Hearing loss that disturbs normal communication is a common sensory disorder worldwide. Half of the cases with congenital impaired hearing are hereditary, which may occur as part of a multi system disease (syndromic hearing impairment) or as a disorder restricted to the ear and vestibular system (non-syndromic hearing impairment). Among the many disorders classified as syndromic hearing loss, the pathology varies widely, but non-syndromic hearing loss is almost exclusively caused by cochlear defect, affected patients suffer from sensorineural hearing loss. Non-syndromic deafness displays extensive genetic heterogeneity. More than 100 loci have been mapped, and 40 of the nuclear genes responsible for non-syndromic deafness have been identified. To date 68 loci for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been mapped and 23 genes have been isolated. A large number of genes with different functions are involved in the etiology of hearing impairment because of the complexity of the inner ear, and various processes and pathways that can lead to the HI phenotype. Identification of genes causing deafness is a crucial first step in understanding the normal function of these genes in the auditory system.
In the present study, thirteen families (A-M) from different regions of Pakistan, segregating non-syndromic recessive hearing loss have been described.
For deafness segregating in family A, evidence of linkage was detected on chromosome 4q, following a genome wide scan. Two-point analysis generated a maximum lod score of 2.75 for marker 04S1645 at recombination fraction 0. Multipoint linkage analysis for the family derived a maximum LOD score of 3.4 with range of markers from D4S2638 to D4S 1600. Examination of haplotype defined a critical region of about 8.2 cM flanked by markers D4S2978 and D4S2367 at cytogenetic band 4q 12-q13.2. This novel hearing impairment region corresponds to a physical map distance of 11.4 Mb and designated as DFNB55. Several of the known genes in DFNB55 region are expressed in the inner ear. All exons and splice junction sites of EPHA5 and REST, located in the critical region of DFNB55, were sequenced in one hearing and two hearing-impaired members of family A and were found to be negative for functional sequence variants.
Family B showed linkage with DFNB8/10 locus on chromosome 21q22.3. The maximum two-point lod score> 1.0 was obtained with several markers. Multipoint linkage analysis resulted in lod score >3 with markers D2lS1246 and D21S266. The region identified in the family B harbors TMPRSS3 gene. All 13 exons of TPMRSS3 gene were sequenced in the DNA samples of the two affected individuals of family B but no functional sequence variant was identified. A non-synonymous sequence variant 157G-A was however observed in exon 3 of TMPRSS3 gene.
Family C linked to DFNB7/11 locus at chromosome 9q13-21. Maximum two point lod score of 2.51 was obtained with marker D9S301 at recombination fraction zero. Multipoint linkage analysis for the family derived a maximum lod score of 3 with two markers D9S301 and D9S976. TMCl, hearing impairment gene at DFNB7/11 locus, was sequenced in two affected individuals of family C but no disease causing mutation was identified.
In family D linkage was observed with marker D7S 1824 linked to DFNB 13 locus, located close to telomere at 149.9 cM on chromosome 7q34. This region was saturated with the additional markers from proximal and distal region of D7S 1824. Multipoint analysis supported linkage to this region with maximum lod score exceeding 3.00 in the interval D7S2450 -D7S2513.
In two of the thirteen families presented in the current study, linkage was established with the DFNB 1 locus harboring GJB2 gene. In family E sequencing of the coding exon of GJB2 gene revealed a G to A transition at nucleotide position 71, resulting in the conversion of tryptophan to premature termination codon (W24X). In family F sequence analysis of the coding exon of the GJB2 gene revealed a deletion of T at nucleotide position 167 (167deIT).
In family G sequencing of all the 24 coding exons and splice junctions of TMCl gene at DFNB7/11 locus, revealed a homozygous A to G transition in exon 13 of all the affected individuals, which was present in heterozygous state in obligate carriers within the family. The A to G transition occurred at nucleotide position 830 (A830G), converting tyrosine to cysteine at amino acid position 277. This novel missense mutation was designated as Y277C.
In family H, linkage was detected on chromosome 18pll at DFNB1910cus. In family I, linkage was established at DFNB43 locus located on chromosome 15q23-25.1. In family J, linkage was detected on chromosome 9q34.3 at DFNB33 locus. In family K, genotyping with microsatellite markers detect linkage to DFNB2 locus on chromosome llq13.5.
In family L, linkage was established with DFNB33 locus harboring MY015 gene. Nine exons of MYO15 gene, reported to contain mutations, were sequenced in affected individuals of the family. Analysis of sequence data revealed the absence of known mutations in family L.
In family M, linkage was established with DFNB37 locus harboring MY06 gene. Five exons of MY06 gene, reported to contain mutations, were sequenced. Analysis of sequence data indicated the absence of known mutations in family M.
In the present study, a novel hearing impaired locus DFNB55 and a novel sequence variant of the TMC1 gene were identified. The identification of a novel non-syndromic recessive deafness locus DFNB55 demonstrates the high degree of locus heterogeneity for hearing impairment, particularly in the Pakistani population. Novel sequence variant, found in TMC1 region, might be critical for function of K + channels in the hair cells.
The work presented in the thesis has been published in the following articles.
1. Saba Irshad, Regie Lyn P. Santos. Dost Muhammad, Kwanghyuk Lee, Nathan McArthur, Sayedul Haque, Wasim Ahmad, Suzanne M. Leal. Localization of a novel autosomal recessive non-syndromic hearing impairment locus (DFNB55) to chromosome 4q 12-4q 13.2. Clinical Genetics (2005) 68, 262 - 267.
2. Regie Lyn P. Santos, Mohammad Wajid, Mohammad Nasim Khan, Nathan McArthur, Thanh L. Pham, Attya Bhani, Kwanghyuk Lee, Saba Irshad, Asif Mir, Kai Yan, Maria H. Chahrour, Muhammad Ansar, Wasim Ahmad, Suzanne M. Leal. Novel Sequence Variants in the TMCl Gene in Pakistani Families with Autosomal Recessive Hearing Impairment. Human Mutation (2005) 26, 396 -404.