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      Steroid 11-Beta-Hydroxylase Deficiency Caused by Compound Heterozygosity for a Novel Mutation, p.G314R, in One CYP11B1 Allele, and a Chimeric CYP11B2/CYP11B1 in the Other Allele

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          Aims: Steroid 11β-hydroxylase deficiency (11β-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 11β-OHD, we performed detailed studies of CYP11B1 in a newly described patient diagnosed with the classical signs of 11β-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 11β-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 11β-hydroxylase. The chimeric CYP11B2/CYP11B1 protein retained 11β-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.

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          Most cited references 27

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          Comparison of DNA sequences with protein sequences.

          The FASTA package of sequence comparison programs has been expanded to include FASTX and FASTY, which compare a DNA sequence to a protein sequence database, translating the DNA sequence in three frames and aligning the translated DNA sequence to each sequence in the protein database, allowing gaps and frameshifts. Also new are TFASTX and TFASTY, which compare a protein sequence to a DNA sequence database, translating each sequence in the DNA database in six frames and scoring alignments with gaps and frameshifts. FASTX and TFASTX allow only frameshifts between codons, while FASTY and TFASTY allow substitutions or frameshifts within a codon. We examined the performance of FASTX and FASTY using different gap-opening, gap-extension, frameshift, and nucleotide substitution penalties. In general, FASTX and FASTY perform equivalently when query sequences contain 0-10% errors. We also evaluated the statistical estimates reported by FASTX and FASTY. These estimates are quite accurate, except when an out-of-frame translation produces a low-complexity protein sequence. We used FASTX to scan the Mycoplasma genitalium, Haemophilus influenzae, and Methanococcus jannaschii genomes for unidentified or misidentified protein-coding genes. We found at least 9 new protein-coding genes in the three genomes and at least 35 genes with potentially incorrect boundaries.
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            A chimaeric 11 beta-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension.

            Glucocorticoid-remediable aldosteronism (GRA), an autosomal dominant disorder, is characterized by hypertension with variable hyperaldosteronism and by high levels of the abnormal adrenal steroids 18-oxocortisol and 18-hydroxycortisol, which are all under control of adrenocorticotropic hormone and suppressible by glucocorticoids. These abnormalities could result from ectopic expression of aldosterone synthase, which is normally expressed only in adrenal glomerulosa, in the adrenal fasciculata. Genes encoding aldosterone synthase and steroid 11 beta-hydroxylase (expressed in both adrenal fasciculata and glomerulosa), which are 95% identical and lie on chromosome 8q (refs 7, 10), are therefore candidate genes for GRA. Here we demonstrate complete linkage of GRA in a large kindred to a gene duplication arising from unequal crossing over, fusing the 5' regulatory region of 11 beta-hydroxylase to the coding sequences of aldosterone synthase (maximum lod score 5.23 for complete linkage, odds ratio of 170,000:1). This mutation can account for all the physiological abnormalities of GRA. Our result represents the demonstration of a mutation causing hypertension in otherwise phenotypically normal animals or humans.
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              High-resolution crystal structure of cytochrome P450cam.

              The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A. While the 1.63 A model confirms our initial analysis based on the 2.6 A model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.

                Author and article information

                Horm Res Paediatr
                Hormone Research in Paediatrics
                S. Karger AG
                August 2005
                10 August 2005
                : 63
                : 6
                : 284-293
                aDepartment of Molecular Genetics, Kochi University Medical School, Nankoku, Kochi, Japan; bDepartment of Pediatrics, Leiden University Medical Center, Leiden, and cDepartment of Endocrinology, University Hospital Groningen, Groningen, The Netherlands
                87074 Horm Res 2005;63:284–293
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 1, References: 40, Pages: 10
                Case Report


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