Background
Over half of all cases of early childhood hearing loss are due to genetic causes; and mutations in the GJB2 gene, encoding the connexin 26 protein, represent the largest proportion of these. A large deletion of 342,000 bases, on chromosome 13, has been found to cause hearing loss. This deletion removes most of the GJB6 gene that encodes the connexin 30 protein (Cx30). Because the deletion is also near the GJB2 gene, which encodes the connexin 26 protein (Cx26), some believe the hearing loss is caused by deleting regulatory elements that control expression of the neighboring GJB2 gene. Regardless of the reason, the deletion, when present in two copies or when combined with a single connexin 26 mutation, causes hearing loss.

As such, all children born with hearing loss, of any severity, are candidates for the Connexin Test. This is true even if there is no family history of hearing loss, as this is the most common presentation. This test alone gives parents the highest likelihood of obtaining a cause for their child's hearing loss. Furthermore, a positive test result can alleviate anxiety associated with the worry that other medical problems may arise in their child due to the association of hearing loss with many other syndromes (e.g. Usher syndrome in which blindness can develop at a later age).

Gene	Name	OMIM#	Locus	Other Conditions
GJB2	Gap junction protein, beta 2 (Connexin 26)	121011	13q12	Autosomal Dominant NSHL, Palmoplantar Keratoderma, KID, Vohwinkel
GJB6	Gap junction protein, beta 6 (Connexin 30), lambda-crystal	604418	13q12	Autosomal Dominant NSHL, Hidrotic ectodermal dysplasia

Epidemiology: 1/2500-1/5000

• Both males and females are affected in equal frequency.
• Certain mutations are found with higher frequency in certain populations (e.g. 35delG in the Caucasian population, 167delT in the Ashkenazi Jewish population and 235delC in the Asian population).
• A higher incidence of the GJB6-D13S1830 deletion has been found in individuals of Spanish, French and Ashkenazi Jewish descent (del Castillo, et al. 2003).

Clinical Features

• Nonsyndromic sensorineural hearing loss, typically congenital but can present in early childhood.
• Mild to profound hearing loss.
• Progressive or non-progressive hearing loss.

Inheritance Pattern: Autosomal recessive

• Typically, each parent is unaffected but is a carrier of a single mutation in the GJB2 gene.
• On average, 25% of the couple's children are expected to inherit both copies of the mutation and be affected.

Test Indications

• Congenital/early childhood mild-to-profound sensorineural hearing loss.

Test Outcome

• The presence of two GJB2 pathogenic variants, two GJB6 deletions, or one GJB6 deletion and one GJB2 pathogenic variant, is considered a positive test.
• The discovery of one mutation in an affected individual is difficult to interpret. Some of these individuals may have hearing loss due to another cause, and be coincidental carriers of the mutation. Others may have a second mutation that has not been detected by the test.

Turn-Around-Times: 3 weeks

Methodology
The first step in testing GJB2 involves screening for the 35delG mutation. If this screen is negative or heterozygous, exon 1 and the coding region of exon 2 of GJB2 are amplified using flanking primer sets. PCR products are sequenced using an ABI fluorescence automatic DNA sequencer. These tests do not detect large deletions or mutations in non-coding regions that could potentially affect the expression of the GJB2 gene.

PCR (polymerase chain reaction) is performed to detect the presence or absence of the GJB6-D13S1830 deletion. This test does not detect point mutations or other deletions in the GJB6 gene.

Analytical and Clinical Sensitivity
This assay is greater than 99.9% accurate in detecting mutations in the GJB2 sequence analyzed. Two GJB2 mutations are detected in approximately 20% of patients with nonsyndromic sensorineural hearing loss. However, a negative result does not rule out a diagnosis of GJB2 hearing loss because there may be other mutations within the GJB2 gene (promoter or other non-coding or regulatory regions) that are not detected by this test. The GJB6-D13S1830 deletion may be present in 2-67% of persons with a single GJB2 mutation, the exact percentage depending on ethnicity. The presence of two copies of the deletion is rare.

Cost and CPT codes:

Connexin Test: GJB2 Sequencing and GJB6-D13S1830 Deletion:

• Cost: $400
• CPT codes: 83894(1), 83898(4), 83904(3), 83909(1), 83912(1)

Testing for known familial mutation:

• Cost: $400
• CPT codes: 83891(1), 83894(1), 83898(1), 83904(1), 83909(1), 83912(1)

If you have any questions, please call the Laboratory for Molecular Medicine at 617-768-8500 or email us at LMM@partners.org

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