals: To summarize the current knowledge of the 8p inversion duplication clinical features, to add observation of our own patients, and to correlate the resulting clinical definition with the new information on the molecular definition of these cases. To define differences, clinical, cytogenetic and molecular, between individuals with 8p inversion duplication with a center of symmetry at 8p23.1, and with the center at 8p23.2 (atypical variant). To define the location of repetitive sequences (MS) on 8p in relation to chromosomal bands in normal chromosome, and location of these sequences on the derivative chromosome with 8p inversion duplication. At the University of British Columbia Department of Medical Genetics, a group of molecular geneticists under the supervision of Dr Stephen Wood is mapping the short arm of chromosome 8. It is through the kind cooperation of this research group, that I was supplied with the molecular data on our patients (Figure 3.), and that I received the megasatellite (MS) cosmid 153G8 to use it as FISH probe in our patients. Page 21 Sex-Average Linkage Map (Genethon) 23.1 D8S504 • D8S264 • D8S1781• D8S1788-D8S1798• D8S518• D8S1742 -D8S561/D8S277 -D8S1819-D8S1706-2 '. 1 2 1 Sex-Average Linkage Map (Wood Lab) D8S356 • D8S264 • D8S1825 * D8S1721/D8S542/D8S503 • D8S516-D8S1755/D8S520 • D8S550• D8S265• D8S552• D8S201 D8S518 D8S67 D8S277 D8S349 D8S252 D8S520 -D8S550 -D8S265 " D8S1640 D8S1993 D8S1619 D8S552 -Figure 3. Ideogram of normal 8p , indicating the position of STS markers used in the molecular analysis of the derivative chromoseomes with 8p inversion duplication. (From T.Nelson, with permission.) Page 22 CHAPTER 2: METHODS. 2.1 Identification of patients. Patients A. and B. were referred through the Ministry for Children and Families of the Province of British Columbia. Their chromosomal studies were performed in the regional service laboratory (Royal Columbian Hospital) by the laboratory staff. Patients C. and D. were referred to the Provincial Medical Genetics Department. Their chromosomal studies were done in the B.C. Children's Hospital Cytogenetic laboratory by the laboratory staff. . Patients A., B.,and C. were examined by the author. File of patient D. was made accessible to the author by Dr.Friedmann with the permission of the patient's family. FISH analysis was performed by the author. 2.2 Fluorescence in situ hybridization with megasatellite cosmid 153G8. 2.21 Probe 153G8-description The probe 153G8 is a cosmid isolated from the LA08NC01 flow sorted chromosome 8 library. This cosmid contains sequences homologous to a novel 4.7 Kb repeat (megasatellite, MS), localized to chromosome 4p and also to chromosome 8p by Kogi et al. (1997). The probe 153G8 was prepared in the UBC medical genetics laboratory of Dr.S.Wood by T. Nelson, and kindly offered for the FISH study on our patients. Cosmid probe 153G8 hybridizes to 4p and 8p subtelomeric regions in normal controls. The purpose of the FISH testing in our patients is to determine the chromosomal location of these repetitive sequences both in normal chromosomes and in 8p inversion duplication. Page 23 2.22 FISH with the cosmid probe 153G8 - procedure. FISH was performed on metaphase lymphocytes, prepared in the service laboratory by standard method [Verma and Babu, 1989]. The method used was according to Current protocols in Human Genetics, 1995, with modifications as specified in the following text. The probes used were Oncor centromeric probe for chromosome 8, together with the megasatellite probe 153G8. In patient C, only the MS probe was used for technical reason. (I had very little probe 153G8 left and was concerned that the commercial probe could obscure the relatively weaker hybridization signal generated by the 153G8.) The 153G8 was labeled with digoxigenin (Boehringer Mannheim) by T.Nelson. The commercial Oncor 8 centromeric probe came prelabelled with digoxigenin. The slides were pretreated in 2x SSC at 37 C for thirty minutes, dehydrated in 70, 80, 90 and 100% ethanol for two minutes each time and air dried. The slides were then denatured in 70% Formamide solution at 74 C for two minutes, and the denaturation was arrested by immersion in 70, 80, 90 and 100% ice cold ethanol and air dried. The probe was prepared by mixing 6 micrograms of the 153G8 probe with 60 micrograms of Oncor Hybrisol VI and denatured by heating at 70 C for five minutes. No preannealing was performed (the EcoKL digested clone was tested for presence of