Identification of Genes Causing Syndromic and Nonsyndromic Hearing Impairment

National Institute On Deafness And Other Communication Disorders

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Abstract

The goals of the Laboratory of Molecular Genetics (LMG), National Institute on Deafness and Other Communication Disorders, national Institutes of Health is to identify and then study the functions of mutated genes associated with human syndromic and nonsyndromic deafness. Our studies begin with the ascertainment of families segregating hearing loss due to monogenic dominant or monogenic recessive mode of inheritance. We then search for linkage of the deafness to 950,000 SNP markers distributed across the human genome followed by exome sequencing to identify potentially causal variants of genes necessary for hearing in a narrow chromosomal linkage interval. This annual report is focused on functional studies of genes necessary for hearing. Staff in the LMG are working on the following projects: 1. Perrault syndrome is a genetically heterogeneous disorder characterized clinically by hearing loss and premature ovarian failure. The LMG staff have been studying a large family with ten deaf individuals (females are infertile). We genetically mapped a novel gene for Perrault syndrome and identified homozygous pathogenic variants. In collaboration with William Newman, MD, PhD, we are preparing a manuscript on the human genetics in which we are describing variants segregating in two unrelated families segregating Perrault Syndrome. In the LMG/NIDCD we are determining the function of this gene in the auditory system. The goal is to understand the function of this newly discovered novel gene for Perrault syndrome. To accomplish this goal we are performing (1) light and electron microscopy localizations of the witl type and mutant Perrault syndrome protein, (2) yeast two hybrid screens (Y2H) to identify protein partners of the novel Perrault syndrome gene, and (3) Y2H analyses to determine if missense mutations associated with Perrault syndrome interfere with its interactions with protein partners. 2. DFNB32/CDC14A: In several consanguineous families segregating recessively inherited nonsyndromic deafness linked to markers for the DFNB32 chromosomal locus, initially we identified 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, result in deafness. Surprisingly, deaf males are also sterile when a variant alters the phosphatase catalytic domain. In comparison to deaf infertile males, deaf females are fertile. Thus, CDC14A is essential for hearing and for male fertility (Imtiaz et al., 2018) The protein substrates of CDC14A phosphatase in the inner ear and sperm are not known. Y2H screens and mass-spec analyses are underway using cells from a mouse engineered with CRISPR/CAS-9 to have two epitope tags on the endogenous CDC14A protein, both wild type and CDC14A with a C278S variants that will trap substrate in the active site. Recently, in a continuing collaboration with Dr. Imtiaz, the N-terminal 50% of CDC14A protein that has the phosphatse domain has been purified, crystalized and the structure of the CDC14A catalytic domain determined. We now know with a new mouse model of Cdc14a that the unfolded C-terminal domain of CDC14A is unnecessary for male fertility but is absolutely essential for normal hearing. These observations are being prepared for submission in 2025. 3. DFNB86/TBC1D24: In 2014, we reported that variants of TBC1D24 are associated with nonsyndromic deafness DFNB86. Concurrently, variants of TBC1D24 were also been reported by other investigators to be associated with seizures, seizures and deafness and DOORS syndrome. Other variants of TBC1D24 are also 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 Tbc1d24, one of which abruptly begins having seizures at P15 and die around P21. The abrupt onset of seizures in mouse is correlated with inclusion of a perfectly conserved alternatively spliced micro-exon encoding eight 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 Dr. Michelle Hastings (University of Michigan), Yasuko Ishibashi MD, co-mentored by Wade Chien, MD, have developed an ASO-based therapy in mouse to circumvent splicing so as to exclude the micro-exon of mouse Tbc1d24 as a proof of principle as a therapy for human epilepsy associated with variants of TBC1D24. 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 protein-protein interactions. One such protein partner of TBc1D24 is KIBRA which interacts with the TLDc domain of TBC1D24. These data were published in Movember 2024 in the Journal of Biological Chemistry (Tona and Inagaki et al 2024). 5. In collaboration with Soami Santiago De Snyder PhD, Wanda Lugo AuD, Elinette Albino PhD from the University of Puerto Rico, Medical Sciences Campus, in San Juan, Puerto Rico, and Carmen Brewer, PhD (NIDCD), Isabelle Roux, Ronna Hertzano MD, PhD, and Rabia Faridi PhD, we prepared an IRB approved human subjects protocol and Informed Consents to do molecular genetics and audiological profiles of childhood hearing loss in Puerto Rico. IRB approval has been obtained in Puerto Rico and is awaiting final NIH IRB approval before ascertainment can begin in Puerto Rico. Exome sequencing and variant identification will be conducted in the LMG with these data shared with Dr. Elinette Albin in Puerto Rico. The function in the inner ear of novel deafness genes discovered as part of this project will be studied in the LMG in collaboration with our Puerto Rican colleagues.