I Genetic Aspects of Hearing Loss in the Limpopo Province of South Africa Rosemary I Kabahuma MBCHB (Makerere University), MMED ENT Surgery (University of Nairobi), MSC Audiological Medicine (University of London) A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Humanities, Department of Speech Pathology and Audiology, University of the Witwatersrand, Johannesburg, South Africa January 2010 II DEDICATION To Almighty God who has by grace made all this possible. To my daughters Constance and Theodora, who experienced the cost of a PhD at an early age and yet remained encouraging, supportive, always believing for their mother. To Ezra whose support gave me the space to fly. ?But he knows the way that I take; when he has tested me, I will come forth as gold.? Job 23:10 III DECLARATION I, Rosemary Ida Kabahuma, do hereby declare that this dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Humanities, Department of Speech Pathology and Audiology, University of Witwatersrand, is my own original work. All assistance I have received has been stated in the acknowledgements. This work has not been submitted before for any degree or examination at this or any other university. I declare that the protocol was cleared by the Committee for Research on Human subjects, Ethics committee clearance certificate protocol number M991005. Rosemary Ida Kabahuma ____ day of ________________ 2010 IV ACKNOWLEDGEMENTS I would like to thank all the many individuals and organizations who, through their support, ensured the successful completion of this project. It is not possible to mention each one by name but the following are singled out: ? My main supervisor, Prof. Claire Penn, Department of Speech Pathology and Audiology, University of Witwatersrand, Johannesburg, South Africa ? My co-supervisor, Prof. Michele Ramsay, Division of Human Genetics, National Health Laboratory Service and School of Pathology, University of Witwatersrand, Johannesburg, South Africa ? Prof. Jackie L Clarke, Faculty of Audiology, Callier Center for Communication Disorders, University of Texas at Dallas, for the constructive criticism and advice ? The eaf students at the Tshilidzini and Bosele Schools for the Deaf who took part in this study ? The parents and guardians of all the subjects for their co-operation ? The school principals and staff at the Tshilidzini and Bosele Schools for the Deaf, who received us warmly and went out of their way to ensure the successful completion of this project ? All the translators at the two Schools for the Deaf who assisted in the completion of the questionnaires ? The nursing staff at the two Schools for the Deaf, for their assistance with phlebotomy and urine testing ? Prof. RF Mueller, Karl Bromelow and Tim Hutchin, formerly of the Deafness Research Team of the Molecular Medicine Unit, St James? University Hospital, V University of Leeds, UK, for the financial support and training in mutation detection techniques during the attachment at Leeds ? Prof. Andrew Read and James O?Sullivan, Department of Medical Genetics, St Mary?s Hospital, University of Manchester, UK, for the invaluable work in the detection of the Waardenburg Syndrome mutations in this study ? Dr Xue Zhong Liu and Xiao Mei of the Research Unit of the Department of Otorhinolaryngology at the University of Miami, USA, for their invaluable training in mutation detection ? Dr Daniels and Mrs Daniels for all their support ? The staff and students of the Department of Speech Pathology and Audiology, University of the Witwatersrand, Johannesburg, South Africa for the assistance with the audiological testing of the subjects ? Ronel Kilian and Philemon Ratshilumela for the audiological testing of the subjects ? The staff and students at the molecular laboratory of the NHLS, University of the Witwatersrand, Johannesburg, South Africa, for the assistance