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Title of Thesis

Khushnooda Ramzan
Institute/University/Department Details
University of the Punjab
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
autosomal recessive deafness, auditory system, syndromic hearing loss, nonsyndromic hearing loss

Deafness, the inability to hear, is the most common sensory deficit in human populations with both genetic and environmental etiologies (McKusick, 1992). It is estimated that it affects 1 in 1000 child births which approximately 60% cases are attributed to genetic factors (Marazita et al.,1 1993). It can be divided into two groups, syndromic and nonsyndromic on the basis of any allied phenotype other than deafness. Nonsyndromic hearing loss (N SHL) transmitted as a recessive trait are the most frequent cause of hereditary deafness and often exhibit the most severe hearing phenotype (Cohen, et al., 1995). As expected from the structural and functional complexity of the inner ear, sensorineural deafness exhibits a high 1egree of genetic heterogeneity. It has been estimated that at least 1 % of 30,000 human protein-coding genes are involved in the hearing process (Friedman and Griffith, 2003). Duri1g the last decade, many deafness loci and the underlying genes have been identified at the speed of sound. Presently, 60 recessive deafness (DFNB) loci and 21 of the corresponding genes have been identified (Hereditary Hearing Loss Homepage). Molecular and genetic information currently available regarding NSHL clearly indicate that a large repertoire of human genes associated with deafness remains to be mapped and identified, and these genes can be used to elucidate the molecular basis of hearing impairment. Consanguineous families have contributed significantly to the identification of mutated genes associate1 with hearing loss (Friedman and Griffith, 2003). Recessively inherited deafness in the Pakistani population is higher than world average due to high consanguinity (Hussain and Bittles, 1998). This population thus provides a valuable genetic resource for the identifica1ion of novel deafness loci/genes.

Seventy-five consanguineous families with three or more affected individuals and showing recessive mode of inheritance were ascertained through the schools and centers for special education from different cities of Pakistan. Clinical histories were obtained for all of the individuals of families. Physical evaluations were performed to rule out any obvious extra auditory Phenotype Pure tone audiometry tests for air and bone conduction were performed at frequencies 250 to 8,000 Hz on affected and unaffected members of these families. Vestibular function was evaluated by testing tandem gait ability and by using the Romberg test. Ocular funduscopy and electroretinography (ERG) was performed to detect the presence of retinopathy if required.

Genomic DNA was extracted from the blood samples of the affected individuals, normal siblings, and other related family members of the selected twenty five enrolled families. Linkage analyses were carried out for the known DFNB loci, by using at least three microsatellite markers for each of the reported deafness loci (Hereditary Hearing Loss Homepage). As a result, six families are found linked to DFNB1. DFNB3. DFNB4/PDS, DFNB8/10, and DFNB9 Mutational analyses were done to find underlying mutations in GJB2, SLA26A4, and TMPRSS3 genes for the families linked to DFNB1. DFNB4, and DFNB8, respectively. The search for a new locus was carried out by the genome wide scan on the seven families which remained unlinked to the known DFNB loci.

Genome wide soon was performed with 388 fluorescent dye-labeled microsatellite markers from ABI PRISM® Linkage Mapping Set, Version 2.5 (Applied Biosystems) spaced at an average of 10cM across the human genome. The microsatellite markers were amplified by the polymerase chain reaction (PCR) on a Gene Amp PCR system 9700 (Perkin Elmer®) and were analyzed on a ABI PRISM® 3100 Genetic Analyzer. The alleles were assigned by using Genescan and Genotyper Software (Applied Biosystems). To have statistical significance of linkage lod scores were calculated by using MLINK of the FASTLINK computer package (Schaffer, 1996).

Genome wide linkage analyses in two large consanguineous families PKDF041 and PKDF 141 segregating recessively inherited, profound congenital deafness led to the mapping of a new lotus for nonsyndromic deafness to a linkage interval of 11 cM on chromosome 5q12.3-q14.1 as a part of this study (Ramzan et al., 2005). DFNB49 was assigned to the new locus reported herein by the Human Genome Organization (HUGO) committee. The mapping of DFNB49 further confirms the heterogeneity underlying nonsyndromic deafness.

To catalog more comprehensive evidences of DFNB49, 700 consanguineous Pakistani families (kindly provided from CEMB DNA bank) segregating congenital, profound recessive deafness were screened. An additional seven out of 700 families were found linked to DFN 49, there by establishing the relative contribution of DFNB49 in the deaf Pakistani population to be ~1.28%, a significant amount considering the extensive genetic heterogeneity of deafness in this population. High resolution mapping of the locus was done by obtaining additional microsatellite markers on chromosome 5q12.3-q14.1 and typing them across all the FNB49 linked families. Fine mapping refined the linkage region of 11 cM to 2.5 cM al d suggested that the disease gene for DFNB49 lies between centromeric boundary at D5S629 (75.89 cM) and telomeric boundary at GATA141B10 (75.89 cM). The refined region spans 2424169 base pairs of DNA sequence. Positional cloning efforts thus helped to reduce the linkage interval to ease the search for the underlying causative DF 49 gene.

Candidate deafness genes in this region include SLC30A5, OCLN, and GTF2H2, encoding solute carrier family 30 (zinc transporter) member 5, occludin, and RNA polymerase II transcription initiation factor, respectively. Sequence analysis of the coding exons of SLC30A5 and OCLN in DNA samples of affected individuals of DFNB49 families revealed no mutation. Localization of locus DFNB49 is a first step towards the identification of a novel gene that is necessary for the development and /or maintenance of normal hearing and has provided further i sight in the molecular basis of deafness.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
285.44 KB
2 1 Introduction 1
1271.49 KB
3 2 Review Of Literature 5
515.85 KB
  2.1 Auditory System 6
  2.2 Molecular Basis And Genetics Of Deafness 25
  2.3 €œLinkage Analysis€ A Valuable Tool For Mapping Disease Genes 65
4 3 Materials & Methods 69
1501.52 KB
  3.1 Field Work 70
  3.2 Lab Work 80
5 4 Results And Discussion 99
556.78 KB
  4.1 Linkage Analysis For Known Autosomal Recessive Deafness ( Dfnb ) Loci 100
  4.2 Mapping & Refinement Of A New Locus For Nonsyndromic Deafness Dfnb49 114
  4.3 Discussion 153
  4.4 References 161