Normal hearing function genetics: have you heard all about it? An integrated approach of genome-wide association studies and transcriptome-wide association studies in three Italian cohorts

Abstract

Introduction: Deepening the genetic mechanisms underlying Normal Hearing Function (NHF) has proven challenging, despite extensive efforts through Genome-Wide Association Studies (GWAS).

Methods: NHF was described as a set of nine quantitative traits (i.e., hearing thresholds at 0.25, 0.5, 1, 2, 4, and 8 kHz, and three pure-tone averages of thresholds at low, medium, and high frequencies). For each trait, GWAS analyses were performed on the Moli-sani cohort (n = 1,209); then, replication analyses were conducted on Carlantino (CAR, n = 261) and Val Borbera (VBI, n = 425) cohorts. Expression levels of the most significantly associated genes were assessed employing single-nucleus RNA sequencing data (snRNA-seq) on human fetal and adult inner ear tissues. Finally, for all nine NHF traits, Transcriptome-Wide Association Studies (TWAS) were performed, combining GWAS summary statistics and pre-computed gene expression weights in 12 brain tissues.

Results: GWAS on the Discovery cohort allowed the detection of 667 SNPs spanning 327 protein coding genes at a p < 10^-5, across the nine NHF traits. Two loci with a p < 5 × 10^-8 were replicated: 1. rs112501869 within SLC1A6 gene, encoding a brain high-affinity glutamate transporter, reached p = 6.21 × 10^-9 in the 0.25 kHz trait. 2. rs73519456 within ASTN2 gene, encoding the Astrotactin protein 2, reached genome-wide significance in three NHF traits: 0.5 kHz (p = 1.86 × 10^-8), PTAL (p = 9.40 × 10^-9), and PTAM (p = 3.64 × 10^-8). SnRNA-seq data analyses revealed a peculiar expression of the ASTN2 gene in the neuronal and dark cells populations, while for SLC1A6 no significant expression was detected. TWAS analyses detected that the ARF4-AS1 gene (eQTL: rs1584327) was statistically significant (p = 4.49 × 10^-6) in the hippocampal tissue for the 0.25 kHz trait.

Conclusion: This study took advantage of three Italian cohorts, deeply characterized from a genetic and audiological point of view. Bioinformatics and biostatistics analyses allowed the identification of three novel candidate genes, namely, SLC1A6, ASTN2, and ARF4-AS1. Functional studies and replication in larger and independent cohorts will be essential to confirm the biological role of these genes in regulating hearing function; however, these results confirm GWAS and TWAS as powerful methods for novel gene discovery, thus paving the way for a deeper understanding of the entangled genetic landscape underlying the auditory system.

Keywords: ARF4-AS1; ASTN2; GWAS; Normal Hearing Function; SLC1A6.

FIGURE 1

Study workflow. This image displays the main steps of this study. Specifically, GWAS analysis, one for each NHF trait, have been performed on the Discovery cohort (Moli-sani). All the association signals with a p < 1 × 10⁻⁵ have been considered for the replication analysis on CAR and VBI cohorts, and then combined. The expression of the most significantly associated genes across the NHF analyzed traits was validated employing human fetal and adult snRNA-seq data (van der Valk et al., 2023). GWAS Meta-analyses summary statistics were then integrated with brain eQTL data in a TWAS analysis. CAR, Carlantino; VBI, Val Borbera.

FIGURE 2

Violin plots showing expression levels of genes prioritized in the GWAS Meta-analyses. The violin plots report the expression levels. (a) SLC1A6, (b) ASTN2 genes in human inner ear tissues, extracted from snRNA-seq data (van der Valk et al., 2023). In the x-axis inner ear cell types are detailed, and in the y-axis, expression values are reported.

FIGURE 3

Manhattan plot of TWAS results at 0.25 kHz in Brain Hippocampus tissue. This Manhattan plot displays the Z-score (y-axis) for the TWAS on the 0.25 kHz trait and the Brain Hippocampus tissue panel. The blue horizontal lines represent the Bonferroni-corrected significance threshold. Significant genes were highlighted with their gene symbol.