THE GENETIC BASIS OF HEMIFACIAL MICROSOMIA Jerome Christopher Siong Teck Lim The Institute of Child Health, University College London Submitted to the University of London for the degree of Doctor of Medicine UMI Number: U592B06 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592B06 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The aetiology of hemifacial microsomia is uncertain; vascular, metabolic, teratogenic and genetic factors have been proposed as aetiological influences. The evidence for a genetic basis comes from observations of the phenotype in individuals carrying chromosomal rearrangements, and from a small number of studies involving families in whom the condition appeared to be transmitted in a Mendelian fashion. Malformations reminiscent of hemifacial microsomia have also been seen in knockout and mutant animal models, and these have given rise to a number of candidate genes for the disease. Two main objectives were defined at the start of this project. The first of these was to expand upon the currently available clinical data by interviewing and examining patients with hemifacial microsomia or isolated microtia who presented to two specialist treatment centres in London, Great Ormond Street Hospital and Mount Vernon Hospital. Alongside this a genome scan was performed on a family in which hemifacial microsomia appeared to be segregating in an autosomal dominant manner. Whilst definite linkage to a single locus could not be inferred from the results of the genome scan, a few regions of interest were identified, and linkage to a large proportion of the genome was excluded. One area of particular interest, 2q32 - 2q37, was analysed in greater detail, and other regions together with their potential disease genes have been highlighted. 2 Table of contents Page Abstract 2 Table of contents 3-6 Figures and tables 7-13 Acknowledgements 14 Introduction 15-16 1.1. Incidence & Epidemiology 17-18 1.2. Clinical features 19 1.2.1. Facial abnormalities 19-20 1.2.2. Ear abnormalities 20-22 1.2.3. Central nervous system abnormalities 22 1.2.4. Eye abnormalities 23 1.2.5. Skeletal abnormalities 23-24 1.2.6. Other abnormalities 24-25 1.3. Nomenclature 26 1.4. Embryology 27-31 1.5. Aetiology 32 1.5.1. Vascular 32-33 1.5.2. Metabolic 33-34 1.5.3. Teratogenic 34-35 1.5.4. Multiple gestations 36-37 1.5.5. Bleeding in pregnancy 37-38 1.5.6. Other aetiological theories 39 3 Page 1.6. Genetic aspects 1.6.1. Mendelian and non-Mendelian inheritance 40-41 1.6.2. Mode of inheritance of hemifacial microsomia 41-43 1.6.3. Investigation of human disease genes 44 1.6.4. Chromosomal rearrangements 44-45 1.6.5. Genetic mapping 46-48 1.6.6. Recombination fractions 48-49 1.6.7. Linkage 49-51 1.6.8. Genetic mapping in hemifacial microsomia in humans 51-52 1.6.9. Investigation of the genetic basis of complex diseases 53-58 1.6.10. Animal models 59-66 Aims of project 67-68 2. Materials and methods 69 2.1. Method of patient data collection 70-71 2.2. Materials 72-75 2.3. Methods of genome scan 2.3.1. Clinical details of patients studied in genome scan 76-78 2.3.2. Polymerase chain reaction 78 2.3.3. Gel electrophoresis 79 2.3.4. Capillary array electrophoresis 79-80 2.3.5. Data analysis 80 4 Page Results of patient data collection 81-82 3.1. Age and sex distribution 83-86 3.2. Laterality 87 3.3. Grade of microtia 88 3.4. Skin tags 89-90 3.5. Abnormalities of the external auditory meatus 91-94 3.6. Facial nerve palsy 95 3.7. Cleft lip and palate 95 3.8. Affected relatives 96-100 3.9. Bleeding in pregnancy 101-102 3.10. Incidence of twinning 102 Discussion of patient data collection 103-104 4.1. Age and sex distribution 105-106 4.2. Laterality 107-110 4.3. Grade of microtia 110-111 4.4. Skin tags 111-112 4.5. Abnormalities of the external auditory meatus 112-113 4.6. Facial nerve palsy 113-114 4.7. Cleft lip and palate 115 4.8. Affected relatives 116-117 4.9. Bleeding in pregnancy 118-119 4.10 Incidence of twinning 119-120 5 Page Results of genome scan 121 5.1. Analysis of candidate loci 122 5.2. Genome search 123 5.2.1. Two point linkage analysis 123-160 5.2.2. Two point lod scores of interest 161-184 5.2.3. Genome-wide multipoint linkage analysis 185-210 5.2.4. Multipoint regions of interest 210-221 Discussion of genome scan 222-224 6.1. Genotyping difficulties 224-225 6.2. Potential sources of error 226-230 6.3. Change of affection status of individual V:7 231-233 6.4. Regions of interest excluded by the results of genome scan 234-237 6.5. Lod