short repetitive sequences and showed minimal non specific hybridization [Nelson, personal communication] ). Ratio for the mix of the two probes was determined by the standard Oncor protocol for mixing unique sequence and commercial probes: 3 micrograms of chromosome # 8 centromeric Oncor probe was added to the denatured 153G8 probe. The commercial probe was not denatured on the premise that the signal from the commercial Page 24 probe was likely to be very strong and thus might overpower the signal from the cosmid probe. For hybridization, 10 micrograms of the prepared mixture was used per slide. Slides were prewarmed so that the probe can be spread more thinly. For hybridization, slides were covered with a coverslip, sealed with the rubber cement, and incubated in moist chamber at 37 C for 24 hours. Post hybridization washes were performed according to manufacturers' specification. The coverslips were removed and the slides were placed in 50% Formamide with 2X SSC , at pH7, in the 43C bath for fifteen minutes, and continuously agitated. This was followed immediately by immersion in 2X SSC at 37 degrees C for eight minutes, again while the jar was agitated continuously, and finished by three washes in IX Phosphate buffered detergent (PBD), two minutes each time. Just before detection, the slides were removed from the buffer solution and the excess buffer was drained. Detection: 60 microliters of Fluorescein (FITC) labelled antidigoxigenin (Oncor) was applied to each slide, and slides were covered with plastic covers and incubated for five minutes at 37C in the dark moist chamber. This was again followed by three washes in IX PBD, two minutes each time. Counterstain: 10 microliters of the counterstain mixture (propium iodide (PI) / Antifade and Antifade) per slide was applied. The slides were covered with coverslips, observed under a Zeiss Axoplan fluorescent microscope, and photographed with 400 ISO Kodak film. Page 25 CHAPTER 3: RESULTS. 3.1 Patient reports: It was my privilege to meet several individuals with 8p inversion duplication during my clinical training and in my practice. Patient A was in a long term care and is now in a group home. Patient B was admitted twice to a Provincial assessment unit for behavioral concerns in mentally handicapped individuals (Willow Clinic). Patient C and D were referred to the B.C. Provincial Medical Genetics Program. Their families kindly gave consent for the patients' participation in this study. The study was approved by the University of British Columbia Research Ethics Committee. 3.11 Patient A. is a 36 year old non ambulant woman, requiring complete nursing care. She is the product of a normal pregnancy and delivery. At the time of her birth, her mother is 20 and her father 23 years old. The birthweight is 3 100 g. Her postnatal history includes severe muscle hypotonia and early developmental delay. Ms. A. is first reported in 1987 byDill et al., and is reexamined for this study. At 36 years of age, Ms. A. is small and thin, with weight and height more than 4 SD below normal. She has a quadrangular head with a high forehead, curly hair, short webbed neck, large ears, antimongoloid slant of the eyes, narrow hooked nose, and a large mouth with prominent lower lip. Her teeth are widely spaced, and her chin is pointed. Her fingers and toes are long and thin, with enlarged interphalangeal joints. She has muscle hypotonia and joint laxity and has had repeated patellar dislocations since the age of 32. She has severe scoliosis. Page 26 Ms. A. has never had seizures. She had a normal menarche with regular menses. Her developmental delay is in the profound range and she has no speech. Cytogenetic diagnosis: ISCNdesignation: 46, XX, inv dup del (8)(:pl2-p23.1::p23.1-qter) de novo The rearrangement on patient A. 's chromosome 8 is of the more frequent type with the center of symmetry at 8p 23.1. Parental chromosomes were normal. Molecular studies could not be done as parents' bloods are not available. 