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2.
Horm Res ; 63(6): 284-93, 2005.
Article in English | MEDLINE | ID: mdl-16024935

ABSTRACT

AIMS: Steroid 11beta-hydroxylase deficiency (11beta-OHD) is the second most common (5-8%) cause of congenital adrenal hyperplasia (CAH), and results from homozygous or compound heterozygous mutations or deletions of the responsible gene CYP11B1. In order to better understand the molecular basis causing 11beta-OHD, we performed detailed studies of CYP11B1 in a newly described patient diagnosed with the classical signs of 11beta-OHD. METHODS: CYP11B1 of the patient was investigated by polymerase chain reaction (PCR), sequencing, restriction fragment length polymorphism (RFLP) analysis, Southern blotting, and transient cell expression. RESULTS: We identified two new mutated alleles in CYP11B1. In one allele CYP11B1 has a g.940G-->C (p.G314R) missense mutation. On the other allele we found a chimeric gene that consists of part of the aldosterone synthase gene (CYP11B2) at exons 1-3 and part of the 11beta-hydroxylase gene (CYP11B1) at exons 4-9. Inin vitro studies, the g.940G-->C (p.G314R) mutation abolished all hydroxylase activity in comparison with the wild-type 11beta-hydroxylase. The chimeric CYP11B2/CYP11B1 protein retained 11beta-hydroxylase enzymatic activity in vitro. CONCLUSION: This case is caused by compound heterozygosity for a nonfunctional missense mutation and a chimeric CYP11B2/CYP11B1 gene with hydroxylase activity that is controlled by the CYP11B2 promoter. The most likely explanation is that the CYP11B2 promoter does not function in the zona fasciculata/reticularis where cortisol is exclusively synthesized.


Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Cytochrome P-450 CYP11B2/genetics , Steroid 11-beta-Hydroxylase/genetics , Adrenal Hyperplasia, Congenital/enzymology , Alleles , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Child, Preschool , Chlorocebus aethiops , Heterozygote , Humans , Male , Molecular Sequence Data , Mutation, Missense , Recombinant Fusion Proteins/genetics , Sequence Alignment , Steroid 11-beta-Hydroxylase/biosynthesis , Zona Fasciculata/enzymology
3.
Endocr Res ; 29(4): 377-81, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14682466

ABSTRACT

11beta-Hydroxylase deficiency (11beta-OHD) inherited in an autosomal recessive manner accounts for about 5-8% of congenital adrenal hyperplasia (CAH). In order to clarify the underlying mechanism causing 11beta-OHD, we have done the molecular genetic analysis on the CYP11B1 gene in a patient diagnosed as 11beta-OHD. The nucleotide sequence of the patient's CYP11B1 revealed a novel nonsense mutation that converts codon 265 CAG (glutamine) to TAG (stop) of exon 4. Restriction fragment length polymorphism (RFLP) data showed that the patient was homozygous for the mutation. The above results confirm that the patient suffers from complete loss of the final step in cortisol biosynthesis pathway because of the nonsense mutation.


Subject(s)
Adrenal Hyperplasia, Congenital/enzymology , Adrenal Hyperplasia, Congenital/genetics , Codon, Nonsense/genetics , Steroid 11-beta-Hydroxylase/genetics , Amino Acid Sequence , Base Sequence , Child , Female , Humans , Hydrocortisone/biosynthesis , Infant, Newborn , Male , Polymorphism, Restriction Fragment Length , Steroid 11-beta-Hydroxylase/chemistry
4.
Horm Res ; 60(5): 255-60, 2003.
Article in English | MEDLINE | ID: mdl-14614232

ABSTRACT

OBJECTIVES: To clarify the underlying molecular mechanism of corticosterone methyl oxidase type II (CMO II) deficiency, Japanese patients newly diagnosed with CMO II deficiency were investigated. METHODS: We analyzed the patients' genomic DNA sequence on all 9 exons of the CYP11B2 gene. In addition, restriction fragment length polymorphism (RFLP) analysis and expression studies were performed. RESULTS: The analysis showed that the patients homozygously retained a missense mutation, Gumacr;GC[435Gly]-->Aumacr;GC[Ser], in the CYP11B2 gene. Expression studies indicated that the steroid 18-hydroxylase/oxidase activities of the mutant enzyme were substantially reduced. CONCLUSION: These results support the hypothesis that this mutation causes CMO II deficiency in the patients, and are in accordance with our theory that the partial loss of P-450(C18) activities causes CMO II deficiency.


Subject(s)
Cytochrome P-450 CYP11B2/genetics , Hypoaldosteronism/congenital , Hypoaldosteronism/genetics , Mutation, Missense/genetics , Amino Acid Substitution , Asian People , Base Sequence , Cytochrome P-450 CYP11B2/deficiency , Cytochrome P-450 CYP11B2/metabolism , Exons/genetics , Humans , Hypoaldosteronism/enzymology , Infant , Infant, Newborn , Male , Pedigree , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA
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