UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ A Family-based Mapping Study of Autosomal Dominant Nonsyndromic Sensorineural Hearing Loss A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati In fulfillment of the requirements for the degree of Master of Science in Medical Genetics Genetic Counseling Program in the Department of Analytical and Diagnostic Sciences of the College of Allied Health Sciences May 18th, 2007 By: Monica A. Giovanni Committee Chair: John H. Greinwald, Jr., MD Abstract Autosomal dominant nonsyndromic hearing loss (ADNSHL) is characterized by postlingual, progressive hearing impairment. This study sought to identify the gene responsible for hereditary nonsyndromic sensorineural hearing loss in a family with multiple generations affected by hearing impairment presenting in the second decade of life. The Affymetrix GeneChip® was used to identify three linkage intervals on chromosomes 4, 10, and 16. The observed hearing loss in this family is not likely due to previously identified deafness-causing genes as no such genes have been reported in the identified intervals. Since preliminary candidate gene sequencing within the regions did not identify any pathogenic mutations, haplotype mapping was employed to further refine the intervals. The intervals on chromosomes 4 and 16 were excluded and the interval on chromosome 10 was narrowed to a 0.4Mb region at 10q22-q23. Future work will employ candidate gene analysis to identify the gene responsible for this family’s hearing impairment. Keywords: family-based linkage analysis, haplotype mapping, PCR based sequence analysis, autosomal dominant nonsyndromic hearing loss (ADNSHL) iii iv Acknowledgements I would like to thank the University of Cincinnati and Cincinnati Children’s hospital Medical Center Genetic Counseling program for supporting this research. I would also like to extend my sincerest thanks to Dr. John Greinwald for serving as my research mentor and advisor; his expertise, guidance, and instruction was an integral part of my learning experience and the completion of this project. Finally, I would like to thank my thesis committee members Dr. Bill Nichols and Judy Johnson for their time and assistance throughout this process. v Table of Contents: List of Tables and Figures…………………………………………………………..……vi Introduction………………………………………………………………………………..1 Methods……………………………………………………………………………………3 Results…………………………………………………………………….……………….6 Discussion………………………………………………………………………………..10 Bibliography……………………………………………………………………………..19 Figures……………………………………………………………………………………24 Tables…...………………………………………………………………………………..34 Appendices……………………………………………………………………………….35 vi Tables, Figures, and Appendices Figure 1. Nonconsanguineous family with apparent autosomal dominant hearing loss across four generations Figure 2. Affymetrix GeneChip® results show linkage interval on Chromosome 4 Figure 3. Ideogram of Chromosome 4 with linkage interval Figure 4. Affymetrix GeneChip® results show linkage interval on Chromosome 10 Figure 5. Ideogram of Chromosome 10 with linkage interval Figure 6. Affymetrix GeneChip® results show linkage interval on Chromosome 16 Figure 7. Ideogram of Chromosome 16 with linkage interval Figure 8. A smaller branch of the family, branch 2, served as the index branch due to the presence of multiple affected and unaffected individuals and the appearance of autosomal dominant transmission of hearing loss Figure 9. Haplotype analysis using chromosome 4 markers excluded this interval due to marker segregation inconsistent with affectation in branch 2 of the family Figure 10. Haplotype analysis using chromosome 16 markers showed plausible marker segregation creating a very small interval within which the disease-causing gene could be positioned. This segregation pattern can be observed across affected individuals in branch 2 of the family (A), however the proposed genotype did not segregate with hearing loss when expanded to include branch 4 of the family (B) Figure 11. Haplotype analysis using chromosome 10 markers showed plausible marker segregation across affected and unaffected individuals in branch 2 of the family Table 1. Subject Inclusion Criteria vii Table 2. Subject Exclusion Criteria Appendix A. Polymorphic markers used to refine the candidate intervals on chromosomes 4, 10, and 16 Appendix B. Candidate genes in the interval of interest on chromosome 10 viii Introduction Hearing impairment is the most commonly diagnosed sensory deficit. It is estimated that 1 in 1000 children is born deaf or will become profoundly deaf before speech is