OtoGenome™ Test for Hearing Loss and Related Syndromes (110 Genes) Details
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Background
Hearing loss has an incidence of 1 in 250 births and over half of isolated childhood hearing loss has a genetic etiology, with many genes involved. Gene variants can be inherited in an autosomal recessive, autosomal dominant, X-linked, or mitochondrial pattern. The comprehensive approach of the OtoGenome™ Test now makes it possible to simultaneously screen 110 genes known to cause both nonsyndromic hearing loss and syndromes that can present as isolated hearing loss, such as Usher, Pendred, Jervell and Lange-Nielsen (JLNS), Branchio-Oto-Renal (BOR), Deafness and Male Infertility (DIS), Perrault, Alport, and Waardenburg syndromes.
The OtoGenome™ Test includes genes associated with:
- Nonsyndromic hearing loss: 84 Genes (see Gene Information)
- Usher syndrome (hearing loss and retinitis pigmentosa): CDH23, CLRN1, ADGRV1, MYO7A, PCDH15, USH1C, USH1G, USH2A, CIB2, HARS, WHRN
- Pendred syndrome/Hearing loss with EVA or Mondini dysplasia: SLC26A4 (PDS)
- Branchio-oto-renal syndrome: EYA1, SIX1
- Waardenburg syndrome: EDN3, EDNRB, MITF, PAX3, SNAI2, SOX10
- Auditory neuropathy/dys-synchrony: OTOF, DFNB59
- Maternally-inherited/Aminoglycoside-induced hearing loss: MTTS1 (tRNAser(UCN)), MTRNR1 (12S rRNA)
- Jervell and Lange-Nielsen syndrome: KCNQ1, KCNE1
- Perrault syndrome: CLPP, HARS2, HSD17B4, LARS2
- Alport syndrome: COL4A5, COL4A3, COL4A4
- Muckle Wells syndrome (Cryopyrin-associated periodic syndrome (CAPS)): NLRP3
- Brown-Vialetto-Van Laere syndrome: SLC52A3, SLC52A2
- CHARGE syndrome: CHD7
- Hearing loss with cone-rode dystrophy: CEP78
- Hearing loss with high myopia: SLITRK6
Determining the etiology of hearing loss is important in determining prognosis (e.g. whether the severity of hearing impairment will worsen), optimal therapeutic interventions (e.g. hearing aids, cochlear implant, sign language) and recurrence risks to future children and other family members. Furthermore, a positive result from this test may predict the onset of syndromic features (e.g. adolescent-onset vision impairment in Usher syndrome, long QT in JLNS, renal abnormalities in BOR, or thyroid abnormalities in Pendred syndrome). On the other hand, a negative result substantially reduces the likelihood that a syndrome is present, alleviating concerns about the future development of these features. Among individuals with sensorineural hearing loss up to 5% have hearing loss associated with Pendred syndrome and up to 10% have Usher syndrome.
LessGene Information
Read More for expanded gene table.
Testing Strategy
The OtoGenome Test is best suited for individuals who have a diagnosis of hearing loss for which an underlying etiology has not yet been identified. For an individual with apparently non-syndromic hearing loss, this panel covers both non-syndromic causes of hearing loss as well as those which can present as non-syndromic. Onset of features associated with a syndromic type of hearing loss can be delayed or variable. Given the genetic heterogeneity of hearing loss, the OtoGenome Test allows for a shortened diagnostic course by analyzing 110 genes in a single test.
Testing may be considered in a reflexive manner, with the Connexin Test (GJB2 sequencing and GJB6 deletion analysis) performed first, and if negative or inconclusive the OtoGenome Test is performed.
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, DFNA5, DFNB59, 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, HARS, HARS2, HGF (excludes exon 12 in NM_000601.4), HSD17B4, ILDR1, KARS, KCNE1, KCNQ1, KCNQ4 (excludes exon 1 in NM_004700.3), KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT (excludes exons 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, 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).
Read More...This assay is performed using the PerkinElmer Sciclone® G3 Workstation combined with the Agilent SureSelect Clinical Research Exome capture kit (#G9496A 5190-7344; targeting coding regions (exons) and canonical splice sites) followed by sequencing on the Illumina NextSeq 550 (High-Output v2 kit). Reads are aligned to the GRCh37 reference sequence using the Burrows-Wheeler Aligner (BWA 0.7.17), and variant calls are made using the Genomic Analysis Tool Kit (GATK v4.0.3.0). Detection of copy number variants (CNVs) encompassing 2 or more exons is performed using next-generation sequencing read data and the VisCap algorithm. CNV analysis is only performed when data meets necessary quality standards and may not be available for all cases. Variant calls are limited bioinformatically to the associated region of interest for the assay (see above for details). Sanger sequencing is used for fill in when bases have <12X coverage.
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 GRCh37 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 <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 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.
This test is 99.93% sensitive (95% CI =99.92-99.94%) to detect variants changing a single base and 96.75% sensitive to detect insertion/deletions (95% CI =96.28-97.22%) within covered regions. Technical positive predictive value for single nucleotide variant changes is 99.42% (95% CI = 99.37-99.48%) and 94.16% (95% CI = 93.34-94.97%) for insertion/deletion changes within covered regions.
LessLIMITATIONS
Copy number variant (CNV) analysis using VisCap algorithm for the full gene panel may not be available for all cases. The CNV analysis used with this test cannot reliably detect copy number variants of less than 2 contiguous exons or mosaicism. Balanced chromosome rearrangements will not be identified. Identification of exon-level copy number variants is dependent on qualities of the targeted regions, including shared homology, exon size.
Copy number variants that do not affect the GJB6 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.
Read More...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 larger 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.
LessAnalytical and Clinical Sensitivity
This test is 99.93% sensitive (95% CI =99.92-99.94%) to detect variants changing a single base and 96.75% sensitive to detect insertion/deletions (95% CI =96.28-97.22%) within covered regions. Technical positive predictive value for single nucleotide variant changes is 99.42% (95% CI = 99.37-99.48%) and 94.16% (95% CI = 93.34-94.97%) for insertion/deletion changes within covered regions.