3.12 Patient B. was first seen at 26 years of age (Figure 2.1). She is the third child of unrelated parents, 28 and 29 years old at B's birth. She has two older sisters, one with Best's vitelline retinopathy. Pregnancy was uneventful, and her birth weight was 3150 gms. At birth, she was hypotonic, and her face was considered to be dysmorphic: she had a wide and flat skull, upturned nose and a large mouth. As a baby, she had feeding difficulties with choking spells. Her development was severely delayed; B walked at five years, was toilet trained at ten years, and her speech did not develop beyond several single words. Pneumoencephalogram at seventeen months showed a large cisterna magna and a dilatation of lateral ventricles, more so on the left. At fourteen years of age, her muscle hypotonia persisted, with brisk deep tendon reflexes and bilateral ankle clonus. A cranial CAT scan confirmed the previous findings of dilated lateral ventricles. In addition, cavum septum pellucidum was found. At twenty six years of age, Ms. B. is ambulant, overweight (95 th centile), with height in the 25th centile and head circumference (58 cm) in 98th centile. (Figure 4.) She has a broad forehead, hypertelorism, short upturned nose, long philtrum, large mouth with Page 27 Figure 4.2. Patient C. at 7 months, 3 years and 6 years of age. Page 28 H = H Figure 5. Case B., recombinant chromosome 8 with typical inversion duplication and the center of symmetry at 8p23.1. Case C , recombinant chromosome 8 with atypical inversion duplication and the center of symmetry at 8p23.2. Page 29 prominent lower lip, high palate, and a malocclusion of the teeth. Her ocular fundi are normal. Her hair is curly and thick with a low posterior hairline. Her neck is short and broad. She has small, slightly puffy hands and feet, with shortened distal phalanges. No skin pigmentation anomalies were found. B. has a residual muscle hypotonia with joint hyperextensibility, resulting in severely valgous knees and feet. Deep tendon reflexes are brisk. Ms B. has a central type obesity, with relatively small breasts and most of the bulk distributed around the waistline. Genital development is that of a mature woman. Menarche was at fourteen years of age. B. is very childlike, affectionate and cheerful. She has a history of temper tantrums. Her communication is limited to single words and several short phrases. She understands simple requests and instructions. Her degree of developmental delay has been assessed as severe. Cytogenetic diagnosis: ISCNdesignation: 46, XX, inv dup del(8)(:pll.2-p23.1::p23.1-qter) de novo. (Figure 5 ) This patient's abnormal chromosome is of the frequent type, with a center of symmetry at 8p23.1. Her parents' chromosomes are normal. Molecular studies (T. Nelson) demonstrated a deletion of the maternal STS markers D8S201 and D8S349. The single copy region between the duplicated segments is not detectable. 3.13 Patient C. is a six year old girl, the first child of healthy, unrelated parents. Pregnancy and delivery were normal, and her birthweight at 39 weeks gestation was 3200 gm. As a baby, she had initial feeding difficulties. Page 30 C was first examined at seven months of age for developmental delay and muscle hypotonia. The following examinations were at three years , and most recently at six years of age (Figure 4.2) At seven months she was a cheerful, attentive infant. Physical examination included height at 75th centile, weight at 50th centile, and head circumference at 75th centile. Her skull was slightly asymmetrical, her hair straight and rather sparse, especially at the frontotemporal regions. Her forehead was high, philtrum and lower lip prominent. Her cheeks were chubby, and her chin small. Mid phalangae on both fifth fingers were shortened. A partial syndactyly between the second and third toes bilaterally, and a mongolian spot in the sacral area were noted. The child had striking truncal hypotonia. The hypotonia persisted on follow up examination at age three , while the lower extremities were becoming hypertonic. The rest of the physical examination was normal. C's parents reported that C. had occasional transient staring spells associated with mild twitching of the extremities, which occurred during the first year of life. An EEG showed no epileptiform activity. CAT scan revealed dilatation of ventricles, more on the left side. At six years of age, her height is at the 50th centile, weight at 10th centile, and head circumference at 75th centile. She has a relatively broad forehead with narrow lower face; her nose is aquiline and mouth large, with thin lips. There is a small gap between her front teeth; she has a high palate and pointed chin. The left half of her face is larger than the right. Her ears are somewhat posteriorly rotated, and her hair is now straight, fine and abundant, with no temporal thinning. C has a mild pectus carinatum, and a mild S-shaped scoliosis. A grade III. precordial systolic