OtoGenome Test for Hearing Loss (110 Genes)

Methodology

This OtoGenome Panel includes 110 genes: ACTG1, ADCY1 (excludes exon 1 in NM_021116.2), ADGRV1, ALMS1 (excludes exon 1 in NM_015120.4), ATP6V1B1, BCS1L, BSND, CABP2, CACNA1D, CATSPER2 (deletion analysis only), CCDC50, CD164 (excludes exon 7 in NM_001142403.1), CDC14A, CDH23, CEACAM16, CEP78 (excludes exon 12 in NM_001098802.1), CHD7, CIB2, CLDN14, CLIC5, CLPP, CLRN1 (excludes exon 1B in NM_174880.1)*, COCH, COL11A2, COL4A3 (excludes exons 36 and 51 in NM_000091.4), COL4A4 (excludes exon 31 in NM_000092.4), COL4A5, DIABLO, DIAPH1 (excludes exon 16 and intron 23 in NM_005219.4), EDN3, EDNRB, EPS8 (excludes exons 3, 10, 16 and 18in NM_004447.5), ESPN (excludes exons 1, 3, 4, 7 and 8 in NM_031475.2), ESRRB, EYA1, EYA4, GIPC3, GJB2, GJB6, GPSM2, GRHL2, GRXCR1, GSDME, HARS1, HARS2, HGF (excludes exon 12 in NM_000601.4), HSD17B4, ILDR1, KARS1, KCNE1, KCNQ1, KCNQ4 (excludes exon 1 in NM_004700.3), KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT (excludes e xons 3B and 6B in NM_001145307.1* and exon 6A in NM_145309.2)*, MARVELD2, MIR96, MITF, MSRB3, MTRNR1 (excludes m.648-m.950), MTTS1,MYH14 (excludes exon 28 in NM_001145809.1), MYH9, MYO15A (excludes exon 2 in NM_016239.3), MYO3A, MYO6, MYO7A, NLRP3, OSBPL2, OTOA (excludes exons 2 and 21-27 in NM_144672.3), OTOF, OTOG (excludes exon 32 in NM_001277269.1), OTOGL, P2RX2 (excludes exon 1 in NM_174873.1), PAX3, PCDH15, PDZD7, PJVK, POU3F4, POU4F3, PRPS1, RDX, RIPOR2, S1PR2, SERPINB6, SIX1, SLC26A4, SLC52A2, SLC52A3, SLITRK6, SMPX, SNAI2, SOX10, STRC (NM_153700.2), SYNE4, TBC1D24, TECTA, TIMM8A, TMC1, TMIE, TMPRSS3, TPRN, TRIOBP, USH1C, USH1G, USH2A (includes deep intronic c.7595-2144A>G variant), WFS1, WHRN. *Exon from an alternate transcript. For additional information on reference sequences and exon coverage, please visit our website (www.partners.org/personalizedmedicine/lmm). Genome sequence is generated from genomic DNA that is fragmented and barcoded followed by sequencing on the Illumina NovaSeq instrument with a minimum coverage of at least 20X for 95%. Reads are aligned to the NCBI reference sequence (GRCh38), using the Burrows-Wheeler Aligner (BWA), and variant calls are made using the Genomic Analysis Tool Kit (GATK). Technical sensitivity of this assay is 99.10% (95% CI: 99.04-99.16%) and positive predictive value is 99.39% (95% CI: 99.37-99.41%). Sequence analysis of the STRC gene is performed by amplifying exons 1-29 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 GRCh38 reference sequence limited to the 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 less than 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) is performed using a probe at GRCh38 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: GrCh38 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 (GrCh38chr15:43892948-43893040) 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 (GrCh38 chr15:43895449-43895542) and CATSPER2 exon 7 (GrCh38chr15:43931196- 43931260). Deletions that do not affect the STRC intron 25 (GrCh38 chr15:43892948 43893040) region will not be identified. Copy number variants that do not affect the GJB6 GrCh38 chr13:21003868-21003954 or the STRC intron 25 (GrCh38 chr15:43892948-43893040) 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 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. Certain exons are excluded due to homology or other technical limitations (see above for excluded regions). Variant classifications are based on ACMG/AMP criteria (Richards et al. 2015) with ClinGen rule specifications (https://www.clinicalgenome.org/working-groups/sequence-variant-interpretation/). Variants are reported according to HGVS nomenclature (www.hgvs.org/mutnomen). Likely benign and benign variants are not included in this report but are available upon request. This test does not routinely detect variants in non-coding regions (aside from the canonical splice sites), triplet repeat expansions, translocations, inversions, and copy number variants encompassing less than 2 consecutive exons. There is reduced detection for large r indels, variants in low complexity regions, and variants in regions with high homology. 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.

Citations

Not provided

All clinically significant variants are confirmed by Sanger sequencing or an alternate assay.