Identification of Genes Causing Syndromic and Nonsyndromic Hearing Impairment

National Institute On Deafness And Other Communication Disorders

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Abstract

The goal of the Laboratory of Molecular Genetics (LMG), Section on Human Genetics is to identify and study the functions of mutated genes associated with human syndromic and nonsyndromic deafness. A new study begins with the ascertainment of large families in which deafness appears to be inherited either as a monogenic dominant or as monogenic recessive trait. We then search for linkage of the deafness to 950,000 SNP markers distributed across the human genome. Staff in the LMG have been working on the following projects, some of which were completed in the past year, have been published recently, are in press or likely to be submitted for publication in the near future. 1. DFNB32/CDC14A: Eleven years ago, DFNB32 was mapped to chromosome 1 by by a research group in Tunisia. The underlying gene for DFNA32 deafness was never reported. In eight of our consanguineous families segregating recessively inherited nonsyndromic deafness linked to markers for the DFNB32 locus, we identified several truncating mutations, a splice site mutation and a missense mutation in CDC14A, a gene located in our refined DFNB32 interval. CRISPR-Cas9 edited alleles of mouse Cdc14a, when homozygous or in compound heterozygosity, result in deafness. Surprisingly, males are also sterile but females have normal fertility but are also deaf. Thus, CDC14A is essential for hearing and for male fertility. As a collaboration with Dr. Katie Kindt, we also constructed zebrafish models of the DFNB32 gene to probe its function. These data were published in Human Molecular Genetics (Imtiaz et al., 2018). CDC14A is a phosphatase. In hair cells, CDC14A is located in the kinocilia and in stereocilia (Imtiaz et al., 2018) The protein substrates of CDC14A phosphatase are not known. Y2H screens and mass-spec analyses are underway to identify substrates of CDC14A as part of a larger project to understand the function of this essential phosphatase in the auditory system. 2. DFNB28/TRIOBP: The gene responsible for human deafness DFNB28 human deafness was identified as TRIOBP (Kitajiri et al., Cell, 2010). TRIOBP encodes three distinct proteins that arise from alternative splicing of TRIOBP transcripts. TRIOBP isoforms are referred to as TRIOBP-1, TRIOBP-4 and TRIOBP-5. Loss of TRIOBP-1 causes embryonic lethality in mouse. The function, if any, of TRIOBP-1 in the inner ear is not known. Simultaneous loss of TRIOBP4 and TRIOBP-5 causes deafness as a result of the inabilityof hair cells to develop stereocilia rootlets. Purified TRIOBP-4 tightly bundles F-actin typical of stereocilia rootlets. At that time, the individual function(s) of TRIOBP-5 is not known. We engineered mice that do not express functional TRIOBP-5 and have wild type expression of TRIOBP-1 and TRIOBP-4. TRIOBP-5 deficient mice develop rootlets. However, the rootlets are unusually dysmorphic, stereocilia are floppy and the reticular lamina, as measured by Atomic Force Microscopy, is significantly less rigid due to the apical loss of TRIOBP-5 in supporting cells surrounding hair cells (Katsuno and Belyantseva et al., 2019). Studies are continuing to understand the function of TRIOBP-4 and TRIOBP-5 in supporting cells and the functions of each of the TRIOBP isoform function elsewhere in hair cells. For example, we are studying a TRIOBP-1 conditional knock out mouse which will us to delete TRIOBP-1 in hair cells or only in supporting cells of the inner ear and determine the phenotype. 3. The LMG is ascertaining Pakistani families segregating Perrault Syndrome, which is characterized by deafness and female infertility. This project is an ongoing collaboration with William Newman, MD, PhD in Manchester, UK and Sheikh Riazuddin, PhD in Lahore, Pakistan. We are also engineering mouse models of the human genes responsible for Perrault syndrome in order to understand their function in the auditory system. 4. DFNB86/TBC1D24: In 2014, we reported that Variants of TBC1D24 are associated with nonsyndromic deafness DFNB86. Variants of TBC1D24 have also been associated with seizures, seizures and deafness and DOORS syndrome. Other variants of TBC1D24 have been associated with Rolandic epilepsy and exercise-induced dystonia (Luthy et al., 2019), expanding the genotype-phenotype range even further. Using CRISPR/Cas9, we have engineered mice with variants of Tbc1d2, one of which abruptly begins having seizures at P15. The abrupt onset of seizures in mouse is correlated with inclusion of a perfectly conserved alternatively spliced micro-exon encoding 8 amino acid residues and harboring a mutation of Tbc1d24 (Tona et al., 2019). The function of the micro-exon is being explored using a conditional knockout variant in which loxP sites were engineered surrounding the micro-exon. As a collaboration with Michelle Hastings, we are also developing a potential ASO-based therapy in mouse to circumvent splicing to include the micro-exon. Additionally, we are working on identifying the protein partners of TBC1D24 in the brain and inner ear and human disease-causing missense mutations of TBC1D24 that are predicted to disrupt such partnerships. 5. Usher syndrome is genetically and clinically heterogeneous. In collaboration with Carmen Brewer, PhD, Andrew Griffith MD, PhD and Wadih Zein MD (NEI) we are studying the natural history of the visual, auditory and vestibular phenotypes of Usher syndrome subjects enrolled at the NIH Clinical Center. All of these Usher subjects have biallelic molecular genetic likely pathogenic variants of the reported Usher genes from sequencing analyses conducted by staff in the LMG. A manuscript is in preparation (Wafa et al., 2019). 6. In collaboration with Adebolajo Adeyemo, MD at the University of Ibadan, Nigeria, Isabelle Roux, PhD in the NIDCD and Andrew J. Griffith MD, PhD, University of Tennessee, staff in the LMG are identifying the pathogenic variants associated with deafness segregating in approximately 60 small Yaruba families living in Nigeria. The deaf proband's gDNA is being whole exam sequenced and evaluated for putative pathogenic variants. A manuscript is under revision.