murmur and split second sound are present: C was found to have a small ventricular septal defect and a bicuspid aortic valve. Abdominal findings are Page 31 unremarkable. Her hands are soft, with clinodactyly of the fifth digit, and moderately prominent finger pads. There are no deep palmar creases. C. wears foot orthoses to compensate for valgosity. Her deep tendon reflexes are brisk on the lower extremities, and Achilles' tendons are somewhat tight. This child has now a vocabulary of eighty words. Her dexterity is yet unclear, though she started to play with dolls recently. C is affectionate and attentive. No seizure like episodes have occurred since infancy, while mild feeding (chewing) difficulties persist. Her developmental delay can be estimated to be moderate at present. Cytogenetic diagnosis: ISCNdesignation: 45XX, invdup?del (8) (:pl2-p23.?2::p23.?2-qter), t(13,14)(p.ll;pll) (Figure 5.) Chromosome painting (Children's Hospital Chromosomal laboratory) has shown all the parts of the recombinant chromosome to be of chromosome 8 origin. The inverted duplication has a center of symmetry at the dark band corresponding to 8p 23.2. This band is wider than on a normal chromosome, indicating that this band is partially duplicated. The inverted segment is large , spanning from the center of symmetry back to the band 8p 12. Parental chromosome are normal, except for mother's balanced Robertsonian 13,14 translocation . The patient has an identical Robertsonian translocation inherited from the mother. Molecular study (T.Nelson) shows that the markers D8S349 and D8S201, distal to 8p23.1, which are usually deleted in the 8p inversion duplication cases with the center of symmetry at 8p23.1, were neither duplicated nor deleted: the child inherited one allele from each parent. Therefore the parental origin of the rearrangement could not be determined. Page 32 Figure 6. Patient D. at the age of 3 months,22 months, 7 years and 15 years. Page 33 Figure 7. Patient D., recombinant chromosome 8, short atypical inverted duplication with the center of symmetry at 8p 23.2. Page 34 Figure 8. Patient D.: Prophase chromosomes 8, demonstrating the short duplication with a wide dark band at 8p23.2 at the center of symmetry. Page 35 3.14 Patient D. is a seventeen year old girl. She is the product of her mother's second pregnancy, the first resulting in a spontaneous loss. The child from the next pregnancy is a girl, who was premature and has a cerebral palsy. The youngest child is a healthy boy. The rest of the family history is normal. D was born after normal pregnancy, with a birthweight of 3200 gms. Her mother was 31 and father 35 years old at the time of her birth. Her development has been somewhat slow, in that she needed remedial education. She is short compared to her family, and has a relatively large head. Magnetic resonance imaging of the brain did not show any anomalies. D. was first referred to endocrinology clinic and from there to genetics, because of her failure to menstruate. This primary amenorrhoea is in contrast with the fact that D's physical maturation started at seven years of age , and that D.'s physique is that of a normal adolescent girl. An abdominal ultrasound demonstrated premenstrual size uterus and normal ovaries. On physical examination at 17 years of age (Figure 6), D.'s height is at the 10th centile, weight at 50th centile, and head circumference at the 98th centile. She has somewhat coarse hair with a widow's peak. The left side of her face is fuller than the right, and her mandible is prominent. Her teeth are normal. She has small hands with clinodactyly on the fifth digits and short nails, and her dermatoglyphic examination revealed ten whorls. She has numerous pigmented nevi on her arms and trunk. . Her breasts are mature, and pubic hair is present. D. is described as an anxious girl, socially awkward, with difficulties in abstract thinking. Her delay was estimated to be mild to borderline normal. Cytogenetic diagnosis: 46 XX, invdup (8)(:p21.71-23. ?2::23. ?2-qter) (Figure 7 and 8). Page 36 Chromosome painting (Childrens Hospital chromosomal laboratory, Dr Kalousek) demonstrated that all parts of the enlarged 8p were of chromosome 8 origin. At the 600 band resolution, the microscopic features are consistent with an atypical inverted duplication with the center of symmetry at 8p 23.2. The derivative chromosome has a dark band at the breakpoint, which by its width may be interpreted as the duplicated 