with DNA extraction and processing of samples. A special thanks to Silke Arndt, Fahmida Essop, Robyn Kerr, Tony Lane and Angela Turner ? Jerry Sigudla, a dedicated research assistant who painstakingly worked to track and compile data ? Sam Ntuli for the assistance with the statistical analysis ? The Mellon Foundation for partially funding this PhD ? The Medical Research Council, South Africa for partially funding this project; and ? The Department of Health and Social Welfare, Limpopo Province, for facilitating my study leave and offering financial support for the attachment to the University of Miami. VI TABLE OF CONTENTS PAGE NO. DEDICATION II DECLARATION III ACKNOWLEDGEMENTS IV TABLE OF CONTENTS VI LIST OF ABBREVIATIONS XIV LIST OF TABLES XVI LIST OF FIGURES XXI ABSTRACT 1 CHAPTER 1: INTRODUCTION 6 1.1 General Introduction 6 1.2 Genes and Populations 11 1.3 The Limpopo Province 14 1.3.1 The Land. 14 1.3.2 Population characteristics 20 1.3.3 Employment 21 1.3.4 Health profile of the people in the Limpopo Province 21 1.3.5 Access to health care in the Limpopo Province 23 1.4 The People, their Cultures and Practices 28 1.4.1 The Venda people and politics 28 1.4.1.1 Betrothal and marriage amongst the Bavhenda 30 1.4.1.2 Attitudes towards disability amongst the Venda 31 1.4.1.3 Ear disease and traditional healing amongst the Venda 31 1.4.2 The Shangaan (Tsonga) people and politics 31 VII 1.4.2.1 Betrothal and marriage among the Shangaan 32 1.4.3 The Pedi people and politics 32 1.4.3.1 Betrothal and marriage among the Pedi 34 1.5 The Rationale for this Study 36 CHAPTER 2: LITERATURE REVIEW AND BACKGROUND INFORMATION I 2.1 Overview of Genetics of Hearing Loss 39 2.1.1 Disease Inheritance 39 2.1.2 Modes of Inheritance 40 2.1.3 Research into Genes for Hearing Loss 43 2.2 Epidemiological Perspectives of Hearing Loss 45 2.2.1 General Considerations in the Aetiology of Hearing Loss 45 2.2.2 Epidemiological Models of Hearing Research. 47 2.2.3 Epidemiological models for SNHL 53 2.2.4 Epidemiology of Hearing Loss in Africa 55 2.2.5 Epidemiological Studies on Genetic Hearing Loss 60 2.3 The Ear in Genetic Hearing Deafness 63 2.3.1 Development of The Ear 63 2.3.2 Overview of the Anatomy of the Mature Inner Ear 66 2.3.3 Gap Junctional Systems of the Human Ear 68 2.3.4 Major Ear Defects in Hereditary Hearing Loss 69 2.3.5 Overview of the Physiology of Hearing 70 CHAPTER 3: LITERATURE REVIEW AND BACKGROUND INFORMATION II 3.1 History of Research in Genetic Deafness 73 3.1.1 History of the Genetics of Hearing Loss 73 3.1.2 Clinical Phenotypes of Genetic Deafness 74 VIII 3.1.3 Histopathologic Phenotypes of Genetic Deafness 76 3.1.4 Molecular Phenotypes in Syndromic Genetic Disease 79 3.1.5 The Human Genome Project 80 3.1.6 Research using the mouse as a model for human deafness 82 3.2 Gene Localization and Auditory Research 86 3.2.1 Genes implicated in Hearing Loss 86 3.2.2 General functional classification of deafness genes 87 3.2.2.1 Genes controlling hair cell structure 88 3.2.2.2 Extracellular matrix genes 88 3.2.2.3 Genes controlling ion homeostasis 89 3.2.2.4 Genes controlling transcription factors 89 3.2.2.5 Miscellaneous genes 90 3.2.3 Overview of Connexins (Cx) and the Gap Junctional Systems of the Ear 91 3.2.4 Gap junction Gene Variants and Hearing Loss 94 3.2.5 GJB2 Mutations and Hearing Loss: Phenotype-Genotype Relationship 94 3.2.6 GJB2 Mutations and Type of Hearing Loss 95 3.2.7 Waardenburg syndrome 96 3.2.7.1 Clinical features of Waardenburg Syndrome 97 3.2.7.2 The Clinical Classification of Waardenburg Syndrome 100 3.2.7.3 Variable penetrance of Waardenburg Syndrome 103 3.2.8 Mitochondrial genes 106 3.2.9 Audiological findings in non-syndromic genetic hearing loss 108 3.2.10 The Future Application of Proteomics and Genomics 109 IX 3.3 Clinical Perspectives 114 3.3.1 Detection of Childhood Hearing Loss 117 