Molecular Genetics Connexin 26 (GJB2) Sequencing and Connexin 30 (GJB6) DeletionGJB2/DFNB1)

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.

Sequencing: Next generation sequencing will analyze the exons or coding regions of the GJB2 gene using Illumina NextSeq 500 technology.  Samples are prepared using hybridization probes to enrich exonic regions.  Promoter, intronic, etc. regions are not assessed on our assay, but may contain variants that impact gene function.

Connexin 30 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.

Blood: EDTA (purple-top tube) or ACD (yellow-top tube)

• Adult:5mL
• Child:5mL
• Infant:2-3mL

Saliva: 2 ORAgene Saliva Kit(s) (OGR-500)
Skin Fibroblast: Punch Biopsy, or 2 T-25 confluent flasks
Prenatal:

• Direct Amniotic Fluid (10-20mL)
• Direct CVS
• Cultured Amnio or CVS (2-T25 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)

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.
• All genetic testing performed on Direct CVS or Direct Amniotic Fluid specimens will be confirmed on cell cultures prepared by Knight Diagnostic Laboratories.Cell cultures will be prepared from the specimen received.Additional charges apply for confirmatory testing.

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.

Weekly

14-21 Days

• 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.

1. Richard et al. Connexin gene pathology. Clin. Exp. Dermatol. 28:397-409 (2003).
2. 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).
3. 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
4. GeneReviews: Smith RJH and Camp GV (http://www.ncbi.nlm.nih.gov/books/NBK1272/#dfnb1.Molecular_Genetics)
5. Rodriguez-Paris J and Schrijver I., 2009 Biochem Biophys Res Commun.;389(2):354-9

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.