8p23.2. The next G positive band is a wide band with the dark appearance of 8p22 , and is followed by a pale band, longer than the normal 8p23.3. This band could represent a segment of the duplicated band 8p21. This duplication is relatively short. Because of the atypical center of symmetry in the derivative chromosome 8,and the unusually short duplication, a direct duplication had been considered in the differential diagnosis: 46, XX, dup (8)(p23.1-p23.3::p23.1-p23.3) Molecular study (T.Nelson) demonstrated the allele D8S349 to be present in triplicate, i.e not deleted, and located within the duplicated region (8p23.1-23.2). The more distal allele D8S201 is present in one maternal and one paternal allele. This supports the FISH analysis in demonstrating the duplication includes areas distal to 23.1 and is consistent with the cytogenetic picture of an atypical variant of inverted duplication. 3.2 Fluorescence in situ hybridization with probe 153G8- results. We have studied the location of repetitive sequences using the cosmid 153G8 (containing the megasatellite MS) as the FISH probe in our four cases of 8p inversion duplication. Cosmid probe 153G8 hybridized to subtelomeric regions on 4p and 8p in Page 37 Figure 9. Fluorescence in situ hybridization with the MS probe 153G8 and the centromeric #8 probe (Oncor) in cases A., B. and D. In patient C. the probe 153G8 alone was used. A single signal at arrow (A and B), and double signal at arrow (C and D). Page 38 normal controls. FISH was performed on metaphase cells from patients A, B, and D., using 153G8 together with Oncor centromeric probe for chromosome 8. The cells from patient C. were examined using 153G8 alone (technical reasons- not enough probe left to risk using with the powerful commercial probe). In patients A.and B., cosmid 153G8 hybridized to the region at the center of symmetry (8p 23.1), i.e. single signal was visible in the middle of the elongated 8p arm on the abnormal chromosome (Figure 9, A and B), i.e. within the 8p 23.1 region.. In patients C.and D. the MS probe 153G8 produced two signals, close together but distinguishable, flanking the center of symmetry. The center of symmetry in these two patients is in the 8p 23.2 region (Figure 9, C and D). This result places again the megasatellite probe in the 8p 23.1 region. CHAPTER 4: DISCUSSION: 4.1 Genotype-phenotype correlation in 8p rearrangements. There are shared features which constitute the Gestalt of the 8p inversion duplication facies: Relative macrocephaly with a prominent forehead, macrostomia, everted lower lip, hypotonia in infancy. The clinical picture of our four patients demonstrates variability within the relative constraint of a syndromic diagnosis. This variability is seen in phenotypic features like vertebral, cardiac and CNS anomalies. It is also seen in the varying degree of developmental delay and neurological deficit, such as spasticity and seizures. The literature data reflect a similar variability. When looking for a possible phenotype - genotype correlation, one is aware that a number of craniofacial dysmorphisms, like large ears, high palate, and low posterior Page 39 hairline, are present in numerous other conditions, and cannot be used for comparison. A syndrome characteristic selected for a comparative analysis should be specific and clearly identifiable. Agenesis of corpus callosum (ACC) and congenital heart anomalies are readily identifiable, but not suitable for comparative analysis at present. ACC, a polygenic anomaly, is known to be associated with 8p duplication [Digilio et al.,1994, Dobyns, 1995]. However, not all patients with 8p inverted duplication reported in the literature were investigated for the anomalies of corpus callosum, so the true incidence of ACC in these patients is not known. Congenital heart disease CHD, mostly of the atrioventricular defect type) is found in half of reported terminal deletion cases. The complexity and the polygenic nature of CHD makes it also unsuitable. I propose that two of the less specific clinical features of 8p inversion duplication may be useful in comparing and correlating variation in cytogenetic diagnosis with the clinical picture. These are 1) the degree of mental handicap, and 2) the infantile hypotonia / late onset spasticity, which is present in some form in all children with 8p inversion duplication and which has characteristics of cerebral palsy. After the cytogenetic diagnosis has been established, the assessment of the