Multiplex Minisequencing of the 21-Hydroxylase Gene as a Rapid Strategy to Confirm Congenital Adrenal Hyperplasia

Background: Congenital adrenal hyperplasia (CAH) is a frequent autosomal recessive disease, with a wide range of clinical manifestations, most commonly attributable to mutations in the 21-hydroxylase gene ( CYP21 ). Large gene deletions, large gene conversions, a small 8-basepair deletion, and eight point mutations in CYP21 account for ∼95% of all enzyme deficiencies. We developed a new strategy for a rapid CYP21 analysis. Methods: DNA samples from 40 CAH patients previously genotyped by direct DNA sequencing were reanalyzed by allele-specific amplification of the functional CYP21 gene followed by a multiplex minisequencing reaction using 13 primers. In addition, a second PCR that amplified a part of exon 3 was used to demonstrate the presence or absence of at least one functional gene. Results: The assay detected the P453S mutation and nine of the most common mutations (P30L, intron 2 splice, Δ8bp, I172N, exon 6 cluster, V281L, F306+t, Q318X, and R356W) caused by microconversions from the CYP21P pseudogene. The concordance was 100% for detecting these mutations, including gene deletions and large gene conversions. The 40 patient DNA samples were analyzed in 1.5 working days by one technician (actual hands-on time, 3.5 h). The material cost for analyzing one sample was approximately €10.00 (US $9.00). Conclusions: This novel mutation screening strategy rapidly detects 90–95% of all mutations associated with CAH and appears applicable as a tool for confirmation of increased 17-hydroxyprogesterone found in neonatal CAH screening.