Interpretation of Test Results

The theoretical sensitivity of GJB2 mutation screening based solely on AS-PCR detection of the 35delG mutation (defined as the subportion of the population with GJB2-related deafness and the 35delG mutation divided by the total population with GJB2-related deafness) has been calculated at 96.9% (range, 95.4% to 98.0%).2 Calculated specificity (defined as the subportion of the population with GJB2-unrelated deafness not coincidentally carrying the 35delG mutation divided by the population with GJB2-unrelated deafness) is equally high at 97.4% (range, 97.0% to 98.0%). The observed sensitivity and specificity of 94% and 97%, respectively, are comparable to these values.2

These calculations assume that the population is randomly mating with respect to GJB2. The existence of population substructure, particularly endogamous subpopulations, results in a decreased proportion of heterozygotes (Wahlund's effect), with an overestimation of sensitivity for the population as a whole. Other assumptions made in these calculations include complete pene-trance, lack of ascertainment bias (i.e., equal referral rates regardless of genotype), and negligible heterozygote selection advantage, spontaneous mutation rate, and migration effects. Deviation of the actual population from Hardy-Weinberg equilibrium due to these factors is likely to be minimal and does not affect the order of magnitude of the figures obtained, with the possible exception of assortative mating among the deaf.2

This mutation screening strategy of 35 delG mutation testing, however, misses GJB2-related deafness caused by non-35delG allele variants. More comprehensive mutation screens are based on DHPLC or direct sequencing. We have tested the sensitivity of DHPLC by screening a panel of 55 individuals carrying 52 combinations of 48 distinct GJB2 sequence variants. Amplicons were analyzed at 62°C, 60°C, and 58°C to increase DHPLC detection efficiency, since all mutations cannot be detected at 62°C (L90P and I230T are not detected at 62°C). DHPLC wave profiles were analyzed for differences in shape and retention time compared to wild-type samples in four separate runs to test detection repeatability. On this basis, we have found DHPLC more sensitive at detecting GJB2 allele variants than SSCP analy-sis,the comparative detection rates being 98.1% and 82.3%, respectively. Other authors have reported the sensitivity of DHPLC to range from 95% to 100%.30-33

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