Next-generation sequencing identified SPATC1L as a possible candidate gene for both early-onset and age-related hearing loss

Abstract

Hereditary hearing loss (HHL) and age-related hearing loss (ARHL) are two major sensory diseases affecting millions of people worldwide. Despite many efforts, additional HHL-genes and ARHL genetic risk factors still need to be identified. To fill this gap a large genomic screening based on next-generation sequencing technologies was performed. Whole exome sequencing in a 3-generation Italian HHL family and targeted re-sequencing in 464 ARHL patients were performed. We detected three variants in SPATC1L: a nonsense allele in an HHL family and a frameshift insertion and a missense variation in two unrelated ARHL patients. In silico molecular modelling of all variants suggested a significant impact on the structural stability of the protein itself, likely leading to deleterious effects and resulting in truncated isoforms. After demonstrating Spatc1l expression in mice inner ear, in vitro functional experiments were performed confirming the results of the molecular modelling studies. Finally, a candidate-gene population-based statistical study in cohorts from Caucasus and Central Asia revealed a statistically significant association of SPATC1L with normal hearing function at low and medium hearing frequencies. Overall, the amount of different genetic data presented here (variants with early-onset and late-onset hearing loss in addition to genetic association with normal hearing function), together with relevant functional evidence, likely suggest a role of SPATC1L in hearing function and loss.

Fig. 1

Pedigree and clinical features of the family and of the two ARHL patients. a Pedigree and audiograms of the Italian family carrying a stop variant in SPATC1L. Filled symbols represent affected individuals. b Audiograms of patients Arhl_1 and Arhl_2: the downward slope indicates that high frequencies are severely affected

Fig. 2

Sequence analysis and molecular modelling of the proteins. a Sequence alignment of human Speriolin-like protein (SPC1L_HUMAN, UniProt ID: Q9H0A9) with mouse Speriolin (SPERI_MOUSE, UniProt ID:Q148B6). b Molecular model of the human speriolin-like protein with potential functional regions. c Average radius of gyration of the four systems. d–g Representative structures of the wild type, c.846C>G (p.(Tyr282*)), c.340_343dupTTCA (p.(Lys115Ilefs*12)) and c.656A>C (p.(Tyr219Ser)) respectively, from molecular dynamics simulations. Here, the locations of the variants are represented as red sticks

Fig. 3

SPATC1L mRNA and protein levels in Hek293 transfected cells and Spatc1l expression in mouse whole cochlea and other tissues at different time points. a qRT-PCR analysis of relative mRNA expression of SPATC1L wild type and mutants after 48 h of transfection in Hek293 cells. Results are expressed as a fold-change of expression levels, and are normalized to the relative amount of the internal standard Neo. Error bars indicate 95% confidence intervals. b Western blot analysis of SPATC1L wild type and mutant proteins. Hsp90 was applied to determine equal loading. c The graph shows expression of Spatc1l in mouse whole cochlea at P3, P8, P12 and 2 months. Results are reported as fold change in gene expression over β actin, used as an internal control. The gene shows an age-related expression. d The graph shows Spatc1l expression at 2 months of age in different mouse tissues, including liver, cochlea, spleen, lung, kidney, brain, testis and heart. Results are reported as fold change in gene expression over β actin, used as an internal control