|Keywords (Extracted from title, table of contents and abstract of thesis)
human hereditary disorders, jammu, kashmir, genetic disorders, hearing loss, genetic heterogeneity, deafness, autosomal recessive hearing impairment, evoked otoacoustic emission (eoaes), syndromic hearing impairment, autosomal dominant, stickler syndrome, muckle-wells syndrome, waardenburg syndrome, branchio-oto-renal syndrome, autosomal recessive, pendred syndrome, usher syndrome, jervell and lange-niesen syndrome, x- linked, norrie disease, alport syndrome, mitochondrial, mouse model
The present study was initiated with the aim to locate families with different genetic disorders living in Azad Jammu and Kashmir, a region neighboring China, Pakistan, India and Afghanistan, which is a gateway to Central Asia that signified its strategic importance. However, during the course of study it was realized that large number of families with various types of genetic disorders are living in the region and practically it is not possible to collect and study all the families in a short time. Therefore, for the work presented here families with hearing impairment were selected.
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 35 genes identified.
In the present study, twelve families (A-L) with syndromic and non-syndromic hearing impairment have been described. In eight families (A-H), with nonsyndromic recessive deafness, affected individuals have prelingual profound hearing impairment. In two families (I & J) affected individuals show severe to profound prelingual hearing loss associated with goiter (Pendred syndrome). In families K and L, patients have profound hearing impairment associated with Retinitis Pigmentosa (Usher syndrome). Linkage in the families was initially searched by using microsatellite markers corresponding to candidate genes involved in related autosomal recessive nonsyndromic deafness phenotypes. Linkage was detected in six families, Le., C-H. In families C-E linkage was established to DFNB1 locus on chromosome 13. In families F it was detected on chromosome 7 at DFNB39 locus. In family G, linkage was established at DFNB7/11 locus on chromosome 9. In family H, the linkage was detected on chromosome 3p21 at DFNB6 locus. In other two families (A and B) linkage to all the known loci was conclusively excluded, thus indicating the involvement of some novel loci, responsible for deafness in these families.
After excluding the disorder from linkage to known hearing loss loci in families A and B, genome-wide scan was performed by using the Mappairs sets of Microsatellite 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, DFNB61, on chromosome 20q13.2-13.32. Twopoint linkage analysis generated LOD score of 2.1 05 with five markers (D20S 1 085, D20S480, D20S100, D20S102, D20S430). Multipoint linkage analysis resulted in a maximum LOD score of 3.31 with several markers in this region. Examination of haplotypes defined a critical region 11.7 cM, which is flanked by markers D20S606 and D20S451. This is the first autosomal rec~ssive hearing loss locus identified on chromosome 20.
In family B, initial genome wide scan revealed six regions (GATAI31D09, AAAAC001, D10S1425, D16S748, D16S3396, D16S2624) where three or four affected subjects were found to be homozygous. Saturation of these regions with additional markers, however, failed to generate a significant LOD score with any of the markers, thus excluding these regions from harboring a causative gene for deafness in family B.
In three 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 and one deletion mutations. In family C, a G to A transition at nucleotide position 231, leads to premature termination codon (W77X). In family D 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 E sequence analysis of GJB2 gene revealed deletion of three base pair starting at nucleotide position 358. This results in deletion of a codon specifying glutamic acid at amino acid position 120 (de1120E).
In family G, sequence analysis of exon 21 of the TMCl gene from affected individuals revealed a missense mutation involving T to G transition at nucleotide position 2004 (T2004G). This missense mutation results in substitution of serine to arginine (S668R) at amino acid position 668.
In family H, sequence analysis of exon 3 of the TMIE gene in affected individuals from the family revealed a C to T transition at nucleotide position 241 (C241T). This missense mutation results in substitution of arginine to cystine at amino acid position 81 (R81 C). Two families I and J showed linkage to Pendred syndrome locus on chromosome 7q31. The loss of hearing in these families was present at birth, whereas the development of goiter was progressive in nature and develops after puberty. The go iter is variable in its severity.
Two families with Usher syndrome were included in the present study. In family K, linkage was established to USHB 1 locus on chromosome 11. In the second family L with Ushre syndrome, linkage was not detected to any of the known Usher syndrome loci, thus indicating the involvement of a novel locus. However, genome-wide search to locate the disease locus is this family was not performed.
Based on the research work, presented in the thesis, the following manuscripts are in preparation stage for submission to "Human Heredity" and "Human Mutation" for publications.
1. Localization of a novel autosomal recessive non-syndromic hearing impairment locus DFNB61 to chromosome 20q13.2-q13.32.
2. Novel Sequence Variants in the TMCl Gene in Pakistani Families with Autosomal Recessive Hearing Impairment