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Meta-Analysis
. 2015 Oct 1;24(19):5655-64.
doi: 10.1093/hmg/ddv279. Epub 2015 Jul 17.

Genome-wide association analysis on normal hearing function identifies PCDH20 and SLC28A3 as candidates for hearing function and loss

Dragana Vuckovic  1 , Sally Dawson  2 , Deborah I Scheffer  3 , Taina Rantanen  4 , Anna Morgan  1 , Mariateresa Di Stazio  1 , Diego Vozzi  5 , Teresa Nutile  6 , Maria P Concas  7 , Ginevra Biino  8 , Lisa Nolan  2 , Aileen Bahl  9 , Anu Loukola  9 , Anne Viljanen  4 , Adrian Davis  2 , Marina Ciullo  6 , David P Corey  3 , Mario Pirastu  7 , Paolo Gasparini  10 , Giorgia Girotto  11
Affiliations
Meta-Analysis

Genome-wide association analysis on normal hearing function identifies PCDH20 and SLC28A3 as candidates for hearing function and loss

Dragana Vuckovic et al. Hum Mol Genet. .

Abstract

Hearing loss and individual differences in normal hearing both have a substantial genetic basis. Although many new genes contributing to deafness have been identified, very little is known about genes/variants modulating the normal range of hearing ability. To fill this gap, we performed a two-stage meta-analysis on hearing thresholds (tested at 0.25, 0.5, 1, 2, 4, 8 kHz) and on pure-tone averages (low-, medium- and high-frequency thresholds grouped) in several isolated populations from Italy and Central Asia (total N = 2636). Here, we detected two genome-wide significant loci close to PCDH20 and SLC28A3 (top hits: rs78043697, P = 4.71E-10 and rs7032430, P = 2.39E-09, respectively). For both loci, we sought replication in two independent cohorts: B58C from the UK (N = 5892) and FITSA from Finland (N = 270). Both loci were successfully replicated at a nominal level of significance (P < 0.05). In order to confirm our quantitative findings, we carried out RT-PCR and reported RNA-Seq data, which showed that both genes are expressed in mouse inner ear, especially in hair cells, further suggesting them as good candidates for modulatory genes in the auditory system. Sequencing data revealed no functional variants in the coding region of PCDH20 or SLC28A3, suggesting that variation in regulatory sequences may affect expression. Overall, these results contribute to a better understanding of the complex mechanisms underlying human hearing function.

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Figures

Figure 1.
Figure 1.
Locus-zoom plot for chromosome 13 locus. The plot shows the locus on chromosome 13 associated with normal hearing functions measured at 2 kHz, highlighting the top SNP rs78043697 and several other significant SNPs in the same region being in high linkage disequilibrium. The results plotted were obtained with a two-stage meta-analysis, focusing on Italian isolates first and then including the communities from the Silk Road. The y-axis shows −log10 of the P-values obtained from the complete meta-analysis of the 2 kHz hearing threshold. The closest gene PCDH20 is shown along the x-axis.
Figure 2.
Figure 2.
Boxplot for rs78043697. The figure displays boxplots for the 2 kHz frequency trait divided by genotype groups for rs78043697 and adjusted by sex, age and relatedness in our largest cohort (INGI-FVG). As the effect for this SNP was observed under a dominant model, the heterozygote genotype TC and the homozygote CC are shown together.
Figure 3.
Figure 3.
Forest plot for rs78043697. The figure shows the forest plot for the top SNP associated with 2 kHz threshold on chromosome 13 after performing the two-stage meta-analysis. The x-axis represents effect sizes (beta coefficients) for trait residuals after sex, age and kinship adjustments. The strongest association can be observed in the INGI-FVG cohort.
Figure 4.
Figure 4.
Boxplot for rs7032430. The figure displays boxplots for the 500 Hz frequency divided by genotype groups for rs7032430 and adjusted by sex, age and relatedness in our largest cohort (INGI-FVG). As shown in the figure, people carrying the AA genotype have higher values and hence worse hearing function.
Figure 5.
Figure 5.
Locus-zoom plot for chromosome 9 locus. The plot shows the locus on chromosome 9 associated with normal hearing function measured at the 500 Hz as well as the genes within the region, including SLC28A3, the strongest candidate for hearing function. The results plotted were obtained with a two-stage meta-analysis, focusing on Italian isolates first and then including the communities from the Silk Road. The y-axis shows −log10 of the P-values obtained from the complete meta-analysis of the 500-Hz hearing threshold.
Figure 6.
Figure 6.
(A) qReal-time analysis of the selected genes (PCDH20, SLC28A3). (B) Semi-quantitative RT-PCR. The plot shows expression for Pcdh20 and Slc28a3, compared with Myo7a and Myo6 (A). The y-axis represents a scale whose unit is the expression of the reference gene Myo7a. Pcdh20 displays a strong expression, whereas Slc28a3 shows lower levels. Semi-quantitative RT-PCR demonstrates that both genes are expressed (B).
Figure 7.
Figure 7.
RNA-seq expression profiles for PCDH20 (A) and SLC28A3 (B). Histograms display the normalized number of reads in hair cells (green) and surrounding cells (purple), in samples from the cochlea (C; dark colors) and utricle (U; light colors) at E16, P0, P4 and P7. The fold change (FC) representing the GFP+/GFP− counts ratio and the multiple test adjusted false discovery rate by the Benjamini–Hochberg procedure are indicated.
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References

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