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dc.contributor.authorRamzan , Memoona-
dc.description.abstractHearing loss inherited as a recessive trait, occurs more frequently than autosomal dominant, mitochondrial or X-linked deafness. Variants of more than 70 genes are associated with nonsyndromic hearing loss inherited as an autosomal recessive disorder but, only a few genes/variants are exclusively associated with moderate-severe hearing loss. Consequently, less is known about the genetic bases of recessively inherited moderate-severe hearing loss despite being more prevalent as compared to profound deafness. Therefore, this study focuses on the genetic understanding of moderate-severe hearing loss in Pakistani individuals. Participants from 26 families were included in this research. All affected individuals predominantly manifested moderate-severe hearing loss. A stepwise experimental approach was used for molecular characterization of the phenotypes of the subjects from sixteen families. Linkage to known deafness genes were excluded for ten families. This was followed by genomewide homozygosity mapping or Whole Exome Sequencing (WES) to identify mutations in any gene which could be associated with the segregating phenotype in the families. Additionally, samples from affected individuals of eight consanguineous families prescreened for GJB2 were directly subjected to WES for the identification of known or novel genes involved in hearing loss. The results of WES for multiplex families revealed that alleles of the reported autosomal recessive deafness causing genes are also the major contributor to the etiology of moderate-severe hearing loss in Pakistan. Eleven known and seven novel variants in the reported deafness genes cosegregated with moderate-severe hearing loss in these families. Variants of SLC26A4, ESPN and OTOF were the three major contributors in this cohort. Variants in ESPN were identified for the first time to cause moderate-severe hearing loss in three different families. Results of WES revealed that for about 19 % of families included in this study, variant in a single gene could not account for hearing loss in all affected individuals of a particular family, suggesting intrafamilial locus heterogeneity. An interesting genetic outcome was observed for family HLGM10. WES revealed two variants, one homozygous mutation in TMC1 and the other in KCNQ4, segregating in two different branches of the family. Variants in both genes are known to cause moderate-severe or profound hearing loss phenotypes. So far, variants in KCNQ4 have been only reported for dominant nonsyndromic v deafness. For family HLGM10, the individuals with the KCNQ4 p.(Pro291Leu) variant in the homozygous condition, had a more severe phenotype with early onset of the hearing loss. In contrast, heterozygous individuals had a mild hearing loss of which they were unaware. Their mild hearing loss also started later, probably in the second decade of life. Therefore, this case demonstrated that p.(Pro291Leu) variant in KCNQ4 can be inherited in a semi-dominant mode with variable severity and age of onset for mono-allelic or bi-allelic forms. Missense variants in two genes, ITSN2 and CLDN9 were identified in two different families as a likely cause of moderate-severe hearing loss in affected individuals. The missense variants in both genes are predicted to affect the conserved regions of respective proteins. However, ITSN2 is located in an overlapping chromosomal position between OTOF and DFNB47. Although genotyping with STR and SNP markers excluded the linkage to both, the regulatory regions of the two genes remain to be explored. CLDN9 was a stronger candidate as its role in etiology of hearing loss was already demonstrated in a murine model. While this thesis was about to be submitted, the association of CLDN9 with human deafness was demonstrated in a small Turkish family. The segregation analysis and homozygosity mapping data supported CLDN9 as the only causative gene in the current family. Further studies were performed to predict the effect of particular missense variant and its importance in causing hearing loss. The in situ hybridization and immunohistochemical studies suggested that both mRNA of CLDN9 and the encoded protein are present in the inner ear. The expression of the gene and localization of protein was observed in both sensory and nonsensory epithelia. The localization of mutant CLDN9 was tested in Madin-Darby Canine Kidney II (MDCK-II) cell lines which suggested that the variant does not affect the targeting of the protein to its specific location. However, the in silico protein modelling suggeststhat the variant in CLDN9 affects the protein to protein cis interaction in the cell. A part of this research was concentrated on the identification of genetic causes of moderate-severe hearing loss in sporadic individuals from Pakistan. A total of twenty isolated cases of hearing loss were included. After the phenotypic evaluation, WES was applied to DNA of all subjects. The results for WES revealed ten novel and five known variants in reported deafness genes. No potential pathogenic variant was identified for three subjects. A missense variant in ESRRB was vi identified for the first time in one subject who had moderate-severe hearing loss. A novel candidate gene BHLHE22 was identified for the phenotype in one individual. It was suspected that few genes would appear as a predominant causative factor for isolated hearing loss due to a founder effect, but variants in multiple genes were identified. It indicates that genetics of deafness in individuals with no previous history of hearing loss is as complex as that of familial cases. Therefore, WES of large cohorts of isolated cases may also serve as a potential source to expand the genetics of hearing loss.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.publisherUniversity of the Punjab , Lahoreen_US
dc.subjectBiological & Medical Sciencesen_US
dc.titleMolecular Studies of Genes Involved in Moderate to Severe Hearing Lossen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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