Molecular Analysis Of Human Hereditary Deafness

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications & trials

Abstract

(1) In the past, we had ascertained families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are studying those families in a combinatorial linkage-based, functional candidate exome sequencing strategy to identify other genetic causes of EVA. To further our knowledge of the transcriptome of the endolymphatic sac, we are collaborating with the NIDCD Genomics and Computational Biology Core led by Dr. Robert Morell. With his help and together with Dr. Erich Boger, we are using long read sequencing (Pac-Bio) to identify the genes expressed in the endolymphatic sac and their isoforms, which could be candidates to carry pathogenic variants in patients and cause EVA. We have identified likely causal variants in two genes which had not been previously associated with nonsyndromic hearing loss and EVA. (2) We used CRISPR/Cas9 genome editing to create several mouse lines segregating alleles of Slc26a4 with missense mutations identified in human patients. These missense mutations encode Slc26a4 protein (pendrin) with significant residual function. We have successfully generated three different lines and are in the process of characterizing their auditory and vestibular phenotypes on different genetic background. The goal of this study is to generate mouse models with hearing loss phenotypes that are less severe than those of the Slc26a4-null line and mimic more closely the phenotype seen in patients. In collaboration with Dr. Michael Hoa and his team, we also continue to study a dox-inducible Slc26a4-insufficient mouse model of EVA which we have previously reported. We are using these lines to explore the molecular and cellular pathogenesis of hearing loss caused by Slc26a4 mutations. In collaboration with Dr. Wade Chien and his team we have identified several viral approaches to infect the cells of the endolymphatic sac for therapeutic purposes. (3) We have identified multiple potential partners of Slc26a4 protein using a yeast two-hybrid approach. We are in the process of confirming and characterizing some of these interactions using both yeast and mammalian cell based assays. We are also studying the cellular distribution of these potential partners in cells of the endolymphatic sac and kidney, and their potential overlap with pendrin localization using both light and electron microscopy with the help of Dr. Ronald S. Petralia and Ya-Xian Wang. We are in the process of developing assays to test those interactions at the functional level. To this effect, we have developed new approaches to culture the endolymphatic sac epithelium and identified strategies to express molecules of interest in these explants. (4) We had identified several reporter mice to help us dissect and study the endolymphatic sac. We shared the approaches we have developed to identify and dissect the endolymphatic sac with the Community (JoVE article). (5) In collaboration with Dr. Adebolajo Adeyemo and his team at the University of Ibadan and together with Dr. Thomas Friedman and his team at NIDCD/NIH, and Dr. Leal and her team at Columbia University Medical Center, we have used a combination of Sanger and Exome sequencing approaches to study the genetic causes of childhood hearing loss in a cohort of families from Yoruba ethno-linguistic group from Nigeria (first manuscript in revision).