Hearing loss is a common sensory disorder that typically illustrates genetic heterogeneity in human populations. The incidence of congenital hearing loss is estimated at 1 in 1,000 births of which approximately 60% of cases are attributed to genetic factors. Genetic hearing impairment can be classified as either syndromic or non-syndromic. Several hundred syndromes, for which hearing impairment is one of the clinical features, have been described. These account for 30% of hearing impairment cases with a genetic etiology. Among non-syndromic hereditary hearing impairments (NSHI), autosomal recessive inheritance predominates and accounts for approximately 75-80% of the cases, while autosomal dominant inheritance is observed in some 15% of cases. Non-syndromic hearing impairment is the most heterogeneous trait known and thus far over 90 loci have been mapped and 37 genes identified.
In the present study, ten families (A-J) from Punjabi and Sindhi speaking population of Pakistan, segregating non-syndromic recessive hearing impairment have been described. The hearing loss in the patients was prelingual, varied from severe to profound, and was not caused by inflammatory middle ear disease or environmental factors.
Linkage in these families was initially searched by using microsatellite markers corresponding to candidate genes involved in related autosomal recessive non-syndromic deafness phenotypes. Linkage was detected in seven families, i.e., D-J. In families D-E linkage was established to DFNBl locus on chromosome 13q12. In family F it was detected on chromosome 3p14-p21 at DFNB6 locus. In family G, linkage was established at DFNB7/11 locus on chromosome 9q13-q21. In family H, the linkage was detected on chromosome 9q32-q34 at DFNB31 locus. In family I, linkage was established at DFNB37 locus on chromosome 6q13. In family J, the linkage was detected on chromosome 17pl1.2 at DFNB3 locus. In other three families (A, B, C) linkage to all the known loci was conclusively excluded, thus indicating the involvement of novel loci, responsible for deafness in these families.
After excluding the disorder from linkage to known hearing loss loci in families A, B and C, genome-wide scan was performed by using the Mappairs sets of Micro satellite markers. In family A, screening of the human genome with markers spaced at 10 cM intervals led to the identification of a new autosomal recessive non-syndromic hearing loss locus, DFNB45, on chromosome lq42.3-q44. Two-point linkage analysis generated LOD score of 2.61 with marker DlS547. Multipoint linkage analysis resulted in a maximum LOD score of 4.23 with marker DlS404 in this region. Examination of haplotypes defined a critical region of 15.28 cM and 20.26 cM according to decode and Marshfield genetic maps, respectively and is flanked by markers DlS1594 and D1S2836. The genetic interval for DFNB45 does not overlap with the regions of the previously mapped deafness loci on chromosome 1.
In family B, significant evidence of linkage to chromosomal region 5qI2.1-qI4.3 was found with two point LOD score of 3.76 and 4.02 with markers D5S629 and D5S2041, respectively. Saturation of the region with additional markers and analysis of haplotypes mapped the gene to an interval of 16.62 cM bounded by markers D5S2107 and D5S1397. This novel locus was designated as DFNB49. However, while the manuscript describing this locus was being written, the same was published by Ramzan et al. (2005).
In family C, initial genome wide scan revealed two regions D2S434 and D13S779 generating LOD score of 2.66 and 2.02, respectively. Saturation of these regions with additional markers, however, failed to generate a significant LOD score with any of the markers.
In two families where linkage was established to DFNB 1 locus, sequencing of the single coding exon of GJB2 gene led to the identification of two nonsense mutations. In family D, a G to A transition at nucleotide position 231, resulted in a premature termination codon (W77X). In family E sequence analysis of the GJB2 gene revealed a G to A transition at nucleotide position 71 (G71A), leading to premature termination codon (W24X).
In family F, sequence analysis of exon 3 of the TMIE gene in affected individuals revealed a C to T transition at nucleotide position 241 (C241 T). This missense mutation resulted in substitution of arginine to cystine at amino acid position 81 (R81C).
In family G, sequence analysis of exon 15 of the TMCI gene from affected individuals revealed a missense mutation involving A to G transition at nucleotide position 1114 (A 1114G). This missense mutation resulted in substitution of valine to methionine (V372M) at amino acid position 372.
In family H, sequence analysis of 12 exons and splice junction sites of the WHRN gene in affected individuals failed to detect a disease causing mutation in the family. However, sequencing data revealed five non-synonymous allelic variants.
In family I, linkage was established with DFNB37 locus harboring MYO6 gene on chromosome 6. Five exons and splice junction sites of MYO6 gene were sequenced. However, mutation screening revealed no disease causing allelic variant in the family.
In family J, linkage was established with DFNB3 locus harboring MYO15 gene on chromosome 17. Nine exons and splice junction sites of MYO 15 gene were sequenced. However, mutation screening failed to detect a disease causing mutation in the family 1.