Direct molecular diagnosis of CYP2 1 mutations in congenital adrenal hyperplasia

Abstract

Abstract The majority of congenital adrenal hy-perplasia (CAH) cases arise from mut-ations in the steroid 21-hydroxylase (CYP21) gene. Without reliance on HLA gene linkage analysis, we have developed primers for differential polymerase chain reaction (PCR) amplification of the CYP21 gene and the non-functional CYP21P gene. Using the amplification created restriction site (ACRS) approach for direct muta-tional detection, a secondary PCR was then performed using a panel of primers specific for each of the 11 known mutations associated with CAn. Subsequent re-striction analysis allowed not only the de-tection but also the determination of the zygosity of the mutations analysed. Ex-isting deletion of the CYP21 gene could also be detected. In the analysis of 20 in-dependent chromosomes in 11 families of CAH patients in Taiwan, four CYP21 mutation types, besides deletion, were de-tected. Interestingly, in five different al-ides, the CYP21P pseudogene contained some polymorphisms generally associated with the CYP21 gene. These results suggest gene conversion events that are occurring in both CYP21P and CYP21 genes. Our combined differential PCR-ACRS pro-tocol is simple and direct and is applicable for prenatal diagnosis of CAH using chor-ionic villi or amniotic cells.