Molecular and Epigenetic Mechanisms of Hereditary Hearing Loss

National Institute On Deafness And Other Communication Disorders

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Abstract

GRHL2 A frameshift mutation in the gene encoding the transcription factor grainyhead-like 2 (GRHL2) was identified as the cause of progressive deafness (DFNA28) segregating in a large pedigree (Peters et al., 2002). We have made the equivalent mutation in mouse (Grhl2-TMinsC/+) to model the cochlear pathology. GRHL2 is necessary for proper development of numerous epithelial tissues. Mice that are homozygous for inactivating alleles of Grhl2 die during embryogenesis, but heterozygous mice develop normally, including having normal cochlear development and initially normal hearing. However, we have discovered that the Grhl2-TMinsC mutation affects age-related hearing loss (AHL) in this mouse model, but the degree and rate of progression is influenced by the mouse strain background. Similarly, the mutation affects susceptibility to noise-induced hearing loss (NIHL), but in strain-specific ways. A goal of our present study is to understand the function of the GRHL2 transcription factor in the auditory system. Finally, we have used shRNA to knock-down specific alternative splice isoforms of GRHL2 and characterized the consequent gene-expression differences in cultured kidney cells. By comparing the intersecting genes identified in these different experiments, we are identifying the GRHL2-regulated pathways that are disrupted by the DFNA28 mutation. A paper describing this work is in preparation. HGF Previously we identified three noncoding mutations of HGF associated with deafness in several families segregating nonsyndromic hearing loss (DFNB39) (Schultz et al., 2009). HGF encodes the multifunctional cytokine hepatocyte growth factor (HGF), which is secreted by mesenchymal cells and recognized by the tyrosine kinase receptor MET, expressed by epithelial cells. HGF is critical for cell signaling, cell motility, growth, and development. There are multiple isoforms of HGF whose expression, we hypothesize, are influenced by the DFNB39 noncoding mutations of HGF. We have created a mouse model which recapitulates a 10-bp intronic deletion found in some human DFNB39 patients. The 10 base pair deletion is located in intron 5 and founder lines retain a neomycin selection cassette, also located in intron 5 (Hgf tmdel10Neo). A comprehensive phenotypic assessment shows no obvious defects in Hgf tmdel10Neo/del10Neo mice other than cochlear abnormalities. Hearing loss was confirmed by ABR and DPOAE analyses. Hgf tmdel10Neo/del10Neo mice show severe-to-profound hearing loss, while heterozygotes Hgftmdel10Neo/+ show normal hearing. The hearing phenotype is changed when the Neomycin cassette is removed by crossing to a ubiquitously expressing Cre-recombinase line (Hgf tmdel10-). Hgftmdel10-/del10- mice show only mild-to-moderate hearing loss. Moreover, unlike their Hgf tmdel10-/+ and Hgf +/+ littermates, Hgf tmdel10-/del10- mice are resistant to noise-induced hearing loss. A full elucidiation of the HGF isoforms, and their relative abundances, produced from Hgf tmdel10Neo and Hgf tmdel10- alleles will further our understanding of the essential functions of HGF in the human and mouse auditory system. A paper describing this work was published in the Morell et al. (2020) Journal of Neuroscience. Further work will concentrate on the regulation and expression of Hgf isoforms, specifically in the inner ear. IsoSeq We conducted IsoSeq of whole cochlea, flow-sorted cochlear hair cells, and isolated single cells from the inner ear. We compared different methods of cDNA generation, library preparation, sequencing, and bioinformatic analyses in order to optimize a procedure for cataloging the isoform content of the mouse inner ear. In total we have generated 573,989 unique isoforms from 20,253 known genes and 16,619 novel genes, for an average 15 isoforms per gene. Of the transcripts, 446,038 (77%) are novel when compared to the GENCODE vM23 reference annotation. The vast majority of unique isoforms differ in 5 and 3 UTR exon usage when compared to the reference, and may harbor important regulatory motifs specific to the inner ear. Our data set also includes 21,103 protein coding exons (CDS) that have no overlap with any CDS in the reference transcriptome. Most interestingly, 268 of these inner ear specific CDS exons are in genes known to cause hearing loss in humans (DFNA/B loci). A paper describing these results is in preparation.