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R G Sutcliffe
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A J Russell
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C R W Edwards
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A M Wallace
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Understanding of the principal pathways of steroid hormone biosynthesis was established over two decades ago through advances in steroid radioisotopic and chromatographic techniques. When the enzymes of individual pathways could be examined in more detail, the dissection of the complex pattern of enzyme activities began. At many points, separate pathways employ precisely the same enzyme for equivalent catalytic steps, e.g. for 21-hydroxylase, 11 β-hydroxylase, aromatase and several dehydrogenases (Orth et al. 1992). A further economy was found for 17α-hydroxylase and 17,20-lyase activities, which co-purify with the same P450c17 polypeptide. This enzyme was later cloned and expressed in tissue culture cells, revealing that, contrary to the enzyme in rat, human and cattle, 17α-hydroxylase cannot convert 17α-hydroxyprogesterone to androstenedione (Bradshaw et al. 1987, Fevold et al. 1989). Further complexity emerged with the existence of multiple tissue-specific forms of 5α-reductase (Wilson et al. 1993), and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases, most of which

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C S Hawes
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M W McBride
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A Petropoulos
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U W Mueller
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R G Sutcliffe
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ABSTRACT

A new monoclonal antibody (FDO26G) is described which was raised against purified human 3β-hydroxysteroid dehydrogenase type I (3β-HSD type I). FDO26G reacted strongly with villous syncytiotrophoblast, weakly with some trophoblast cells in chorion laeve, and not at all with extravillous trophoblast in cytotrophoblast cell islands and decidual trophoblast. All these types of trophoblast reacted strongly with monoclonal antibody FDO161G, which has previously been shown to react with 3β-HSD type I and, like FDO26G, reacts strongly with adrenal cells.

Mapping experiments using a combination of lacZ fusion polypeptides and synthetic peptides located the FDO26G epitope to residues 354–366 at the C-terminal end of the molecule, a sequence that is identical in the type I and type II forms of the enzyme. The epitope contains a consensus for a casein kinase-II site with serine 359 as the candidate phosphorylation site. This suggested that the lack of reactivity of FDO26G to 3β-HSD in extravillous trophoblast might be due to phosphorylation at serine 359. Peptide 354–366 was synthesized with phosphoserine at residue 359 and its binding to FDO26G was compared with that of the unphosphorylated peptide. FDO26G bound the phosphopeptide at least as strongly as the unphosphorylated peptide. It is concluded that the lack of staining of extravillous trophoblast by FDO26G is due to the presence of a different sequence at residues 354–366 and that a hitherto unidentified third isoform of human 3β-HSD is expressed in these cells.

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A J Russell
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A M Wallace
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M G Forest
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M D C Donaldson
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C R W Edwards
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R G Sutcliffe
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ABSTRACT

A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). Circulating concentrations of Δ5 steroids and Δ5 urinary steroid metabolites were elevated and remained elevated after orchidectomy. There was no evidence of salt loss, plasma renin being within normal limits, and no detectable glucocorticoid abnormality. The coding sequences of the genes for 3β-HSD types I and II were amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis (DGGE) and manual and automatic DNA sequencing. A mutation in the gene for 3β-HSD type II was observed at codon 173 (CTA→CGA), leading in the affected patient to a homozygous substitution in which the leucine at residue 173 was altered to an arginine (L173R). The propositus's 2-year-old XX sister was also homozygous for L173R and showed the biochemical characteristics of partial 3β-HSD deficiency without clinical symptoms or signs. The mutation segregated as an autosomal recessive. Three related heterozygous adult females showed evidence of a small over-production of Δ5 steroids and steroid metabolites and a variable reduction in ovarian function. Concentrations of Δ5 steroids and steroid metabolites in the heterozygous father of the propositus were within the normal range.

These data are discussed in relation to the endocrine causes of pseudohermaphroditism and hirsutism. Evidence for tight linkage between the genes for 3β-HSD types I and II was obtained using a microsatellite polymorphism in the third intron of the gene for 3β-HSD type II and synonymous and non-synonymous mutations and polymorphisms in the gene for 3β-HSD type I. The latter polymorphisms were located 88 bp apart at the 3′ end of the type I coding sequence and could be physically resolved as haplotypes using DGGE. The application of DGGE to the analysis of mutations in members of a multigene family is discussed.