severity of the developmental delay and of the motor handicap can be useful in the genotype-phenotype correlation. The evidence from the literature suggests that the severe phenotype is present in all reported patients, not showing any correlation with the size of the duplication, as long as the band 21 is duplicated. The influence of the terminal deletion is unclear. Recently, several authors reported patients with various 8p duplications, and without severe mental handicap [Engelen et al.,1995, Dhooge et al., 1994, Barber et al., 1998, Sklower Brooks et al., 1998]. These reports all suggest that duplication of bands 8p 22>ter and of 8p23.1 may be associated with learning disability, but not with a severe handicap. These observations strongly suggest that the duplication of the band 8p21 is the Page 40 feature responsible for the severe phenotype in 8p inversion duplication. It is of interest that a neurofibrilin precursor light protein gene (OMLM #162 280), coding for one of the components necessary for development of cellular and axonal structure of motor neurons, has been mapped to 8p21. The neurofibrilin precursor gene was suggested as the candidate gene for the agenesis of corpus callosum by Digilio et al., in 1994. Its relation to the neurological symptomatology in 8p inversion duplication deserves further attention. 4.2 Analysis of the genotype - phenotype correlation in our patients. 4.21 Patients A. and B. both have the more typical 8p inversion duplication with the center of symmetry at 8p 23.1. Both have a large duplication. The absence of a single copy region in B. is unusual. The two women share important clinical features: a large skull, a large mouth with downturned lower lip, severe-to-profound degree of mental handicap, and muscle hypotonia with brisk ankle reflexes. Both women have attained sexual maturity and menstruated. These two patients have a different body size: A. is very small and thin, 4 SD below average in weight and height. B. is somewhat shorter than her normal sisters , within the 2SD range. She is very overweight. 4.22 Patient C has the atypical inversion duplication 8p(l 1.2-23.2). The duplication is large, spanning almost the entire short arm back to 8pl2. Because of the large size of the duplication, the mirror configuration is clearly defined. The dark band, corresponding to partially duplicated 8p23.2, is located in the center of symmetry. Fluorescence in situ hybridization with the MS probe 153G8 shows two signals in the middle of the elongated 8p, flanking the center of symmetry (Fig 9 c). Page 41 Genotyping shows that a single maternal and a single paternal allele D8S201 and D8S349 have been inherited. Duplication or deletion was not detected at these sites, and parent of origin could not be determined. So far, all typical cases of inversion duplication had a terminal deletion shown by various markers: D8S7, D8S201, D8S349, Defensin I. gene [Mitchell, et al,1993]. The three patients previously reported with the atypical 8p inversion duplication [Feldman et al., 1993, Hoo et al, 1995], have not been examined using these markers. The finding that in our patient the D8S349 and D8S201 are not deleted needs clarification. The most plausible explanation for the non-deleted STS markers in patient C. would be that there is a terminal deletion, but smaller than the deletions in the typical 8p inversion duplication, distal to D8S201 and D8S349. Absence of a terminal deletion has been seen as typical of a 8p tandem duplication in Engelen's cases (1996). The FISH probe used in Engelen's case is defined as originating from the 8p23-ter. While the cytogenetic findings in Engelen's patients were more typical for a tandem duplication, in patient C, where the deletion is also absent, the cytogenetic characteristics show the inverted features of the duplication quite clearly. Considering Cs duplication on 8p which is clearly inverted, by analogy one would expect a very severe phenotype. This is not the case. C. presents with a picture of moderate developmental delay and a mild neurological deficit, in contrast to the severe to profound delay reported in majority of other cases of the typical 8p inversion duplication. She walked before her third birthday, and at six years of age she has a vocabulary of about eighty words. Her heel cords are somewhat tight and the deep tendon reflexes are brisk.. It can be argued that in her case the presumed subtelomeric deletion, smaller than in the typical 