3.3.1.1 Targeted Screening 119 3.3.1.2 Universal Neonatal Hearing Screening 120 3.3.2 Principles of Assessment 126 3.3.3. Audiological assessment 129 3.3.3.1 Immitance testing 129 3.3.3.2 Evoked otoacoustic emissions 129 3.3.3.3 Auditory brainstem response testing 129 3.3.3.4 Auditory steady-state response testing 130 3.3.3.5 Audiometry 130 3.3.3.6 Audioprofiles 131 3.3.3.7 Description of hearing loss 132 3.3.4 Assessment and Investigations 134 3.3.4.1 History 135 3.3.4.2 Clinical examination 137 3.3.4.3 Ophthalmology 138 3.3.4.4 Serology 138 3.3.4.5 Haematology and Biochemistry 139 3.3.4.6 Thyroid tests 139 3.3.4.7 Immunology 139 3.3.4.8 Metabolic screen 139 3.3.4.9 Urinalysis 140 3.3.4.10 Electrocardiography 140 3.3.4.11 Radiology 140 X 3.3.4.12 Audiology 141 3.3.4.13 Vestibular investigations 141 3.3.4.14 Clinical photographs 142 3.3.4.15 Genetic testing 142 3.3.4.16 Referral to geneticist 145 3.3.5 Aetiological Diagnosis 148 3.3.6 Intervention for the hearing impaired Child 149 CHAPTER 4: METHODOLOGY 4.1 Problem Statement, Research Question and Purpose of the Study 152 4.2 Aim and Objectives 153 4.3 Study Design 154 4.3.1 Reference Population 157 4.3.2 Setting (Schools for the Deaf) 158 4.3.3 Study Population 159 4.3.4. Inclusion Criteria 160 4.3.5 Exclusion Criteria 160 4.3.6 Limitations of the Study 161 4.3.6.1 Language 161 4.3.6.2 Sample Size 161 4.3.6.3 The Use of Questionnaires 162 4.3.6.4 Attrition 163 4.3.6.5 Pedigrees and family testing 163 4.3.6.6 Unavailability of Investigative Facilities 163 4.3.7 Ethical Considerations 163 4.3.8 Ethics Approval 164 XI 4.4 Methods and Procedures 164 4.4.1 Equipment 164 4.4.2 Audiological Evaluation 164 4.4.3 Procedures 166 4.4.3.1 Phase1 166 4.4.3.2 Phase 2a 168 4.4.3.3 Phase 2b 170 4.5 Methods used for Mutation Detection 174 4.5.1 Specimen Collection 174 4.5.2 DNA Extraction 174 4.5.3 Mutation Detection 175 4.6 Data analysis 180 4.6.1 Mapping techniques used for epidemiological analysis 180 4.6.2 Statistical Analysis 181 4.6.3 The Null Hypothesis (H0) 182 CHAPTER 5: RESULTS 5.1 Demographic Information of Subjects 183 5.1.1 Phase I 183 5.1.2 Phase 2 188 5.2 Geographical Distribution of Hearing Loss 191 5.2.1 Phase I 191 5.2.2 Phase 2 192 5.3 Type and Degree of Hearing Impairment 199 5.3.1 Tympanometry and Transient otoacoustic emissions 199 5.3.2 Audiometry 200 XII 5.4 Aetiological Investigation of Hearing Disorders 204 5.4.1 Family History of Hearing Loss Among the Subjects 204 5.4.2 Consanguinity Among Parents 205 5.4.3 Urinalysis Results 211 5.4.4 Reported Pregnancy and Perinatal history 211 5.4.5 Reported Medical Conditions Among the Subjects 211 5.5 Mutation Detection 215 5.5.1 GJB2 215 5.5.2 Waardenburg Syndrome 218 5.5.3 Mitochondrial Mutations 219 5.6 Clinical signs in Hearing Loss 223 5.6.1 Eye Findings Among the Subjects 223 5.6.2 Skeletal Findings Among the Subjects 223 5.6.3 Ear, Nose and Throat Findings Among the Subjects 224 5.6.4 Other Systemic Findings Among the Subjects 225 5.7 Tests of association and Binary logistic regression analysis 227 5.7.1 Calculation of crude odds ratio 233 5.7.2 Interpretation of the crude odds ratio 233 5.7.3 Assessment of the fitted logistic regression model 233 5.7.4 Interpretation of the odds ratio for family history 233 5.7.5 The Hosmer-Lemeshow goodness-of-fit test 234 5.7.6 Magnitude of area under ROC curve 235 5.7.7 Plot of sensitivity/specificity vs probability cut-off point 236 XIII CHAPTER 6: DISCUSSION, CONCLUSION AND RECOMMENDATIONS 6.1 Discussion 238 6.1.1 Geographical Distribution of Hearing Loss in Limpopo 238 6.1.2 Accounting for Bias in this Study 243 6.1.2.1 Bias due to migratory labour practice 243 6.1.2.2 Bias due to non-random admission into schools 244 6.1.2.3 Bias due to proximity to the schools 244 6.1.2.4 Bias due to varying population density within the province 244 6.1.3 Type and Degree of