Identification of Genes Involved in Hereditary Hearing Impairment and Alopecias

Abstract

A study of rare monogenetic disorders in humans is often L.'1valuable for discovery of the previously unknown genes and physiological pathways. In Pakistan, due to strict social customs, inbreeding has led to unique large fanlilies with un-described phenotypes. These families represent the opportunity to clinically describe new entities, and large family size allows the variability and natural history to be documented. The families also provide an ideal solution to the linkage problem, as individual families contain enough power to allow the disease genes to be localized, the first step in defining the defective genes. For the present study, Pakistani families with autosomal recessive disorders, Hearing Impairment or Alopecias, have been studied as described below. In the present investigation, five highly consangumeous families (A-E) with hereditary hearing impairment and four families (F-I) with hereditary alopecias have been studied from Punjabi, Sindhi and Balochi speaking population of Pakistan. In all these families, a classical linkage analysis approach was used to identify the underlying genes. In family A, screening of the human genome led to the identification of a novel autosomal recessive non-syndromic hearing impairment locus, DFNB62, on chromosome 12p13.2-pll.23 . Significant evidence of linkage to this chromosomal region was found with two-point LOD score of 4.0 and multipoint LOD score of 5.3. Haplotype analysis located the DFNB62 locus in 22.4 cM (15.0 Mb) region flanked by markers D12S358 and D12S1042. DFNB62 represents the second autosomal recessive non-syndromic hearing impairment (ARNSHI) locus that mapped on chromosome 12p13.2. In family B, linkage data obtained from genome scan and saturation of several chromosomal regions with additional markers failed to define a region harboring a causative gene for deafness. In three families (C, D, E), linkage was established to known hearing impairment loci. Family C showed linkage to DFNB9 locus on chromosome 2p23.3 containing OTOF gene. In families D and E, linkage was established to DFNB8/10 on chromosome 21q22.3. The entire coding region, as well as intron-exon boundaries of TMPRSS3 gene was sequenced in affected and unaffected individuals of both the families, but the efforts failed to detect the functional sequence variants. In family F with hereditary hypotrichosis, linkage was established to AH/LAH2 locus on chromosome 3q27. The entire coding region, as well as intron-exon boundaries of LIPH gene were sequenced in all the three affected and one normal individuals of the family. Sequence analysis of a candidate gene Lipase-H (LIPH) revealed a novel five base pair deletion mutation (c.346-350delATATA) in exon 2 of the gene leading to frame shift and downstream premature termination codon. The deletion mutation identified in the family is the second mutation identified in LIPH gene. In family G with alopecia and mental retardation syndrome, hairs were completely absent by birth from all areas of body of the affected individuals. Candidate gene mapping showed linkage of this family to micro satellite markers linked to APMR1 and APMR2 loci on chromosome 3q26.2-q28. A maximum two-point LOD score of 1.74 (8=0.0) was obtained with nine markers. Multipoint linkage analysis resulted in a maximum LOD score of 3.17 with markers D3S2433, D3S2427 and D3S3676 which supports the linkage. Haplotype analysis located the APMR locus between markers D3S3622 and D3S3596, spanning 32.53 cM (20.14 Mb) region on chromosome 3q26.2-q28. Sequence analysis of five candidate genes, LIPH, ETS variant gene 5, TNFSFIO, AP2MI and CamK2 from DNA samples of two affected and one unaffected individuals of the family failed to reveal pathogenic sequence variants. In family H with hypotrichosis, initial genome wide scan revealed five chromosomal regions including D1S1151 at 1p36.22, D3S1544 at 3q12.2, D8S1832 at 8q24.13, D10S1700 at 10q26.3 and D12S291 at 12q12, which were found to be homozygous in all the three affected members of the family. Saturation of these five 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 hypotrichosis in family H. The family I was tested for linkage by using polymorphic microsatellite markers linked to hairless (HR) gene on chromosome 8p21.2. Genotyping of five members including two affected and three unaffected individuals with polymorphic micro satellite markers D8S322, D8S282, D8S560, D8S298, established linkage to the HR locus. Subsequently, the entire coding region as well as intron-exon boundaries of the HR gene was sequenced in · two affected individuals, but failed to identify functional sequence variant suggesting that the mutation is probably present in the regulatory sequence of the gene. The work presented in the thesis has been published in the following articles. 1. Ali G, Santos RL, John P, Wambangco MA, Lee K, Ahmad W, Leal S. The mapping of DFNB62, a new locus for autosomal recessive non-syndromic hearing impairment, to chromosome 12p13.2-pl1.23. Clinical Genetics 69(5):429-33, 2006. 2. Ali G, Chishti MS, Raza SI, John P, Ahmad W. A mutation in the lipase H (LIP H) gene underlie autosomal recessive hypotrichosis. Human Genetics 121(3-4):319- 325,2007.