8p inversion duplication, resulted in better developmental outcome. She has a congenital heart defect. Whether in her case the congenital heart disease (ventricular septal defect) is related to the presumed deletion, remains a speculation. Page 42 4.23 Patient D. has a mild phenotype and an unusual presentation. She is not obviously dysmorphic, and is only mildly developmentally delayed. The problem that brought her to the geneticist's attention is a referral from an endocrinologist. Ms.D. has a primary amenorrhoea at seventeen years of age, in spite of early signs of puberty at seven, and ongoing maturation for the following ten years. Her maturation is externally completed, pelvic ultrasound demonstrated ovaries to be present and the uterus to be of prepubertal size. Rather inconspicuous clinical features (large forehead, triangular face, and borderline normal developmental delay) in this patient are reminiscent of the phenotypes reported with 8p tandem duplication. The cytogenetic characteristics of her duplicated chromosome have been disputed, and the diagnosis of direct duplication has been suggested. Examined in prophase, the configuration of the bands is more consistent with the diagnosis of an atypical inversion duplication with the center of symmetry at 8p23.2, similar to our patient C. In this case, the duplication is very short. (Figure 7 and 8). Our interpretation is that the duplication terminates very closely to the band 8p 22, i.e. only a small part of the band 8p21, the subband 8p21.3, is included in the duplication. When one considers that this patient has no signs of cerebral palsy ( normal reflexes, no tight heel cords or hypotonia), it is possible to hypothesize that her phenotype is related to the shortness of the duplication, and that the duplication does not include the gene which is associated with the CNS malformation and / or the neurological deficit in most patients with 8p inversion duplication. Presuming that the cytogenetic diagnosis of inverted duplication will be confirmed by further studies, the small size of the duplication, with only a small segment of the band 8p21 present., is obviously significant in mitigating this patient's phenotype. The role of the presumed small terminal deletion in this patient remains unclear. Page 43 The cause of D.'s amenorrhoea could be associated with altered function of LHRH gene, OMTJV1 # 162280, located in 8p21 region [Bruskewich et al.,1996]. The fluorescence in situ hybridization, using the MS probe 153G8, shows two signals, close together and approximately flanking the center of symmetry. The FISH signals are in a configuration very similar to that seen in patient C, except that the signals are not in the middle of the 8p arm, because the duplication is so short. The grouping of the signals around the center of symmetry suggests that the duplication is indeed inverted, but does not prove it conclusively. For such proof, it would be necessary to perform a multicolored FISH with probes known to be on 8p. The sequence of the signals then would provide the conclusive diagnosis of either direct or inverted duplication. The maternal markers D8S201 and D8S349 are not deleted, and indeed there are two copies of the maternal allele D8S349 in this patient. This suggests that the locus D8S349 is within the duplicated region. A deletion distal to the two markers used is not ruled out. To demonstrate a terminal deletion and so to support the diagnosis of inverted duplication, it would be necessary to use markers distal to D8S349 and D8S201. 4.3 Repetitive sequences (MS 153G8) located in the 8p23.1 region The megasatellite is defined as a 4.7 kb EcoRI unit that is tandemly repeated [Kogi et al., 1997]. The cosmid 153G8 is isolated from chromosome 8 specific cosmid library and contains megasatellite sequences. These megasatellite sequences span about 15 kb, and are embedded within a large reiterated sequence (LRS). There are at least four locations of the LRS containing the MS within a 2-3 Mb region of distal 8p. The orientation of these sequences is unknown (Nelson, personal communication). The cosmid probe 153G8 hybridizes to the corresponding repetitive sequences (megasatellite) present both on 8p and 4p. Page 44 4.31 The FISH result in the patients A. and B. with the typical 8p inversion duplication (center of symmetry at 8p23.1) demonstrates the location of repetitive sequences in the 8p 23.1 region. This location is consistent with the hypothesis that repetitive sequences may facilitate recombination in 8p inversion duplication.. 