Hearing Loss in Limpopo 245 6.1.4 Aetiology of Learing Loss in Limpopo 248 6.1.5 Influence of Consanguinity on Genetic Hearing Loss in Limpopo 255 6.1.6 Mode of Inheritance of Hearing Loss in the Study Population 258 6.1.7 Significance of the Candidate Genes for Deafness in the Limpopo 259 6.1.7.1 GJB2 (Connexin26) 260 6.1.7.2 Common Mitochondrial Mutations 263 6.1.7.3 Waardenburg syndrome 264 6.1.8 Nosological Entities of Hearing Loss in Limpopo 264 6.2 Conclusions 268 6.2.1 High risk areas for hearing loss in the Limpopo province 268 6.2.2 Clinical Perspectives 269 6.2.3 Genetic Perspectives 269 6.2.4 Policy Issues 271 6.3 Recommendations 277 7.1 References 281 7. .2 Appendices 304 XIV LIST OF ABBREVIATIONS HL Hearing Level StatsSA Statistics South Africa DNA Deoxyribose nucleic acid EcoG Electrochocleography GP general practioner OME Otitis media with effusion PTA Pure tone average ECG Electrocardiogram MRI Magnetic resonance imaging TORCH Toxoplasmosis, Rubella, Cytomegalovirus, Herpes CSF Cerebral spinal fluid CT Computerised tomography CME Continued medical education SEN Special education needs ENT Ear Nose and Throat SNHL Sensorineural hearing loss TEOAEs Transient evoked otoacoustic emissions ART Acoustic reflex threshold PMHC Pietersburg Mankweng Hospital Complex ENG Electronystagmography NHLS National Health Laboratory Services PCR Polymerized chain reaction WS Waardenburg syndrome XV ARNSHL Autosomal recessive nonsyndromic hearing loss NSSNHL Nonsyndromic sensorineural hearing loss NSAHL Nonsyndromic autosomal hearing loss Cx26 Connexin 26 DOH Department of Health WHO World Health Organisation BP Base Pair(s) EDHI early detection of hearing impairment XVI LIST OF TABLES Table number Page no. CHAPTER 1 Table 1.1: Population of Limpopo Province by home language and district 20 Table 1.2: Limpopo Province population in five ? year age groups according to race 20 Table 1.3: Disabled population by district in the Limpopo Province 21 Table 1.4: Public sector human resource data, Limpopo Province 27 Table 1.5: Home area of students at Tshilidzini School, August 1997 36 CHAPTER 2 Table 2.1: Features of some epidemiological methods in use 49 Table 2.2: The domains and measures of auditory dysfunction (adapted from Davis et al 1983) 52 Table 2.3: Prevalence of hearing loss in childhood (after Davidson et al 1989) 56 Table 2.4: Depicting time of appearance of ear features 64 CHAPTER 3 Table 3.1: Gene expression in the human ear 93-94 Table 3.2: Classes and genes identified for Waardenburg syndrome 101 Table 3.3: Phenotypic penetrance of selected Waardenburg syndrome traits 104 Table 3.4: Penetrance of pigmentary abnormalities WS patients with and without hearing lossin relation to syndrome type 104 Table 3.5: The degree of hearing loss and the frequency of pigmentary abnormalities in relation to syndrome type 105 Table 3.6a: Audiological manifestation of the autosomal dominant nonsyndromic hearing impairment genes 109 Table 3.6b: Audiological manifestation of the autosomal recessive nonsyndromic hearing impairment genes 110 XVII Table 3.6c: Audiological manifestation of the X-linked nonsyndromic hearing impairment genes 111 Table 3.6d: Audiological manifestation of the mitochondrial nonsyndromic hearing impairment genes 111 Table 3.7: Evaluation strategy of hearing loss 135 CHAPTER 5 Table 5.1: Demographic information of subjects, Phase 1 183 Table 5.2: Age of detection, by parents, of hearing loss among subjects, Phase 1 188 Table 5.3: Demographic information of subjects, Phase 2 189 Table 5.4: Age of detection, by parents, of hearing loss among subjects, Phase 1 189 Table 5.5: Geographical distribution of hearing loss according to district, Limpopo Province, both schools Phase 1 191 Table 5.6: Geographical distribution of hearing loss according to district, Limpopo Province, both schools Phase 2 192 