Gene duplications in 21-hydroxylase deficiency: the importance of accurate molecular diagnosis in carrier detection and prenatal diagnosis

BACKGROUND: The detection of 21-OH deficiency (21OHD) carriers in the general population requires that misinterpretations of apparently severe mutations in alleles carrying duplicated genes be avoided. Prenatal treatment prevents virilization in female fetuses and genetic counseling may be offered to couples in which one partner is either a patient or a carrier. This paper proposes a semiquantitative PCR method involving primer extension that distinguishes the severe point mutation Q318X in single gene copy alleles from the normal/nondeficient variant in gene-duplicated alleles. SAMPLES AND METHODS: DNA from 65 individuals carrying Q318X variants, that of 85 partners of 21OHD carriers or patients, and one fetal sample (as well as the DNA of his family) were analyzed. 21OHD alleles were studied by gene-specific PCR/allele-specific oligonucleotides hybridization for common mutations, Southern analysis, complementary direct sequencing and microsatellite typing. Primer extension analysis of the Q318X variants using fluorescent dideoxynucleotides was performed on CYP21A2 gene-specific PCR-amplified DNA samples from controls, patients, potential carriers and prenatal samples. RESULTS: Different fluorescence patterns were seen for the severe mutation (single gene copy) and the nondeficient (gene-duplicated) alleles carrying Q318X. The normal/mutant fluorescence peak (N/M) ratio was < 1 in all heterozygous carriers (mean 0.83; min. 0.70; max. 0.95). In all normal individuals carrying the gene-duplicated Q318X normal variant, the N/M ratio was > 1 (mean 1.69; min. 1.44; max. 2.02). CONCLUSION: The proposed method discriminated between the severe Q318X mutation and the normal Q318X variant in gene duplication, and could be a useful complementary tool in prenatal diagnosis and carrier detection.