IDENTIFICATION OF A NOVEL SPLICING MUTATION AND 1-BP DELETION IN THE 17-ALPHA-HYDROXYLASE GENE OF JAPANESE PATIENTS WITH 17-ALPHA-HYDROXYLASE DEFICIENCY

We report studies of two unrelated Japanese patients with 17 alpha-hyd roxylase deficiency caused by mutations of the 17 alpha-hydroxylase (C YP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and deter mined their nucleotide sequences. Patient 1 had novel, compound hetero zygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intro n 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp dow nstream from the exon 1-3 boundary. The other allele had a missense mu tation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mut ations abolished the 17 alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismat ch oligonucleotide showed that the patient's mother and brother carrie d the splice-site mutation, but not the missense mutation. Patient 2 w as homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exo n 2. This 1-bp deletion produces a frameshift in translation and intro duces a premature stop codon (TAG) proximal to the highly conserved he me iron-binding cysteine at codon 442 in microsomal cytochrome P450 st eroid 17 alpha-hydroxylase (P450c17). RFLP analysis showed that the mo ther was heterozygous for the mutation.