Table 5.7: Comparison of municipal wards considering high risk areas for hearing loss 193 Table 5.8: Municipalities with highest geographical distribution of hearing loss, both schools Phase I 193 Table 5.9: Municipalities showing the highest geographical distribution of hearing loss according to school, Phase I 194 Table 5.10: The geographical distribution of hearing loss according to Municipalities municipalities normalized to African population, both schools: Phase 2 194 Table 5.11: Tympanometric results 199 Table 5.12: Cross tabulation of Tympanometric results between ears 199 Table 5.13: Abnormalities for ear with abnormal tympanogram 200 Table 5.14: Severity of hearing impairment, best ear average 0.5-4kHz, Tshilidzini, Phase 2 201 XVIII Table 5.15: Audiogram configuration among subjects, Tshilidzini, Phase 2 201 Table 5.16: Asymmetry of hearing impairment among subjects, Tshilidzini, Phase 2 201 Table 5.17: Severity of hearing impairment , best ear average 0.5-4kHz, Bosele, Phase 2 201 Table 5.18: Audiogram configuration among subjects, Bosele, Phase 2 201 Table 5.19: Asymmetry of hearing impairment among subjects, Bosele, Phase 2 202 Table 5.20: Severity of hearing impairment, best ear average 0.5-4kHz, both schools, Phase 2 202 Table 5.21: Audiogram configuration among subjects, both schools, Phase 2 202 Table 5.22: Asymmetry of Hearing Impairment among subjects, both schools, Phase 2 202 Table 5.23: Family History of hearing loss among subjects, Phase 2 203 Table 5.24: Distribution of family history of hearing loss according to municipality, Limpopo Province, both schools, normalized to African Population, Phase2 203 Table 5.25: Cross tabulation of consanguinity of parents by municipality, Bosele School, Phase 2 207 Table 5.26: Cross tabulation of consanguinity of parents by municipality, Tshilidzini School, Phase 2 210 Table 5.27: Cross tabulation of consanguinity of parents by municipality, Bosele School, Phase 2 212 Table 5.28: Cross tabulation of consanguinity of parents by municipality, Tshilidzini School, Phase 2 212 Table 5.29: History of consanguinity of parents by school, Phase 2 213 Table 5.30: Cross tabulation of consanguinity of parents by family history of hearing loss, Phase 2 213 Table 5.31: Cross tabulation of language group by consanguinity of parent 213 Table 5.32: Results of urinalysis among participants 213 Table 5.33: Cross tabulation of consanguinity of parents by relative with hearing loss 214 XIX Table 5.34: Cross tabulation of consanguinity of parents by relative with hearing loss 214 Table 5.35: History of maternal problems during pregnancy and labour 214 Table 5.36: History of other medical conditions among participants 214 Table 5.37: GJB2 variations observed in a deaf population from the Limpopo Province of South Africa. 215 Table 5.38: Cross tabulation of GJB2 variations and language group in a South African control population (n=74). 219 Table 5.39: GJB2 (Cx26) variations: genotype versus allele frequency as observed in a South African population. 219 Table 5.40: GJB2 (Cx26) variations tested for Hardy-Weinberg equilibrium: Position g.3318-34 220 Table 5.41: GJB2 (Cx26) variations tested for Hardy-Weinberg equilibrium: Position g.3318-15 221 Table 5.42: Cross tabulation of consanguinity of parents by base variation: Position g.3318-34 222 Table 5.43: Cross tabulation of family history of hearing loss by base variation: Position g.3318-34 222 Table 5.44: Cross tabulation of ethnic group by base variation: Position g.3318-34 222 Table 5.45 : Levels of significance of results following cross tabulation of participants? age at detection with other variables 227 Table 5.46 : Levels of significance of results following cross tabulation of risk factors for hearing loss with other variables. 228 Table 5.47 : Levels of significance of results following cross tabulation of consanguinity of parents with other variables. 