scores relative to candidate genes suggested by animal 238-240 models 6.6. Regions of interest warranting further investigation 241-249 6.7. Future work 250-251 Reference list 252-273 Appendix A Patient information and consent forms 274-279 Appendix B Patient data collection form 280-282 6 Figures and Tables Page Figure 1 Schematic drawing of the pharyngeal arches 27 Figure 2 Lateral view of the head of an embryo showing the six 28 auricular hillocks Figure 3 Schematic representation of the development of the middle 29 ear structures Figure 4 Schematic representation of the derivatives of the first three 31 branchial arches Figure 5 Pedigree of family R exhibiting autosomal dominant 77 hemifacial microsomia Figure 6 Year of birth distribution of microtia patients 84 Figure 7 Year of birth distribution of hemifacial microsomia patients 85 Figure 8 The pedigree for the family with hemifacial microsomia 97 affecting three generations. Figure 9 The haplotypes for the markers from the Research Genetics 162 Marker Set version 8 analysed in the region 5q34-5q35 Figure 10 The haplotypes for other markers from the Research Genetics 164 Marker Set version 8 analysed in the region 5q34-5q35 Figure 11 The haplotypes for the markers from the Research Genetics 166 Marker Set version 8 analysed in the region 3q25 Figure 12 The haplotypes for the markers from the Research Genetics 168 Marker Set version 8 analysed in the region 5pl4 7 Page Figure 13 The haplotypes for the markers from the Research Genetics 170 Marker Set version 8 analysed in the region 1 lq25 Figure 14 The haplotypes for the markers from the Research Genetics 172 Marker Set version 8 analysed in the region 13q34 Figure 15 The haplotypes for the markers from the Research Genetics 174 Marker Set version 8 analysed in the region 2q32-2q37 Figure 16 The haplotypes for the markers from the Research Genetics 176 Marker Set version 8 analysed in the region 8q24 Figure 17 The haplotypes for the markers from the Research Genetics 178 Marker Set version 8 analysed in the region 16pl 2 Figure 18 The haplotypes for the markers from the Research Genetics 180 Marker Set version 8 analysed in the region 3p25 Figure 19 The haplotypes for the markers from the Research Genetics 182 Marker Set version 8 analysed in the region 8q22 Figure 20 The haplotypes for the markers from the Research Genetics 184 Marker Set version 8 analysed in the region 10q22 Figure 21 Chromosome 1 multipoint lod score results 187 Figure 22 Chromosome 2 multipoint lod score results 188 Figure 23 Multipoint lod scores for the Research Genetics markers used 189 in the telomeric region of 2q 8 Page Chromosome 3 multipoint lod score results 190 Chromosome 4 multipoint lod score results 191 Chromosome 5 multipoint lod score results 192 Chromosome 6 multipoint lod score results 193 Chromosome 7 multipoint lod score results 194 Chromosome 8 multipoint lod score results 195 Chromosome 9 multipoint lod score results 196 Chromosome 10 multipoint od score results 197 Chromosome 11 multipoint od score results 198 Chromosome 12 multipoint od score results 199 Chromosome 13 multipoint od score results 200 Chromosome 14 multipoint od score results 201 Chromosome 15 multipoint od score results 202 Chromosome 16 multipoint od score results 203 Chromosome 17 multipoint od score results 204 Chromosome 18 multipoint od score results 205 Chromosome 19 multipoint od score results 206 Chromosome 20 multipoint od score results 207 Chromosome 21 multipoint od score results 208 Chromosome 22 multipoint od score results 209 Page Figure 44 Schematic illustration of map locations of microsatellite 211 markers for 2q32 - 2q37 Figure 45 The haplotypes for the additional markers analysed in the 213 region 2q32 - 2q37 in addition to those from the Research Genetics Marker Set version 8 Figure 46 Plot of the multipoint scores for the additional markers analysed 214 in the region 2q32 - 2q37 Figure 47 The haplotypes for the markers from the Research Genetics 216 Marker Set version 8 analysed in the region 8p21 Figure 48 The haplotypes for the markers from the Research Genetics 218 Marker Set version 8 analysed in the region 14q23 Figure 49 The haplotypes for the markers from the Research Genetics 220 Marker Set version 8 analysed in the region 15ql 1 — 15ql2 Figure 50 Output from Genetic Profiler analysis of data showing multiple 227 “stutter” peaks Figure 51 Output from Genetic Profiler demonstrating spectral overlap 228 Figure 52 The relative positions of markers