Background:
DFNB1 is an autosomal recessive disorder characterized by congenital non-progressive hearing loss that is moderate to profound. No other systemic findings are associated with DFNB1, thus it is designated a non-syndromic hereditary hearing loss. Approximately 98% of cases of DFNB1 have been shown to be caused by mutations in the GJB2 gene (connexin 26). GJB2 encodes the gap junction protein, connexin 26, located on chromosome 13q12.1. Incidence of DFNB1 is estimated to be 1-2 in 10,000 live births. Mutations in connexin 26 have also been shown to cause the autosomal dominant nonsyndromic hearing loss DFNA3, as well as keratitis-ichthyosis-deafness syndrome, palmoplantar keratoderma with deafness, and hystrix-like ichthyosis-deafness syndrome (Richard, 2003).
A 342-kb deletion in (GJB6), which encodes the gap-junction protein, connexin 30 (Cx30) has also been associated with DFNB1 (del Castillo I et al., 2002). The frequency of this variant, D13S1830 in the US population is 1.6% to 4% and it is considered the most common variant in GJB6 to be linked to DFNB1 (GeneReviews: Smith RJH and Camp GV). The pathogenic mechanism involving this deletion in GJB6 is attributed to the loss of cis-acting regulatory elements that affect the expression of GJB2 rather than a digenic mechanism of inheritance (Rodriguez-Paris J and Schrijver I., 2009). No other variant in GJB6 is tested.
Reason for Referral:
Non-syndromic congenital non-progressive hearing loss that is moderate to profound.
Methodology:
Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs <50 bp, the analytical sensitivity is >87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.
Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmental duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.
This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.
Connexin 30 (GJB6) assay: The testing for connexin 30/GJB6 deletion, del (GJB6-D13S1830), is done by PCR-based amplification using primers that are specific for the proximal and distal breakpoints. The presence or absence of PCR product is used to detect the variant. (Del Castillo et al., 2002, Wu et al. 2003)
Test reporting follows the American College of Medical Genetics (ACMG) guidelines.
Specimen Requirements:
Blood: EDTA (purple-top tube) or ACD (yellow-top tube)
- Adult:5mL
- Child:5mL
- Infant:2-3mL
Saliva: 2 ORAgene™ Saliva Collection Kits (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Assisted Saliva: 4 ORAgene™ Assisted Saliva Collection Kits (OGR-575) used according to manufacturer instructions. Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.
Skin Fibroblast: Punch Biopsy (Cell cultures will be prepared at KDL and used for testing), or 2 T-25 confluent flasks.
DNA: 5-10µg at a minimum of 50-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).
Prenatal:
- Direct Amniotic Fluid (10-20mL)
- Direct CVS
- Direct POC
- Cultured Amniocytes (2-T25 flasks)
- Cultured CVS (2-T25 flasks)
- Cultured Fetal Tissue: Product of Conception (2-T25 flasks)
- Cord Blood (1-2mL)
Notice Regarding Molecular Genetic Testing on Prenatal Specimens:Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood (or saliva) in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.
For routine testing of blood and saliva (or DNA extracted from them), KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed within the above windows, please contact our lab.
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including ethnicity, clinical history, and family history.
Test Performed (Days):
Weekly
Turn Around Time:
14-21 Days
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold, but not frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
References:
- Richard et al. Connexin gene pathology. Clin. Exp. Dermatol. 28:397-409 (2003).
- Wu et al. Use of a multiplex PCR/sequencing strategy to detect both connexin 30 (GJB6) 342 kb deletion and connexin 26 (GJB2) mutations in cases of childhood deafness. Am. J. Med. Genet. 121A:102-108 (2003).
- Del Castillo et al. A Deletion Involving the Connexin 30 Gene in Nonsyndromic Hearing Impairment. N Engl J Med 2002; 346:243-249 January 24, 2002
- GeneReviews: Smith RJH and Camp GV (http://www.ncbi.nlm.nih.gov/books/NBK1272/#dfnb1.Molecular_Genetics)
- Rodriguez-Paris J and Schrijver I., 2009 Biochem Biophys Res Commun.;389(2):354-9
Additional Info:
Prior to any genetic testing we recommend genetic counseling. To receive forms and information about prenatal diagnostic testing, please contact Client Services at (855) 535-1522.