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

Frontiers in Genetics, vol. 16, pp. 1522338

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.

Authors 20

1. Aurora Santin

   University of Trieste · IRCCS Materno Infantile Burlo Garofolo

   Affiliation as printed

   Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy

   Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

2. Giulia Pianigiani corresponding

   IRCCS Materno Infantile Burlo Garofolo

   Affiliation as printed

   Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

3. Alessandro Gialluisi

   Istituto Neurologico Mediterraneo · LUM Jean Monnet University

   Affiliation as printed

   Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

   Department of Medicine and Surgery, LUM University, Casamassima, Italy

4. Alessandro Pecori

   IRCCS Materno Infantile Burlo Garofolo

   Affiliation as printed

   Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

5. Beatrice Spedicati

   University of Trieste · IRCCS Materno Infantile Burlo Garofolo

   Affiliation as printed

   Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy

   Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

6. Simona Costanzo

   Istituto Neurologico Mediterraneo

   Affiliation as printed

   Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

7. Mariarosaria Persichillo

   Istituto Neurologico Mediterraneo

   Affiliation as printed

   Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

8. Francesca Bracone

   Istituto Neurologico Mediterraneo

   Affiliation as printed

   Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

9. Giuseppe Giovanni Nardone

   University of Trieste

   Affiliation as printed

   Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy

10. Paola Tesolin

    IRCCS Materno Infantile Burlo Garofolo

    Affiliation as printed

    Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

11. Stefania Lenarduzzi

    IRCCS Materno Infantile Burlo Garofolo

    Affiliation as printed

    Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

12. Anna Morgan

    IRCCS Materno Infantile Burlo Garofolo

    Affiliation as printed

    Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

13. Amalia De Curtis

    Istituto Neurologico Mediterraneo

    Affiliation as printed

    Neuromed Biobanking Centre, Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

14. Wouter H. van der Valk Aachen Exobiology Leiden Department of Otorhinolaryngology and Head & Neck Surgery Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW)

    Leiden University Medical Center · Novo Nordisk Foundation Center for Stem Cell Medicine

    Affiliation as printed

    Exobiology Leiden, Department of Otorhinolaryngology and Head and Neck Surgery, Leiden University Medical Center, Leiden, Netherlands

    The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden, Netherlands

15. Francis Rousset

    University of Geneva

    Affiliation as printed

    The Inner Ear and Olfaction Lab, Department of Clinical Neurosciences, Faculty of Medicine, University of Geneva, Geneva, Switzerland

16. Marta Roccio

    University of Zurich · University Hospital Zurich

    Affiliation as printed

    Inner Ear Stem Cell Laboratory, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Zurich (USZ), Zurich, Switzerland

    University of Zurich (UZH), Zurich, Switzerland

17. Heiko Locher Aachen Exobiology Leiden Department of Otorhinolaryngology and Head & Neck Surgery Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW)

    Leiden University Medical Center · Novo Nordisk Foundation Center for Stem Cell Medicine

    Affiliation as printed

    Exobiology Leiden, Department of Otorhinolaryngology and Head and Neck Surgery, Leiden University Medical Center, Leiden, Netherlands

    The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden, Netherlands

18. Licia Iacoviello

    Istituto Neurologico Mediterraneo · LUM Jean Monnet University

    Affiliation as printed

    Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, Italy

    Department of Medicine and Surgery, LUM University, Casamassima, Italy

19. Maria Pina Concas

    IRCCS Materno Infantile Burlo Garofolo

    Affiliation as printed

    Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy

20. Giorgia Girotto

    University of Trieste · IRCCS Materno Infantile Burlo Garofolo

    Affiliation as printed

    Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy

    Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy