Deafness or hearing loss can be due to genetic or environmental causes or a combination of both. The hearing loss disorders attributable to genetic causes, are classified as syndromic or non-syndromic. Among the many disorders classified as syndromic hearing loss, the pathology varies widely, but, in non-syndromic hearing loss, the defect is generally sensorineural. Seventy percent of deafness due to genetic causes is classified as non-syndromic. Autosomal recessive non-syndromic deafness is genetically heterogeneous and is the most common form of inherited hearing loss. Autosomal recessive genes are responsible for about 77% of the cases of hereditary non-syndromic deafness, with over 60 loci and 21 different genes identified to date. The high degree of genetic heterogeneity of deafness reflects the great diversity of specialized proteins that are required to make sense of sound, and continuing discovery of common and rare mutations associated with deafness in humans has provided many serendipitous points of entry into the biology of hearing.
In the present study, thirteen families (A-M) demonstrating autosomal recessive form of non-syndromic hearing loss were ascertained from remote areas of Punjab province in Pakistan. Clinical findings in each family were consistent with the diagnosis of autosomal recessive non-syndromic hearing loss (ARNSHL). The affected individuals in the families had prelingual severe to profound hearing loss with no associated features of syndromic or acquired form of deafness. Initially, linkage in the families was searched by using polymorphic microsatellite markers corresponding to the candidate genes involved in related autosomal recessive non-syndromic deafness phenotypes. Linkage with known loci was detected in eight families Le. E-H and J-M. In families E-H, linkage was established to DFNB110cus, supporting the involvement of GJB2 gene causing deafness. In family J, linkage was detected to DFNB 12 locus on chromosome 10q21-q22. In family K, linkage was established to locus DFNB42 on chromosome 3q1331-q223. In family L, linkage was established to DFNB7/11 locus on chromosome 9q13-q21, whereas family M showed the linkage to DFNB3 locus on chromosome 17pl1.2. In five families (A-D and 1) linkage to the known loci was conclusively excluded, indicating the involvement of novel loci responsible for deafuess in these families. After excluding the disorder in five families (A-D and I) from linkage to the known deafness chromosomal regions, genome-wide scan was carried out to identify the disease loci.
In family A, genome-wide search led to identification of a novel hearing loss locus (DFNB46) on chromosome 18pl1.32-pl1.31. A maximum two-point LOD score of 2.09 was obtained at marker D18S976 and multipoint LOD score of 3.8 was obtained at markers D18S481 and D18S1370. Saturation of the region with additional markers and examination of haplotypes defined a critical region of about 18.7 cM on Marshfield sex-averaged genetic map and delimited by markers GATA178F11 and D18S452. The exons and splice junction sites of the genes ZFP161, MRCL2, MRLC3, CETN1, MYOM1, located in the genetic interval of DFNB46, were sequenced but putatively causative variants were not detected.
In three families (B, C, D) analysis of the genome scan identified a novel hearing loss locus DFNB43, which maps to chromosome 15q24.1-q25.2. The maximum two point combined LOD score of 5.14 was obtained with marker D15S1023. Analysis of the linkage intervals identified in these families revealed the segregation of a shared region of 7.10 cM among all the affected individuals. The shared linkage interval of DFNB43 identified in these families is flanked by markers D15S205 and D15S818.
Four families (E-H) in the present study were found linked to DFNB 1 locus on chromosomes 13q12 that harbors GJB2 gene. Sequence analysis of the coding exon of the GJB2 gene in the affected individuals of all the four families revealed a G to A transition at nucleotide position 71, leading to premature termination codon (W24X).
In family I, a novel locus DFNB54 for autosomal recessive non-syndromic deafness was mapped to chromosome Ip34.2-p32.1. A maximum two-point LOD score of2.55 with marker D1S2700 and multipoint LOD score of 3.42 with marker D1S1661 was obtained. The' observed recombination events localized the locus to an interval of 18.36 cM between the markers D1S2706 and D1S1596. The critical linkage interval of DFNB54, identified in family I, overlaps with the linkage interval of autosomal dominant deafness locus (DFNA2). Sequence analysis of exon 1 of the BSND gene, mapped to the DFNB54 linkage region, revealed a novel missense mutation involving T to C transition at nucleotide number 34 (34T >C), which resulted in substitution of theronine to isolucine at amino acid position 12 (T12I). Clinical investigations examination of the affected individuals in family I ruled out the presence of clinical features, which are hallmark of the Barter syndrome.
In family L, linkage was detected to DFNB7/11 locus on chromosome 9q13-q21. The TMCI gene responsible for DFNB7/11 hearing loss was sequenced to detect mutation. Sequence analysis of exon 16 of the gene in affected individuals revealed a novel missense mutation involving G to A transition at nucleotide position 1334 (c.1334G>A), resulting in an arginine-to-histidine substitution at amino acid position 445 (R445H).
To identify a mutation in the affected individuals of family M, linked to DFNB3 on chromosome 17p11.2. Myosin 15 gene was screened for reported mutations in exons 3, 4, 28, 29, 30, 39, 40, 43 and 44. However, no disease causing mutation was detected, suggesting the presence of a novel mutation in the family M.
The high genetic heterogeneity of hearing disorders would need the completion of the mapping genetic loci for support in diagnosis and for a deeper understanding of the basic mechanisms of the biology of the ear. While strong evidence of linkage of hearing loss to three new loci has been presented in the present study, distribution of the recombination events in the families has only allowed assigning the genes to large genetic intervals. This being the case, the map locations will have to be substantially refined to identify the mutated gene. An alternative approach is to identify candidate gene mapping to regions, which could be tested for disease specific mutations.
The work presented in the thesis has been published or in preparation for submission in the following articles:
1. Asif Mir, Muhammad Ansar, Maria H. Chahrour, Thanh L. Pham, Muhammad Wajid, Sayedul Haque, Kai Yan, Wasim Ahmad, Suzanne M. Leal (2005) Mapping of a novel autosomal recessive nonsyndromic deafness locus (DFNB46) to chromosome 18p 11.32-p 11.31 Am J Med Genet 133:23-26
2. Regie Lyn P. Santos, Muhammad Wajid, Muhammad Nasim Khan, Nathan McArthur, Thanh L.Pham, Attya Bhatti, Saba Irshad, Asif Mir, Kai Yan, Maria H. Charhrour, Muhammad Ansar, Wasim Ahmad and Suzanne M. Leal (2005) Novel sequence variants in the TMCI gene in Pakistani families with autosomal recessive hearing impairment. Human Mutation 26(4):396
3. Asif Mir, Regie Lyn P. Santos, Muhammad Ansar, Wasim Ahmad and Suzanne M. Leal. Mapping of DFNB43, a gene for a non-syndromic autosomal recessive deafness, to chromosome 15q24.1-q25.2 (In preparation).
4. Asif Mir, Regie Lyn P. Santos, Muhammad Ansar, Suzanne M. Leal and Wasim Ahmad. Localization of a novel autosomal recessive non-syndromic hearing impairment locus (DFNB54) to Ip34.2-p32.1 in consanguineous kindred from Pakistan (In preparation).