Rare-variant association analysis reveals known and new age-related hearing loss genes

Abstract

Age-related (AR) hearing loss (HL) is a prevalent sensory deficit in the elderly population. Several studies showed that common variants increase ARHL susceptibility. Here, we demonstrate that rare-variants play a crucial role in ARHL etiology. We analyzed exome and imputed data from white-European UK Biobank volunteers, performing both single-variant and rare-variant aggregate association analyses using self-reported ARHL phenotypes. We identified and replicated associations between ARHL and rare-variants in KLHDC7B, PDCD6, MYO6, SYNJ2, and TECTA. PUS7L and EYA4 also revealed rare-variant associations with ARHL. EYA4, MYO6, and TECTA are all known to underline Mendelian nonsyndromic HL. PDCD6, a new HL gene, plays an important role in apoptosis and has widespread inner ear expression, particularly in the inner hair cells. An unreplicated common variant association was previously observed for KHLDC7B, here we demonstrate that rare-variants in this gene also play a role in ARHL etiology. Additionally, the first replicated association between SYNJ2 and ARHL was detected. Analysis of common variants revealed several previously reported, i.e., ARHGEF28, and new, i.e., PIK3R3, ARHL associations, as well as ones we replicate here for the first time, i.e., BAIAP2L2, CRIP3, KLHDC7B, MAST2, and SLC22A7. It was also observed that the odds ratios for rare-variant ARHL associations, were higher than those for common variants. In conclusion, we demonstrate the vital role rare-variants, including those in Mendelian nonsyndromic HL genes, play in the etiology of ARHL.

Fig. 1. An illustration of samples used in the analysis.

We included in the analysis 416,522 samples after excluding individuals who are not white-European, do not meet the inclusion criteria, or provided inconsistent answers. Blue dashed lines indicates the replication sample which was used for the discovery and mega-analysis. Associations identified in the discovery sample are only replicated using the exome replication sample and not the secondary-replication sample. The replication sample consists of individuals included in UK Biobank Release 1. For replication, permutation is used to estimate empirical p-values adjusting for the number of variants/genes and phenotypes brought to replication. There is no overlap of individuals in the discovery, replication, and secondary-replication samples. All samples were used in the single-variant analysis and only those samples highlighted in light blue were used in the rare-variant aggregate analysis. There was no available replication sample for the mega-analysis of rare-variant aggregated association testing.

Fig. 2. Manhattan plots for the discovery and mega-sample rare-variant aggregate association analysis.

Results for the discovery sample are shown for the analysis of H-aid (A), H-diff (B), H-noise (C), and H-both (D). Results for the mega-sample are shown for the analysis of H-aid (E), H-diff (F), H-noise (G), and H-both (H). For each Manhattan plot, the blue dots display the result for the analysis of predicted loss of function (pLoF) variants and the orange dots for missense and pLoF variants. The threshold for exome-wide significance (p < 2.5 × 10⁻⁶) is indicated by a red dotted line. Genes that reached exome-wide significance are annotated.

Fig. 3. Expression of PDCD6/Pdcd6 in the human and mouse inner ear.

Pdcd6 expression levels (normalized counts) from RNA sequencing of hair cells (GFP+) and surrounding cells (GFP-) from the cochleae and utricles of mice at four developmental stages: E16, P0, P4, and P7. Pdcd6 is expressed in cochlea and utricle cells over all four developmental stages (A). Pdcd6 expression levels (top panel: red = high expression, yellow = low expression) and location in the cochlear floor epithelium at P7 developmental stage (bottom panel: colors indicate the cell type). The scale bar represents gene expression based on log transformed and normalized expression data. Abbreviations for bottom panel: BM basilar membrane cells, DC dieter cells, Hensen Hensen cells, IHC inner hair cells, IPC inner pillar cells, ISC inner sulcus cells, LGER lateral great epithelial ridge cells, MGER medial greater epithelial ridge cells, MLGER medial lateral greater epithelial ridge cells, OHC outer hair cells, OPC outer pillar cells, OS outer sulcus cells, Glia glial cells (B). RNA expression of Pdcd6 obtained from microarray data based on perfect match and mismatch probe differences (PM/MM) in spiral ganglion neurons and vestibular ganglion neurons from mice at six developmental stages: E12, E13, E16, P0, P06, and P15 (C). PDCD6 expression in the adult human inner ear. PDCD6 expression data (normalized counts) were obtained from RNA sequencing of adult human inner ear tissues (D).