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Title of Thesis


Zubair Mohiuddin Ahmed
Institute/University/Department Details
University Of The Punjab/ National Centre Of Excellence In Molecular Biology
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
genetic, syndromic deafness, usher syndrome, ear, childhood deafness, hereditary hearing loss, ush1, ush2, ush3

The aetiology of childhood deafness is markedly diverse and involves numerous environmental and genetic factors. Hereditary hearing loss is divided into tow group, syndromic and nonsyndromic. Hundreds of syndromes associated with hearing loss have been described (Gorlin 1995) including Usher syndrome, in which hearing loss occurs with night blindness. There are three clinical subtypes of Usher syndrome, designated as USH1, USH2 and USH3. Usher syndrome type I, the most common of three clinical subtypes(astuto et at. 2000), is characterized by congenital profound sensorineural hearing loss, vestibular areflexia and retinitis pigmentosa with an onset near to puberty (Smith et al. 1994). Seven loci of USH1 (USH1A- USH1G) are known and genes for USH1B, USH1C, USH1D and USH1F have been identified (Hereditary Hearing Loss Homepage).

Previous studies have shown that USH1F is linked t markers at 10q21-22 and spans a 15cM interval centromeric to the DFNB12 and USH1D loci (Wayne et al. 1997). In the present study, three families, PKSR17a PKSR54a with inherited profound, congenital deafness, were linked to USH1F. Haplotype analysis of these three families reduced the linkage interval for USH1F to 1cM at 10q21.1 bounded by the markers D10S1642 and D120S596 (Ahmed et al 2001). The physical map of overlapping Bacterial Artificial Chormosomes (BACs) of the USH1F linkage interval was constructed suing the human genomic sequence available form NCBI and Sanger’s and it was observed that 10BACs span the whole linkage region of USH1F.

In previous studies, mouse models have in identifying genes for USH1B and USH1D(Weil et .at 1995; Liu et al. 1997a, 1997b; Bloz et al.2001; Bork et al.2001). Conserved synteny between human and mouse chromosome 10 suggested that Pcdh 15,the gene mutated in ames waltzer (av) mice, might be the murine model for USH1F and /or DFNB23 (Alagramam et al.2001) Two novel mutations, R3X and IVS27-2A>G, in PCDH15 were found in two of the above three Pakistani families (PKSE17a PKSR8b). A northern blot probed with the PCDH15 cytoplasmic domain shoed expression in the retina, consttent with its pathogenetic role in the retinitis pigmentosa associated with USH1F. Immunostaining with polyclonal antibodies against the peptides form cytoplasmic domain of protocadherin 15 demonstrated its expression in the inner and outer hair cells stereocilia of the inner ear. In the retina protocadherin 15 expressions is localized in rods and cones. This work has been published in July 2001 issue of the American Journal of Human Genetics (Ahmed et al.2001).

Human chromosome 11 harbors two Usher type I loci, USH1B and USH1C, which encode myosin VIIA and harmonin, respectively. The USHIC locus overlaps the reported critical interval for nonsyndromic deafness locus DFNB18. In the present study a splice site mutation, IVS12+5G>C, in USH1C gene which is associated with nonsyndromic recessive deafness segregating in the original DFNB18 family S-11/12 was discovered. An exon trapping assay was used with a construct containing genomic fragment harboring exon 11 and exon 12 USH1C with IVS12+5G>C substitution. This construct produced wild type spliced RNA containing exons 11 and 12 and RNA that skipped exon 12. The results of this study further demonstrated that mutations of USH1C can cause both Usher syndrome type IC and nonsyndromic recessive deafness (DFNB18).

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 00 Contents
143.64 KB
2 0 Introduction 1
42.76 KB
3 1 Unit 1 Literature survey 5
613.37 KB
  1.0 Molecular and genetic basis of usher syndrome 5
  1.1 Clinical classification 7
  1.2 Usher Loci and Mouse Models 13
  1.3 USH1A 13
  1.4 USH1B/DFNB2/DFNA11 & Shaker-1 13
  1.5 USH1C/DFNB18 21
  1.6 USH1D/DFNB12 and Waltzer(v) 25
  1.7 USH1E 30
  1.8 USHIF/DFNB23and Ames Waltzer(av) 30
  1.9 USH1G/DFNA22/DFNA26 31
  1.10 USH2A 31
  1.11 USH2B 31
  1.12 USH2C 35
  1.13 USH3A 36
  2 Structure of the Ear 40
  2.1 The Human ear 41
  3 Material & Methods 47
  3.1 DNA extraction 48
  3.2 From Blood Samples 48
  3.3 Form Buccal Swabs 48
  3.4 From BAC plasmid 49
  3.5 Isolation of BAC DNA via Miniprep 49
  3.6 Mix preparation of DNA 50
  3.7 Linkage analysis 51
  3.8 Genotyping 52
  3.9 Automated Fluorescent Genotyping 53
  3.10 Radioactive genotyping 54
4 2 Unit 2 Mapping families to the USH1F/DFNB23 locus, linkage interval refinement, physical map of the region and clinical evaluation of the three linked families 80
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  1.1 Introduction 81
  1.2 Results 81
  1.3 Genotyping to screen of linkage to USH1F/DFNB23 81
  1.4 Refinement of linkage region and haplotype analysis 84
  1.5 Allele frequencies and two point –Lod sore calculation 86
  1.6 Physical Mapping 86
  1.7 Finger print BAC Map of USH1F/DFNB23 93
  1.8 Computer analysis of genomic DNA with in the refined USH1F/DFNB23 linkage region 95
  1.9 Known Genes & ESTs within the USH1F/DFNB23 refined region 96
  1.10 Mouse Model for USH1F/DFNB23 locus 97
  2 Identification and characterization of PCDH15 106
  2.1 Introduction 106
  2.2 Results 107
  2.3 Cloning 107
  2.4 Phylogenetic analysis of PCDH15 109
  2.5 Mutational screening of PCDH15 112
  2.6 Characterization of PCDH15 113
  2.7 PCDH15 m RNA expression 118
  2.8 Protein Expression 120
  2.9 Immunohistochemistry 112
  2.10 organ of corti staining 122
  3 Nonsyndromic Recessive Deafness DFNB18 and Usher syndrome type IC Are Mutations of USHIC 130
  3.1 Introduction 131
  3.2 Genotyping to screen for linkage to USH1C/DFNB18 131
  3.3 Clinical evaluation 133
  3.4 Results 134
  3.5 LocusDFNB18 134
  3.6 Locus USH1C 134
  3.7 Exon Trapping 136
  3.8 Discussion 140
5 3 References 142
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