Identification of a novel splicing mutation and 1-bp deletion in the 17alpha-hydroxylase gene of Japanese patients with 17alpha-hydroxylase deficiency.
We report studies of two unrelated Japanese patients with 17alpha-hydroxylase deficiency caused by mutations of the 17alpha-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 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 downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient's mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17alpha-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.
Yamaguchi
H
H
Third Department of Internal Medicine, Miyazaki Medical College, Kiyotake, Japan.
Nakazato
M
M
Miyazato
M
M
Toshimori
H
H
Oki
S
S
Shimizu
K
K
Suiko
M
M
Kangawa
K
K
Matsukura
S
S
D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital; D000328:Adult; D004252:DNA Mutational Analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D007564:Japan; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D012326:RNA Splicing / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*
D013254:Steroid 17-alpha-Hydroxylase