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) is to identify and study the functions of mutated genes associated with human syndromic and nonsyndromic deafness. Our studies begin with the ascertainment of families with members who appear to be segregating deafness 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 and exome sequence to identify potentially causal variants. Staff in the LMG are working on the following projects: 1. DFNB32/CDC14A: In several consanguineous families segregating recessively inherited nonsyndromic deafness linked to markers for the DFNB32 locus, 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 but 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, both wild type and CDC14A with a C278S variants that will trap substrate in the active site. 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 and is being studied. Simultaneous loss of TRIOBP4 and TRIOBP-5 causes deafness as a result of the inability of hair cells to develop stereocilia rootlets. Purified TRIOBP-4 tightly bundles F-actin typical of stereocilia rootlets. 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 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). Further studies are underway to understand the functions of each TRIOBP isoform in supporting cells and hair cells. 3. The LMG is presently ascertaining Pakistani families segregating syndromic forms of deafness. 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 Tbc1d24, 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 Dr. Michelle Hastings (University of Michigan), Yasuko Ishibashi MD, co-mentored by Wade Chien, MD, are developing an ASO-based therapy in mouse to circumvent splicing so as to exclude 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 protein-protein interactions. Some TBC1D24 mouse mutants don't show hearing loss despite being identical to human deafness variants of TBC1D24. We are exploring if modifiers of these hearing -associated variants in the genetic background of mouse permit normal hearing. 5. Usher syndrome is genetically and clinically heterogeneous. In collaboration with Robert Hufnagel MD, PhD (NEI) 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. (Wafa et al., 2019). These studies are continuing with recent observations that variants of LRP2 can be associated with an Usher-like syndrome of deaf-blindness (Faridi et al. 2023 Clinical Genetics). 6. In collaboration with Adebolajo Adeyemo, MD at the University of Ibadan, Nigeria, Isabelle Roux, PhD and staff in the LMG identified pathogenic variants associated with deafness segregating in approximately 60 small Yaruba families living in Nigeria. The deaf proband's gDNA were exome sequenced and evaluated for putative pathogenic variants. This study was published recently (Adeyamo et al. Genomic analysis of childhood hearing loss in the Yoruba population of Nigeria. 2022 European Journal of Human Genetics 30: 42-52.) 7. In collaboration with Drs. Gregory Alushin (Rockefeller University) and Jonathan Bird (University of Florida at Gainesville), LMG staff including Mr. Arik Shams as a post-bac and Dr. Bird, post-doctoral fellow at the time, purified the motor domain of wild type myosin 15 and myosin 15 incorporating an amino acid substitution in the D-loop of the motor domain, which is a recessive variant associated with deafness in the Jordan mouse model. The team examined the interaction with F-actin using cryo-electron microscopy. Myosin 15 was found to enhance polymerization of F-actin by a novel mechanism. This project resulted in a manuscript under revision at Nature Communications by Moreland et al.,(2023) from the Bird laboratory entitled Myosin-driven nucleation of actin filaments promotes stereocilia development critical for hearing and a published paper (Gong et al., 2022) 8. 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, PhD, Rabia Faridi, PhD and Brenda Quinones Flores, LMG staff, have prepared an IRB approved human subjects protocol of the molecular genetics and audiological profiles of childhood hearing loss in Puerto Rico. Once approved in Puerto Rico and by the NIH IRB, ascertainment will begin. 9. Each year, several large families segregating deafness are ascertained in Pakistan by Sheikh Riazuddin, PhD, Lahore, Pakistan and his team. gDNA and clinical data including audiograms are provided to the LMG/NIDCD/NIH for molecular geentic analyses to identify the variants of the genes likely causing deafness. 10. A possible study of syndromic deafness segregating in families living in Nigeria is being discussed with our collaborator, Dr. A. Adeyemo as a continuation of our study with him published recently (see reference #1 below)