Biological insights from multi-omic analysis of 31 genomic risk loci for adult hearing difficulty

Abstract

Age-related hearing impairment (ARHI), one of the most common medical conditions, is strongly heritable, yet its genetic causes remain largely unknown. We conducted a meta-analysis of GWAS summary statistics from multiple hearing-related traits in the UK Biobank (n = up to 330,759) and identified 31 genome-wide significant risk loci for self-reported hearing difficulty (p < 5x10-8), of which eight have not been reported previously in the peer-reviewed literature. We investigated the regulatory and cell specific expression for these loci by generating mRNA-seq, ATAC-seq, and single-cell RNA-seq from cells in the mouse cochlea. Risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. Regions of the human genome homologous to open chromatin in epithelial cells from the mouse were strongly enriched for heritable risk for hearing difficulty, even after adjusting for baseline effects of evolutionary conservation and cell-type non-specific regulatory regions. Epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. Of these, 39 were expressed robustly in mouse cochlea and 16 were enriched specifically in sensory hair cells. These results reveal new risk loci and risk genes for hearing difficulty and suggest an important role for altered gene regulation in the cochlear sensory epithelium.

Fig 1. Genome-wide association studies of hearing-related traits in the UK Biobank.

a. Heritability of top 9 hearing-related traits in the UK Biobank. b. Genetic correlations among the four most significantly heritable hearing-related traits and between these traits and 14 non-hearing traits that are significantly correlated with the hearing traits. c. Manhattan plot for genetic associations with hearing difficulty in the UK Biobank, following meta-analysis across the four hearing-related traits.

Fig 2. Heritable risk for hearing difficulty is enriched near genes expressed in the cochlea.

Black vertical lines indicate the -log10(p-value) for the enrichment of hearing difficulty risk near genes expressed in each cochlear cell type. Gray vertical lines indicate -log10(p-value) for genes expressed in each of 5,674 non-cochlear cell types. Labels are provided for significantly enriched cell types (p-value < 1e-4). The density plot represents the frequency distribution of p-values from non-cochlear cell types, computed using the density() function in R.

Fig 3. Heritable risk for hearing difficulty is enriched at open chromatin regions in cochlear epithelial cells.

a. Fluorescence-activated cell sorting (FACS) of cochlear cells. Cochlear cells were labeled with a CD326 antibody conjugated to Allophycocyanin (APC), and sorted two ways as CD326 (+) and CD326 (-). b. Overlap of open chromatin regions identified by ATAC-seq of epithelial vs. non-epithelial cells in the mouse cochlea. c-e. Open chromatin peaks near cell type-specific marker genes: Epcam (b), Pou3f4 (b), and Sox2 (c). f. -log10(p-value) for enrichment of hearing difficulty risk in regions of the human genome homologous to open chromatin in epithelial and non-epithelial cells from mouse cochlea (black vertical lines) and in non-cochlear cell types from ENCODE (gray lines) https://umgear.org/p?l=3a70e6e7.

Fig 4. Epigenomic fine-mapping predicts distal target genes for hearing difficulty risk loci.

Genetic associations and epigenomic annotations at chr3q26.3 (a) and chr1q23.3 (b). From top to bottom, genome browser tracks indicate: -log10(p-values) for association with hearing difficulty; fine-mapped SNPs in strong LD with an LD-independent lead SNP and located <500bp from a region homologous to a cochlear open chromatin region based on ATAC-seq; -log10(p-values) for chromatin interactions between the locations of the fine-mapped SNPs and distal regions, based on the minimum chromatin interaction p-value in each 40kb region from Hi-C of 20 non-cochlear human tissues and cell types[44]; locations of UCSC knownGene gene models.

Fig 5. Single-cell RNA-seq of mouse cochlea reveals cell type-specific expression patterns of hearing difficulty risk genes.

a. t-distributed stochastic neighbor embedding (t-SNE) plot of 3,411 cells in the postnatal day 2 mouse cochlea colored by Louvain modularity clusters corresponding to 12 cell types. b-e. t-SNE plots colored by the expression of selected hearing difficulty risk genes expressed selectively in cochlear cell types: LMX1A in a subset of epithelial Oc90 cells (b); ARHGEF28 in hair cells (c), SOX2 in supporting cells (d), and BAIAP2L2 in hair cells (e). f. Dot plot showing the average expression and percent of cells with non-zero counts for each cochlea-expressed risk gene in each of the 12 cochlear cell types.