The identification of deafness genes is an essential step in understanding the molecular mechanism of hearing and hearing loss. Although significant advances have been made, there is no doubt that many more genes await discovery. Identifying these genes and characterizing the proteins they encode will increase our knowledge of the molecular processes involved in the auditory system and will improve our understanding of how such processes can become altered and lead to hearing impairment. A large number of dominant, recessive and sex-linked loci for non-syndromic deafness have been mapped in the human population. Non-syndromic deafness is paradigm of genetic heterogeneity. More than 100 loci have been mapped, and 40 of the nuclear genes responsible for non-syndromic deafness have been identified. Autosomal recessive non-syndromic hearing loss is clinically, the most important group because it accounts for about 80% of non-syndromic deafness. This extreme genetic heterogeneity suggests that there are many different processes that can malfunction within inner ear to cause hearing loss.
Genetic studies of hearing loss have been successful in isolated populations and consanguineous families. Present study is based on ten families (A-J) from remote areas of Pakistan, segregating non-syndromic recessive hearing loss. The patients hearing loss was prelingual, varied from severe to profound, and was not caused by inflammatory middle ear disease or environmental factors. Linkage in the families was initially searched by using polymorphic micro satellite markers located within the genetic intervals of the known hearing loss loci. Linkage to these known loci was conclusively excluded in three families (A, B, C) by the detection of heterozygotes for the microsatellite markers in the affected individuals. Seven other families (D, E, F, G, H, I, J) showed linkage to establish known deafness loci.
After excluding the disorder in the three families (A, B, C) from linkage to the known deafness chromosomal regions, a genome-wide screening was undertaken using a panel of 390 fluorescently labeled simple tandem repeat (STRP) markers located on autosomes and X and Y chromosomes. In family A, genome-wide search with micro satellite markers detected the linkage between a novel hearing loss locus DFNB68 and markers on chromosome 19p 13.3-13.11. A maximum two-point LOD score of 3.28 and multipoint LOD score of 4.58 was obtained at marker D19S586. Saturation of the region with additional markers and examination of haplotypes defined a critical region of about 23.66 cM flanked by markers D 19S 1 034 and D19S199.
In family B genome scan revealed a potential autosomal recessive deafness locus on chromosome 12q23 .1-q23.3. Two-point and multipoint analysis generated LOD scores of 1.43 and 1.82, respectively. Expected LOD score that could be obtained in this family was 2.05. Haplotype analysis placed the region in a 10.55 cM genetic interval between the markers D12S1063 and D12S353. In family C, the linkage data obtained from genome scan and further saturation of the two chromosomal regions (ATA78D02 and D9S1120) with additional markers failed to define a region harboring a causative gene for deafness.
In family D linkage was established with DFNB6 locus harboring TMIE gene. All the 4 exons of TMIE gene were PCR amplified and sequenced directly in an ABI Prism 310 automated DNA sequencer. A novel sequence variant (c.92A>G) was identified in exon 1 of the TMIE gene, resulting in the substitution of glutamic acid to glycine (p.E31 G).
In family E, sequencing of the coding exon of connexin 26 gene, which is responsible for hearing loss at DFNB 1 locus, revealed a compound heterozygote mutation in the affected individual. These individuals carried a paternally derived G-to-A transition at nucleotide position 380 (c.380G>A), resulting in arginine to histidine (p.R127H) and a maternally derived G-to-A transition at nucleotide position 457 (c.457G>A), resulting in a missense mutation of valine to isoleucine (p.V153I).
Family F showed linkage to DFNB3 locus on chromosome 17p 11.2 harboring MYO15A gene. Nine exons and splice junction sites of MYO15A gene were PCR amplified and sequenced directly. Mutation screening of the nine exons of MYO15A gene failed to detect any known or novel pathogenic allelic variant in this family. The four other families G, H, I and J showed linkage to DFNB2, DFNB17, DFNB9 and DFNB510ci on chromosome 11q13.5, 7q31, 2p22-p23, and 14q12, respectively.