A missense mutation of Ile¹⁷² → Asn or Arg³⁵⁶ → Trp causes steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common recessive genetic disease caused mainly by steroid 21-hydroxylase (P450c21) deficiency. Many forms of CAH exist resulting from various mutations of the CYP21B gene. We sequenced CYP21B cDNA from a normal person and its genes from a patient with simple virilizing CAH. When comparing several CYP21B sequences, we found it was polymorphic. In the patient, a single base substitution replaced Ile¹⁷² (ATC) with Asn (AAC) in one allele while Arg³⁵⁶ (CGG) was converted to Trp (TGG) in the other. A normal P450c21 cDNA clone was transfected into COS-1 cells to produce 21-hydroxylase activity toward its substrates, progesterone and 17-hydroxyprogesterone. Mutants corresponding to Asn¹⁷² or Trp³⁵⁶ mutation were constructed by site-directed mutagenesis of the normal c21 cDNA clone. They failed to produce active enzyme toward either substrate upon transfection into COS-1 cells, demonstrating that these mutations caused CAH. Aligning sequences with other P450s, Ile¹⁷² could be located in the membrane anchoring domain and Arg³⁵⁶ in the substrate-binding site of P450c21. Both mutations are present in the CYP21A1P pseudogene, suggesting that they may be transferred from CYP21A1P by gene conversion events.