Comprehensive DFNB1 and STRC Panel

Background

The Comprehensive DFNB1 and STRC Panel assesses for the presence of variants in the GJB2 (DFNB1 locus) and the STRC genes, two of the major contributors to autosomal recessive nonsyndromic hearing loss. Both sequencing and the most common copy number variants in these genes are identified, including the contiguous gene deletion of STRC and CATSPER2, associated with Deafness and Male Infertility syndrome.

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 that have been reported across these genes.

Pathogenic variants impacting the GJB2 gene (DFNB1 locus) are the most common cause of autosomal recessive nonsyndromic hearing loss, accounting for as much as 50% of cases (Smith 1998 PMID: 20301449). The hearing loss is often congenital and can range from mild to profound. Progression has been documented in a significant proportion of individuals (Kenna 2010 PMID: 20083784, Chan 2010 PMID: 20154630). The pathogenic variant spectrum at the DFNB1 locus includes sequence variants in the GJB2 gene and upstream large deletions which have been shown to directly decrease expression of GJB2. Several previously reported pathogenic deletions in this region, including the common GJB6-D13S1830 and GJB6-D13S1854 that truncate the upstream GJB6 gene are detected by this test. However, sequence variants in the GJB6 gene have not been associated with nonsyndromic hearing loss and are not included.

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.

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Gene Information

*Long range PCR discriminates against pseudogene locus

Gene/locus	Sequencing (NGS)	Copy Number (ddPCR)
GJB2	X
DFNB1 deletions (including GJB6)		X
STRC	X*	X
CATSPER2		X

DFNB1 deletions detected by assay

Deletion	Genomic coordinates (GRCh37/hg19)	Size	Reference
del(GJB6-D13S1830)	Chr13: g.(20,797,177_21,105,945)del	309 kb	del Castillo et al. (2002)
del(GJB6-D13S1854)	Chr13:g.(20,802,727_21,034,768)del	232 kb	del Castillo et al. (2002)
del(131-kb)	Chr13:g.(20,939,344_21,070,698)del	131 kb	Wilch et al. (2010)
del(179-kb)	Chr13:g.(20,921,711_21,101,115)del	179 kb	Tayoun et al. (2016)
del(101-kb)	Chr13:g.(20,757,021_20,858,394)del	101 kb	Bliznetz et al. (2014)

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Testing Strategy

This panel can be utilized as a first-tier test to identify the two most common causes of autosomal recessive sensorineural hearing loss: DFNB1 (GJB2 and GJB6 genes) and the STRC gene. The test includes deletion analysis of the CATSPER2 gene to identify the contiguous gene deletions of STRC and CATSPER2, which is associated with autosomal recessive hearing loss and male infertility. The phenotypic spectrum of DFNB1 ranges from mild to profound, and the hearing loss associated with STRC is typically mild to moderate in severity. Therefore, it is particularly useful for individuals with apparently nonsyndromic mild to moderate sensorineural hearing loss.

Methodology

This test is comprehensive for identifying pathogenic variants in the DFNB1 locus (GJB2 gene) and the STRC gene. It includes sequencing of the coding regions and splice sites of exons 1 and 2 of GJB2 (DFNB1), and long-range sequencing of the entire coding region of STRC. In addition, it detects common deletions in both DFNB1 and STRC, including the contiguous gene deletion of STRC and CATSPER2 associated with Deafness and Male Infertility.

Deletion analysis is performed via droplet digital PCR. It is designed to detect previously reported large deletions in the DFNB1 region (Abou Tayoun PMID: 26444186) including D13S1830 (309kb), D1321854 (232 kb), and the deletions reported by Wilch 2010 (PMID: 20236118) and Feldman 2009 (PMID: 19101659). This test is also designed to identify whole gene deletions of STRC and the contiguous gene deletion of STRC and CATSPER2. Small single exon and multi exon deletions of STRC and CATSPER2 will not be identified unless they overlap with the probed regions.

The sequencing assay is performed by amplifying exons 1 and 2 of the GJB2 (NM_004004.5) gene and 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.

Droplet digital PCR (ddPCR) for the detection of DFNB1 deletions is performed using a probe at GRCh37 chr13:21003868-21003954 to test for the presence or absence of the previously reported deletions in the DFNB1 (GJB6 gene) region, including the GJB6-D13S1854 309kb deletion, the GJB6-D13S1854 232kb deletion, and the deletions reported by Wilch 2010 (PMID: 20236118) and Feldman 2009 (PMID: 19101659). Any deletions that are identified are further clarified using the ddPCR probes at the following locations: GRCh37 chr13:21089281-21089358, chr13:21055271-21055336, chr13:20794699-20794765, chr13:20805056-20805133.

Droplet digital PCR (ddPCR) is performed to screen for common large deletions in STRC and CATSPER2 genes. It is performed using a probe at STRC intron 25 (GRCh37chr15:43892948-43893040) to test for the presence of a deletion in STRC. Any deletions that are identified are further clarified using ddPCR probes at the following locations: STRC exon 23 (GRCh37chr15:43895449-43895542) and CATSPER2 exon 7 (GRCh37chr15:43931196-43931260). Deletions that do not affect the STRC intron 25 (GRCh37chr15:43892948-43893040) region will not be identified.

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LIMITATIONS

Copy number variants that do not encompass this test's DFNB1 deletions target probe (GRCh37 chr13:21003868-21003954) or the STRC intron 25 (GRCh37chr15:43892948-43893040)probed regions 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 GJB6 or CATSPER2 genes. It does not include deletion analysis of the GJB2 coding region. It will not detect variants in non-coding regions, aside from exon 1 of GJB2, the canonical splice sites of GJB2 and STRC, and the GJB6 deletions described above. 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.

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Analytical and Clinical Sensitivity

Gene	# Truth Variants	True Positives	False Negatives	False Positives	Sensitivity (TP/TP+FN)	Sensitivity 95% CI	PPV (TP/TP+FP)	PPV 95% CI
STRC	156	156	0	0	100.00%	97.6-100%	100.00%	97.6-100%
GJB2	44	44	0	0	100.00%	91.9-100%	100.00%	91.9-100%
Total	200	200	0	0	100.00%	98.1-100%	100.00%	98.1-100%

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LMM Tests

Background

Gene Information

Testing Strategy

Methodology

LIMITATIONS

Analytical and Clinical Sensitivity

Hearing Loss and Related Syndromes Gene Association

Questions? Contact Us.

Understanding the Genetics of Deafness

Common Causes of Hearing Loss

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