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:
Sanger Sequencing: Sequencing is carried out by amplification of all exons and intron/exon boundaries followed by bi-directional Sanger sequencing. All nucleotide changes are analyzed within the context of current databases and literature to predict pathogenicity.
Alternatively, NGS could be performed if Sanger sequencing is unavailable. Next generation sequencing (NGS) will analyze the exons or coding regions of the genes using Illumina NextSeq 500/550 technology and preparing samples using hybridization probes to enrich exonic regions. This assay does not assess regions of insufficient coverage, introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and regions of high GC or polynucleotide repeats, but may contain variants that impact gene function.
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: 1-2µg 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 CVS or Amniotic Fluid Specimens:
- Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.
For routine testing of blood, saliva and buccal swabs, 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 outside of 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.