acquired. Another 1 in 1000 children will become deaf or develop a significant hearing impairment before adulthood (Morton 1991). Roughly 50% of all cases of hearing loss can be attributed to genetic causes (Marazita et al., 1993). Seventy percent of cases of hearing loss with a genetic etiology appear in isolation and are considered nonsyndromic (Sundstrom et al., 1999). The most common mode of inheritance for all hereditary hearing impairment is autosomal recessive, accounting for an estimated 80% of all cases. Autosomal dominant inheritance accounts for roughly 20% of cases and the remaining cases (less than 1%) show X-linked or mitochondrial inheritance (Petersen & Willems, 2006). Research into nonsyndromic hearing impairment has established that there are at least 50-100 nonsyndromic deafness-causing genes (Zbar et al., 1998). Autosomal dominant nonsyndromic hearing loss (ADNSHL) exhibits a wide range in age of onset with some individuals presenting with congenital or early onset hearing impairment, while other individuals develop hearing impairment in their forties and fifties which can be misdiagnosed as presbycusis, or age-related hearing loss. In general, ADNSHL is characterized by hearing impairment beginning in the second or third decade of life after the development of normal speech, or postlingually, and is progressive in nature. ADNSHL genes have been localized using extended family pedigrees in which a single deafness-causing gene segregates. ADNSHL genetic loci are termed DFNA (DFN = deafness, A = dominant), followed by a number. The first gene for ADNSHL, DFNA1, was localized in 1992. Since that time, close to 50 loci have been 1 mapped and 20 genes identified for ADNSHL (Yan et al., 2006; Hereditary Hearing Loss Homepage: http://webh01.ua.ac.be/hhh/). These genes have been found to encode a wide variety of proteins, including transcription factors, components of the extracellular matrix and cytoskeleton, ion channels, and proteins of unknown functions (Sundstrom et al., 1999). Deafness research has focused on genetic mapping studies in an effort to elucidate the genes responsible for hearing loss. The mapping of recessive genes is often plagued with challenges including finding families large enough for linkage analyses and ascertaining families with some degree of consanguinity (Sundstrom et al., 1999, Strachan & Read, 2004). Mapping autosomal dominant genes can be less complicated as individuals affected by autosomal dominant conditions are usually seen with a higher frequency in families as compared to recessive conditions, making it easier to ascertain multiple affected individuals in a bloodline (Sundstrom et al., 1999). By definition, a dominant genetic mutation should manifest in an individual heterozygous at that genetic locus (Strachan & Read, 2004). Dominant family studies are often complicated by nonpenetrance, in which some individuals heterozygous for a gene mutation lack symptoms of the disease. Thus, it is possible for an autosomal dominant condition to appear to skip generations such that an individual can have both an affected parent and an affected child without showing any symptoms of the condition themselves (Strachan & Read, 2004). In ascertaining large families for mapping studies, some degree of nonpenetrance can be expected in a multigenerational pedigree. This study sought to identify the genetic cause of nonsyndromic sensorineural hearing loss that exhibits apparent autosomal dominant inheritance in a large 2 nonconsanguineous family. In this investigation, linkage studies were performed using DNA extracted from blood samples of affected and unaffected members of a family with ADNSHL to establish candidate intervals within which the disease-causing gene was likely located. After we identified three intervals in which linkage appeared likely, we selected three functional candidate genes within the intervals. PCR based sequencing did not identify any disease causing mutations in these three candidate genes. Haplotype mapping techniques were then employed with the aim of refining the intervals of interest for more targeted candidate gene analysis to identify the deafness-causing gene in this family. Methods Study Subjects This study enrolled members of a large, nonconsanguineous family of northern European descent with no recognized Jewish heritage. Multiple generations in this family experienced progressive hearing impairment with onset in the second decade of life (see Figure 1). The subjects were enrolled at Cincinnati Children’s Hospital Medical Center based on their family