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CJ Kenyon
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M Panarelli
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L Zagato
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L Torielli
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RP Heeley
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CD Holloway
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R Fraser
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G Casari
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RG Sutcliffe
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G Bianchi
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The Milan hypertensive strain of rat (MHS) displays abnormalities in both renal function and adrenocortical activity. While the pressor role of the former has been studied in detail, the role of the latter has not yet been clearly evaluated. In the present study, glucocorticoid receptor (GR) binding characteristics in liver cytosol from adult MHS and Milan normotensive controls (MNS) have been investigated. Dexamethasone, aldosterone and corticosterone were bound with lower affinity to cytosol of MHS rats compared with that of MNS rats. This pattern of binding could explain the raised plasma corticosterone concentrations and adrenocortical hypertrophy previously noted in MHS. The coding sequence of MHS and MNS GR genes have been determined. The MHS gene differed in four respects from that of MNS: three silent point mutations and a polymorphic microsatellite region in exon 2. The latter polymorphism has been used in cosegregation studies of F2 hybrids of MHS x MNS. The MHS GR genotype was associated with hypercalciuria and lower blood pressure in female rats and lower body weight in male rats. Although the effect on blood pressure is small, it is consistent with the affinity data. MHS GR genotype cosegregated with lower blood pressure in F2 rats and displayed a lower affinity in binding studies. In conclusion, GR polymorphism may be responsible for differences of adrenocortical function between MHS and MNS. This may lead to a reduction in the blood pressure difference between the two strains.

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G C Inglis
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C J Kenyon
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C Szpirer
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K Klinga-Levan
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R G Sutcliffe
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J M C Connell
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ABSTRACT

Mouse hepatoma × rat hepatocyte hybrids that segregate rat chromosomes were used to determine the chromosomal location of the rat genes encoding 11 β-hydroxylase and aldosterone synthase (Cyp11b1 and Cyp11b2 respectively). By means of species-specific restriction fragments and microsatellite markers both genes were mapped to rat chromosome 7. The Cyp11b1 microsatellite marker was subsequently found to vary in length between and within rat strains. Furthermore, we compared the sequences of Cyp11b1 markers in two genetically hypertensive strains of rat with their normotensive counterparts. Previous studies have indicated that 11β-hydroxylase activities in Milan and Lyon hypertensive strains are different from their respective genetic controls. The Cyp11b1 microsatellite regions from Lyon hypotensive and normotensive strains of rat were similar and were both shorter by 15 bases than that of the Lyon hypertensive strain. The Cyp11b1 marker in Milan hypertensive (MHS) and normotensive (MNS) strains differ from all the Lyon strains and from each other. The MHS marker is 12 bases shorter than that of MNS rats. These differences in microsatellite length may provide useful polymorphic markers in co-segregation studies of genetic hypertension in rats.

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M W McBride
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A J Russell
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K Vass
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K Frank-Raue
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N J Craig
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N Morrison
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E Boyd
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C Szpirer
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R G Sutcliffe
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ABSTRACT

Four hirsute females from a family exhibiting idiopathic dominant hirsutism were examined. Basal blood levels of Δ5 and Δ4 steroids were within the normal range, but ACTH stimulation led to increases in 17-hydroxypregnenolone and dehydroepiandrosterone that were significantly above control levels. Using polymorphic genetic markers, the genes for cytochrome P450c17 encoded by CYP17, and the type I and II forms of 3β-hydroxysteroid dehydrogenase (3β-HSD) were found not to segregate with hirsutism in this family, though a base substitution was detected in the 3′ end of exon 1 of the gene for 3β-HSD type I in three of the four patients investigated.

Analysis of PCR amplification products by denaturing gradient gel electrophoresis (DGGE) and sequencing revealed a novel homologue of exon 3 of 3β-HSD. DNA of one of the affected patients was used to create a genomic library in λ gem11 and clones containing the novel homologue were obtained and partially sequenced. The equivalent clone was obtained from a genomic library of an unrelated normal individual. The sequences of the clones from patient and control were identical and homologous to exons 2–4 of human 3β-HSD types I and II. No difference was found in the PCR primer sites that flanked the exon 3 homologue which led to its detection on DGGE gels. In both clones, stop codons and deletions were identified in the exon 4 homologue, leading to the deduction that the sequence comes from a pseudogene, which we call 3β-HSD Ψ1. The pseudogene mapped to chromosome 1p13. It was concluded that dominantly inherited idiopathic hirsutism in this rare kindred was not due to deficiencies in 3β-HSD types I, II, or Ψ, or of CYP17.

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B B Mendonça
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A J Russell
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M Vasconcelos-Leite
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IJ P Arnhold
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W Bloise
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B L Wajchenberg
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W Nicolau
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R G Sutcliffe
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A M Wallace
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ABSTRACT

A mutation (A82T) is described in the coding sequence of the gene for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) type II that is associated with variable clinical consequences. Four homozygotes are described, all of which showed elevated levels of Δ5 steroids consistent with 3ß-HSD deficiency. Two males from a consanguineous family were found to be homozygous for A82T and were affected with pseudohermaphroditism. They differed in their degree of mild salt loss. In the same family a female was found to be homozygous for A82T, but was clinically normal and had no history of premature pubarche or of abnormal menstrual cycles. However, in an apparently unrelated family, the A82T mutation was found in a female affected with premature pubarche. This is the first report of a proven mutation in 3ß-HSD type II associated with premature pubarche.

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