4.32 The FISH in patients C and D, resulting in two signals around the center of symmetry at 8p23.2, again demonstrates presence of repetitive sequences in the 8p23.1 region, and supports the cytogenetic diagnosis of the atypical variant in that it shows the duplication includes the region distal to 8p 23.1 CHAPTER 5: CONCLUSIONS: 5.1 The phenotype of 8p inversion duplication. 8p inversion duplication has a distinct constellation of clinical features, none of which is pathognomonic for the disorder. The clinical phenotype in all cases consists of craniofacial dysmorphism (prominent forehead, upturned nose, large mouth with everted lower lip), macrocephaly, muscle hypotonia with feeding difficulties in infancy, CNS anomalies (agenesis of corpus callosum, ventriculomegaly), and severe developmental delay. This combination of features would invariably prompt a clinician to arrange for chromosomal analysis. Given the clinical presentation, the clinician could direct the cytogeneticist to evaluate chromosome 8p. Page 45 5.2 Phenotype of the 8p inversion duplication variant (center at 8p23.2). There are no detailed data in the literature on phenotype of inversion duplication with the center of symmetry at 8p23.2 (the atypical variant). The report on three cases with the atypical variant are brief, and there is no discernible difference from the phenotype of "classic" 8p inversion duplication. Detailed evaluation of our patients show that the developmental deficit may be milder than in the typical cases, while the phenotypic features are unchanged. This conclusion is based on a case observation and would need to be corroborated by studying more patients. Molecular analysis has provided an additional finding. In both patients with the atypical variant the subtelomeric markers D8S349 and D8S201, deleted in the common form of 8p inversion duplication, are not deleted. This suggests that that the center of symmetry is more distal and is consistent with cytogenetic and FISH analysis with probe 153G8. In these patients we predict that there are also distal deletions which would be consistent with the model of origin of 8p inversion duplication we favor. To show the deletion in these patients, more distal markers need to be tested. Considering the better developmental outcome, the extent of the deletion could be the determining variable. 5.3 Atypical variant of 8p inversion duplication with a short deletion. Patient D. has the atypical variant of 8p inversion duplication, where the center of symmetry is at 8p23.2. In her case the duplication is very short, terminating just beyond the duplicated 8p22 in the region of 8p21.3. Both her cytogenetic and her clinical features, i.e. non dysmorphic face and borderline normal development, are unusual. This phenotype resembles the clinical picture of patients reported with a direct terminal duplication on 8p22-ter. In uncertain cases, differentiation between an inverted and a direct duplication requires dual color FISH to establish the sequence of the duplicated loci. Page 46 The mild phenotype and the short duplication suggest that in this patient, the chromosomal region associated with the severe phenotype in 8p inverted duplication (region within 8p21) is not duplicated. 5.4 FISH analysis with cosmid probe 153G8. FISH with the probe 153G8, which contains repetitive sequences (megasatellite, MS), demonstrated that the megasatellite is located in the 8p23.1 region. In the atypical variant of 8p inversion duplication, the finding of two signals flanking the center of symmetry again confirms the location of MS at 8p 23.1, and supports the cytogenetic diagnosis of inverted duplication with the center of symmetry distal to 8p 23.1. 5.5 Areas for future research: Determination of a terminal deletion in atypical 8p inversion duplication, by identifying suitable markers distal to D8S349 and D8S201. Identification of suitable markers on 8p with which to examine cytogenetically unusual 8p duplications by multicolored FISH, in order to differentiate between a tandem and inverted duplication in uncertain cases. In cases of 8p inversion duplication where the duplication is very short, correlating of the neurological status of the patient with the extent of 8p21 duplication may bring more data about the region critical for the phenotype. Page 47 B I B L I O G R A P H Y . 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