Background

Pathogenic copy number variants in STRC are estimated to cause between 1-5% of hearing loss (Vona 2015 PMID: 26011646, Yokota 2019 PMID: 30867468, Francey 2012 PMID: 22147502). The STRC gene is associated with autosomal recessive hearing loss that is typically mild to moderate. The pathogenic variant spectrum includes sequence variants, large deletions of the STRC gene, and contiguous gene deletions that also encompass the CATSPER2 gene. The contiguous gene deletion of STRC  and CATSPER2 is responsible for autosomal recessive Deafness and Male Infertility syndrome. The STRC gene is highly homologous to a pseudogene (99.6% of coding regions and 98.9% including introns), thus requiring long range sequencing and appropriately targeted deletion assays to accurately detect variants across the entire gene (Mandelker 2014 PMID: 25157971). The common large deletions have an estimated carrier frequency of up to 2.5% in the different populations (Vona 2015, Yokota 2019), suggesting that STRC related hearing loss may be underreported, in part due to technical limitations in detecting STRC variants in regions with high pseudogene homology. This panel addresses this limitation through a specialized assay that has been validated to detect variants across all exons of STRC and eliminate pseudogene contamination.

This test combines customized workflows on two different platforms (NGS and ddPCR) that address the analytical and technical challenges associated with capturing the broad spectrum of disease-causing variants affecting STRC.

Gene Information

*Long Range PCR discriminates against pseudogene locus

Testing Strategy

This test is appropriate for patients with hearing loss who have a heterozygous deletion involving STRC and require full gene sequencing. It also may be an appropriate test for patients who require confirmation of a STRC or STRC and CATSPER2 deletion identified on microarray.

Methodology

This test is comprehensive for identifying pathogenic variants in the STRC gene. It includes long-range sequencing of the entire coding region (exons 1-29) ofSTRC(NM_153700.2). In addition, it detects common whole gene deletions of  STRC and the contiguous gene deletion of STRC and CATSPER2 associated with Deafness and Male Infertility.

This assay is performed by amplifying exons 1-29 of the STRC (NM_153700.2) gene loci using long range polymerase chain reaction. Primers proven to discriminate between STRC and its known pseudogene locus are used. The PCR products are then subjected to next generation sequencing library preparation using seqWell’s plexWell™ kit. Samples are pooled and sequenced on the Illumina NextSeq 550 Mid Output Kit (150 bp paired end reads). Reads are aligned to the GRCh37 reference sequence limited to the GJB2 and STRC gene loci only using the Burrows-Wheeler Aligner (BWA 0.7.17), and variant calls are made using the Genomic Analysis Tool Kit – HaplotypeCaller (GATK v4.0.3.0). Sanger sequencing is used to fill in when bases have <200x coverage. All clinically significant variants are confirmed by Sanger sequencing or droplet digital PCR; variants classified as likely benign or benign are not confirmed.

LIMITATIONS

Copy number variants that do not affect the  STRC  intron 25 (GRCh37chr15:43892948-43893040)probed region will not be detected by this assay.CATSPER2  deletions will only be reported if an  STRC  deletion was also identified. Duplications in  STRC  and/or  CATSPER2  will not be reported because exact breakpoints cannot be determined by this testing methodology and duplications in these genes are not known to be associated with hearing loss and/or male infertility.

This test does not include sequencing of the CATSPER2 genes. It will not detect variants in non-coding regions, aside the canonical splice sites STRC. There is reduced sensitivity for larger indels and variants in low complexity regions. It will not detect triplet repeat expansions, translocations, inversions, gene-pseudogene conversions, or other complex rearrangements. Low level mosaic variants may not be identified.

This test was developed, and its performance characteristics determined by the Laboratory for Molecular Medicine at Partners HealthCare Personalized Medicine (LMM, 65 Landsdowne St, Cambridge, MA 02139; 617-768-8500; CLIA#22D1005307). It has not been cleared or approved by the U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary

Analytical and Clinical Sensitivity