The goal of the Laboratory of Molecular Genetics (LMG), Section on Human Genetics is to identify and study the function of mutated genes for human hereditary 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. During the past year we ascertained several large families segregating deafness, mapped novel deafness loci and have identified variants in the causative gene. Staff in the LMG have been working on the following projects, some of which were completed in the past year and have been published, are in press or are likely to be published in the near future. 1. Staff in the LMG mapped a locus for dominantly inherited, progressive deafness to chromosome XX and reported the DFNA27 locus on chromosome 4q (Peters et al., 2008). However, until recently we were unable to identify the variant responsible for DFNA27 deafness. The variant responsible for DFNA27 deafness is a single nucleotide change in an intron upstream of a novel micro-exon of REST, which encodes a transcription repressor. A wild type micro-exon introduces a translation stop codon in all three reading frames of REST and thus inactivates the repressor function of REST. In hair cells REST repressor must be inactive or hair cells die quickly after birth. In collaboration with Botond Banfi's laboratory at the University of Iowa, we reported in CELL (174: 536-548, July 2018) that the DFNA27 intronic variant introduces an upstream acceptor splice site for the micro-exon of REST, which circumvents the inactivation of REST repressor function. A mouse model demonstrates that functional REST repressor kills hair cells resulting in deafness. REST repressor function requires histone deacetylase (HDAC) as part of itse repressor complex. HDAC inhibitors rescues hair cells and hearing in a mouse that is deleted for the micro-exon, a potential treatment for subjects with this particular form of hereditary deafness. We do not yet know the extent to which DFNA27 is associated with the more common form of late onset progressive deafness. 2. 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. The protein substrates of CDC14A are not known. A Y2H screen 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. 3. 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. 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. The individual function of TRIOBP-5 is not known. We have engineered mice that do not express functional TRIOBP-5 and they are deaf but develop rootlets. However, the rootlets are dysmorphic, stereocilia are floppy and the reticular lamina is less rigid due to the apical loss of TRIOBP-5 in supporting cells surrounding hair cells. A manuscript is in preparation by Drs. Belyantseva, Frolenkov and Kitajiri. 4. The LMG is ascertaining 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. We are also engineering mouse models of the human genes responsible for Perrault syndrome in order to understand their function in the auditory system. 5. In 2014, we reported that Variants of TBC1D25 are associated with nonsyndromic deafness DFNB86. Variants of TBC1D24 have also been associated with sezures, seizures and deafness and DOORS syndrome. 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 is correlated with inclusion of a perfectly conserved alternatively spliced micro-exon of Tbc1d24. MRI defects in the hippocampus of Tbc1d24 mutant mice have been documented by the NIH MIF. A manuscript is being prepared by Dr. Risa Tona. 6. 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. These Usher subjects have known biallelic molecular genetic variants determined by analyses conducted by staff in the LMG. As part of this project, several Usher-like subjects appear not to have pathogenic variants in the reported Usher genes. Whole exome sequencing (WES) is being undertaken in the LMG to identify novel Usher genes or possible novel regulatory variants of the reported Usher syndrome genes.
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