228 Table 5.48: Levels of significance of results following cross tabulation of family history of hearing loss with other variables. 229 Table 5.49: Levels of significance of results following cross tabulation of degree of first affected relative with other variables. 229 XX Table 5.50: Levels of significance of results following cross tabulation of degree of second affected relative with other variables. 230 Table 5.51 : Levels of significance of results following cross tabulation of language group with other variables. 230 Table 5.52: Levels of significance of results following cross tabulation of GJB2 variation C>T at position -34 with other variables. 231 Table 5.53 : Levels of significance of results following cross tabulation of GJB2 variation C>T at position -15 with other variables. 231 Table 5.54 : Levels of significance of results following cross tabulation of participants? home address with other variables. 232 Table 5.55 Results of binary logistic regression analysis 232 Table 5.56 Logistic model for consanguinity of parents 234 Table 5.57 Hosmer-Lemeshow goodness-of-fit test 235 XXI LIST OF FIGURES Figure number Page no. CHAPTER 1 Fig. 1.1: Aetiological classification of genetic hearing loss 7 Fig. 1.2: Location map of the study areas within the map of South Africa 16 Fig. 1.3: Map of the Limpopo Province showing the districts and municipal boundaries 17 Fig. 1.4: The Baobab tree, the Icon of Limpopo Province 18 Fig. 1.5: The Land of the legends ? Lake Fundudzi, Venda 18 Fig. 1.6: The arid landscape of No-Body and Moria regions, the headquarters of the ZCC church whose star logo seen in the background is etched in the mountainside. 18 Fig. 1.7: A group following a climbing trail in the mountains in Agatha 18 Fig. 1.8: The Tzaneen Dam with the Drakensberg mountain range in the background 19 Fig. 1.9: Polishing the homestead floor with fresh cow dung in a Giyani village 19 Fig. 1.10: Sharing a meal, the typical homestead arrangement seen in the background 19 Fig. 1.11: A Shangaan (Tsonga) girl greeting visitors to the homestead in a Giyani village 19 Fig. 1.12: One of the reception areas inside the Pietersburg Provincial Hospital 25 Fig. 1.13: Ear, nose and throat outpatient clinic at the Pietersburg Provincial Hospital 26 Fig. 1.14: A Venda woman in full traditional attire 35 Fig. 1.15: Shangaan women dance group 35 Fig. 1.16: Pedi women?s dance group from Mashashane in traditional wear 35 CHAPTER 2 Fig. 2.1: Pedigree showing autosomal dominant inheritance 41 Fig. 2.2: Pedigree showing autosomal recessive inheritance 41 Fig. 2.3: Pedigree showing dominant X-linked inheritance 42 Fig. 2.4: Pedigree showing recessive X-linked inheritance 42 Fig. 2.5: Pedigree showing mitochondrial inheritance 43 XXII Fig. 2.6: The relationship between genetic and environmental factors in causation of hearing loss as a function of age (adapted from Davis et al 1983a) 51 Fig. 2.7: Epidemiological model of hearing function 54 Fig. 2.8: Schematic drawing of inner ear development in mammals (after Varela-Nieto et al (2004). 66 Fig. 2.9: Diagram showing the structure and gene expression of the human ear 67 CHAPTER 3 Fig. 3.1a: The progress of deafness gene discovery from 1994-2001 85 Fig. 3.1b: Total number of deafness gene identified annually 1986-2007 86 Fig. 3.2: Aetiological surveys among 3,064 children in Southern Africa 106 Fig. 3.3 Realistic relationship of need, demand and supply in the health services 116 Fig. 3.4: The ideal relationship of need, demand and supply in the ideal health service 117 Fig. 3.5: Medical assessment of the hearing impaired child 128 Fig. 3.6: The areas addressed in a hearing impaired child?s management protocol 149 Fig. 3.7: Components of a paediatric audiological medicine service 151 CHAPTER 4 Fig. 4.1: Parents and Teachers at Prayer in the