Genome-wide SNP genotyping identifies the Stereocilin (STRC) gene as a major contributor to pediatric bilateral sensorineural hearing impairment

Abstract

Hearing loss is the most prevalent sensory perception deficit in humans, affecting 1/500 newborns, can be syndromic or nonsyndromic and is genetically heterogeneous. Nearly 80% of inherited nonsyndromic bilateral sensorineural hearing loss (NBSNHI) is autosomal recessive. Although many causal genes have been identified, most are minor contributors, except for GJB2, which accounts for nearly 50% of all recessive cases of severe to profound congenital NBSNHI in some populations. More than 60% of children with a NBSNHI do not have an identifiable genetic cause. To identify genetic contributors, we genotyped 659 GJB2 mutation negative pediatric probands with NBSNHI and assayed for copy number variants (CNVs). After identifying 8 mild-moderate NBSNHI probands with a Chr15q15.3 deletion encompassing the Stereocilin (STRC) gene amongst this cohort, sequencing of STRC was undertaken in these probands as well as 50 probands and 14 siblings with mild-moderate NBSNHI and 40 probands with moderately severe-profound NBSNHI who were GJB2 mutation negative. The existence of a STRC pseudogene that is 99.6% homologous to the STRC coding region has made the sequencing interpretation complicated. We identified 7/50 probands in the mild-moderate cohort to have biallelic alterations in STRC, not including the 8 previously identified deletions. We also identified 2/40 probands to have biallelic alterations in the moderately severe-profound NBSNHI cohort, notably no large deletions in combination with another variant were found in this cohort. The data suggest that STRC may be a common contributor to NBSNHI among GJB2 mutation negative probands, especially in those with mild to moderate hearing impairment.

Figure 1. SNP genotyping array data of GJB2 mutation negative cohort for Chr15q15.3 with successive Illumina array platform resolution for HLS433P

(A) SNP array data for all probands in the array cohort visually depicted in the UCSC Genome Browser, Build hg18 with a genomic window of 41.6Mb to 41.85Mb on Chr15q15.3. The light red horizontal bars represent the heterozygous deletions and the dark red bars represent the homozygous deletions separated by array platform and calling algorithm used to detect the CNV. Research = PennCNV and Clinical = CNV Workshop. The SNP probes for each Illumina SNP Genotyping array platform are depicted as vertical black bars below the deletion CNVs followed by the UCSC gene track, Database of Genomic Variants track and the Segmental Duplication track from the UCSC Genome Browser, Build hg18. (B–D) Increased probe resolution for the Illumina SNP genotyping platform using HLS433P genotyping data visualized in Beadstudio. (B) Illumina 610k SNP array data filtered to the intersecting markers on the Illumina 550k SNP array that was not called by PennCNV. (C) Illumina 610K SNP array data unfiltered reveals a larger deletion window that was detected by CNV Workshop. (D) Illumina Omni1-Quad SNP array confirms a homozygous deletion and further defines the deletion CNV breakpoints for HLS433P.

Figure 2. Copy number determination using Sanger sequencing in regions of the gene with divergent base pairs

(A) Alignment of STRC and pSTRC for a subset of the exon 24 coding sequence displaying 3/56 divergent base pairs used for copy number determination. The white squares with Ns depict the divergent base pair sequences between the two copies used to determine copy number. (B–C) Chromatograms of control sample with normal copy number by array used as reference file and batch control-baseline STRC specific peak heights for each base pair. (B) Control sample amplified using unique primers for exon 24. (C) Control sample amplified using generic primers for exon 24 and the peak height ratios for STRC to pSTRC as quantified using Mutation Surveyor’s area under the peak function described in methods. (D) Heterozygous deletion by array (HLS461P) has a decrease in STRC sequence to about 50% of control at each of the divergent base pairs. (D) Homozygous deletion by array (HLS433P) has a complete absence of STRC sequence.

Figure 3. The schematic representation of STRC and pSTRC sequence variation and mutational sequencing results

Black boxes represent coding exons with 100% homology between the two gene copies while the green boxes represent exons with at least 1 base pair divergence between the two gene copies in or around the coding region. The individual divergent base pairs (n=56) in or around the coding sequence that were used to determine copy number by Sanger sequencing are represented above the corresponding exons by black circles. White boxes are the untranslated regions and horizontal black lines are the introns. Variants not seen in the homozygous state in controls screened here are listed above the corresponding exons where they were found. The white arrows below the gene graphic represent the various sized deletions that were detected by Sanger sequencing. Defined deletion breakpoints are indicated with a vertical black bar and undefined breakpoints are indicated with an open arrowhead. * Indicates a sequence variant that was not seen in a homozygous or compound heterozygous state and so the contribution of these variants to hearing loss cannot be determined. ^a Indicates a variant that was present in a homozygous or compound heterozygous state in one or more probands but was also seen in one Yoruban control from the 1000 genome database and so its potential pathogenicity cannot be clearly defined at this time.