hall ? Bosele School 172 Fig. 4.2: Translator (Nurse) explaining the questionnaire to the parents ? Bosele School 172 Fig. 4.3: Parents waiting for assistance in completing the questionnaire- Bosele School 172 Fig. 4.4: Subjects waiting their turn at Bosele School 172 Fig. 4.5: Doctor completing a subject?s medical examination form - Bosele School 173 Fig. 4.6: Doctor examines subject?s ear at Bosele School 173 Fig. 4.7: TEOAE (transient otoacoustic emission) station - Bosele School 173 Fig. 4.8: Sound-proofed testing room, Tshilidzini School 173 CHAPTER 5 Fig. 5.1a: Box and Whisker plot showing the ages (in years) of the participants, Phase 1, both schools 184 XXIII Fig. 5.1b: Box and Whisker plot showing the ages (in years) of the participants, Phase 1, Tshilidzini School 184 Fig. 5.1c: Box and Whisker plot showing the ages (in years) of the participants, Phase 1, Bosele School 185 Fig. 5.2a: Box and Whisker plot showing the distance of the participants homes from school in kms, Phase 1, both schools 186 Fig. 5.2b: Box and Whisker plot showing the distance of the participants homes from school in kms, Phase 1, Bosele School 186 Fig. 5.2c: Box and Whisker plot showing the distance of the participants homes from school in kms, Phase 1, Tshilidzini School 187 Fig. 5.3: Box and Whisker plot showing the ages (in years) of the participants, Phase 2, both schools 190 Fig. 5.4: Geographical distribution of hearing loss according to municipality, Limpopo Province, both schools Phase 1 195 Fig. 5.5: Geographical distribution of hearing loss according to municipality, Limpopo Province, both schools Phase 2 195 Fig. 5.6: Spatial distribution of Hearing Loss according to municipality, Limpopo Province, Phase 1 196 Fig. 5.7 Spatial distribution of Hearing Loss according to municipality, Limpopo Province, normalized to African Population, Phase 2 196 Fig. 5.8: Spatial distribution of hearing loss in the Limpopo Province according to language group, Phase 1 197 Fig. 5.9: Spatial distribution of hearing loss in the Limpopo Province according to language group, Phase 1 198 Fig. 5.10: A Spatial distribution of subjects according to family history of Hearing loss per local municipality, Limpopo province, Phase 1 206 Fig. 5.11: Spatial distribution of subjects without a family history of hearing loss per local municipality, Limpopo Province, Phase 2 207 XXIV Fig. 5.12: Spatial distribution of subjects with a family history of hearing loss per local municipality, Limpopo Province, Phase 2 208 Fig. 5.13: Spatial distribution of subjects with a family history of hearing loss per local municipality, Limpopo Province, Normalized to African population, Phase 2 208 Fig. 5.14: Spatial distribution of subjects with a history of consanguinity among parents, per local municipality, Limpopo Province, Phase 2 209 Fig. 5.15: Spatial distribution of subjects without a history of consanguinity among parents, per local municipality, Limpopo Province, Phase 2 209 Fig. 5.16: Spatial distribution of subjects with unknown history of consanguinity per local municipality, Limpopo Province, Phase 2 210 Fig. 5.17: Gel electrophoresis showing size of PCR fragment (GJB2) 216 Fig. 5.18: Gel electrophoresis (GJB2) following Fermentas SsiI enzyme digest (cutting at position g.3318-15) 216 Fig. 5.19: Gel electrophoresis (GJB2) following Bsml enzyme digest (cutting at position g.3318-34) 216 Fig. 5.20:Electropherograms showing GJB2 variation T A at position -6 C T variation at position -15 217 Fig. 5.21: Electropherogram showing GJB2 variant at GJB2 position -34 217 Figure 5.22a: Clinical features Waardenburg syndrome Type I. 226 Figure 5.22b: Patchy depigmentation of the skin in participant with WS type I 226 Figure 5.23:The ROC (receiver operating characteristic) curve 236 Figure 5.24: Plot of sensitivity/Specificity versus probability cut-off point 237