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Original Studies |
Departments of Endocrinology (R.M.C., M.A., J.S., H.F.E.-M.) and Molecular Genetics (D.T., J.L.S.M.), Hospital Ramón y Cajal, Madrid 28034, Spain
Address all correspondence and requests for reprints to: Héctor F. Escobar-Morreale, M.D., Ph.D., Department of Endocrinology, Hospital Ramón y Cajal, Carretera de Colmenar Km 9'100, 28034 Madrid, Spain. E-mail: hector.escobar{at}uam.es
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Abstract |
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Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients.
In the present study we have screened, using heteroduplex analysis, the
genes encoding StAR and SF-1 as well as DAX-1 and CYP11A
for mutations in genomic DNA from 19 women presenting with hirsutism
and increased serum androgen levels. When variants were found, analysis
was extended to a larger group of hyperandrogenic patients and
nonaffected women. Two variants were identified in the SF-1 gene. A
G
C change in exon 6, resulting in an Arg365Pro mutation,
was found in 1 of 45 patients, but not in controls. Also, a
Gly146Ala missense mutation, resulting from a GC change
in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected
individuals. We identified a CT base pair change at position -33 of
the StAR gene. Three of 48 patients and 3 of 43 controls presented this
variant. No mutations were found in coding regions of the StAR gene.
Analysis of CYP11A-coding regions identified a GA
change in exon 3, resulting in a Val179Ile missense
mutation. This mutation was found in 1 of 29 patients studied and was
not present in 50 controls. Finally, analysis of DAX-1 showed no
variant in any of the women studied.
In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Hirsutism results from androgen excess at the pilosebaceous unit and is considered a clinical marker of hyperandrogenism in women. In nearly all hirsute women, abnormalities in adrenal and/or ovarian steroidogenesis can be demonstrated, suggesting that both glands contribute to some extent to the androgen excess responsible for hirsutism (3, 4).
The enzymes required for the synthesis of androgens are cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A; P450c17, encoded by CYP17; 3ßhydroxysteroid dehydrogenase; and several isoenzymes of 17ß-hydroxysteroid dehydrogenase. The adrenal and the ovary share most of these enzymes. Moreover, the activities of P450scc, P450c17, 3ß-hydroxysteroid dehydrogenase, and 17ß-hydroxysteroid dehydrogenase type 5 are increased in thecal cells from PCOS patients maintained in primary culture, an abnormality that can be considered a primary event in the pathogenesis of this disorder (5, 6).
The genes encoding for the enzymes involved in androgen synthesis have been studied in women with hyperandrogenism, but no definite role for any of the variants found has been established to date. Polymorphisms in the regulatory regions of CYP11A and CYP17 do not appear to be related to hyperandrogenism (7, 8, 9, 10). The coding regions of CYP11A have not been studied, and the existence of nonclassical 3ß-hydroxysteroid dehydrogenase deficiency has never been demonstrated at the molecular genetic level (11). Furthermore, no association of variants in any of the genes encoding the 17ß-hydroxysteroid dehydrogenase isoenzymes with hyperandrogenism have been reported to date. Therefore, the genes encoding other factors involved in the regulation of adrenal and ovarian androgen synthesis should be considered candidates to explain the etiology of hyperandrogenism.
The initial step in adrenal and ovarian androgen synthesis is the conversion of cholesterol to pregnenolone. This conversion is catalyzed by the mitochondrial enzyme P450scc and requires transport of cholesterol into the mitochondria, which is mediated by the steroidogenic acute regulatory protein (StAR). Dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene-1 (DAX-1) is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of StAR gene, CYP11A, and DAX-1, among others.
Recently, it has been shown that inactivating mutations in DAX-1 and StAR genes are the cause of adrenal hypoplasia congenita and lipoid congenital adrenal hyperplasia, respectively (12, 13), and an inactivating mutation in SF-1 resulted in sex reversal and adrenal failure in a 46,XY subject (14). To date, inactivating CYP11A mutations have not been described.
Conversely, variants in the genes encoding for these factors, resulting in gain of function, or loss of repression, of StAR, P450scc, or other steroidogenic enzymes, might contribute to androgen excess in hyperandrogenic women. To test this hypothesis, we have screened the genes encoding StAR, SF-1, DAX-1, and CYP11A for mutations in genomic DNA from patients presenting with hirsutism and increased androgen levels.
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Subjects and Methods |
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Nineteen hyperandrogenic hirsute women [mean ± SD: age, 22.3 ± 6.2 yr; body mass index, 26.7 ± 6.8 kg/m2; modified Ferriman-Gallwey score (15), 17.1 ± 5.3] were initially screened for mutations.
Serum samples were obtained during the follicular phase of the menstrual cycle. All of the patients presented hyperandrogenemia, as defined by increased serum concentrations of one or more of the following androgens: total testosterone above 2.30 nmol/L, calculated free testosterone (16) above 35 pmol/L, androstenedione above 16.0 nmol/L, and dehydroepiandrosterone sulfate above 9.9 µmol/L. The mean ± SD circulating levels of these androgens were 2.9 ± 1.4 nmol/L, 61 ± 52 pmol/L, 12.8 ± 4.5 nmol/L, and 9.7 ± 4.7 µmol/L, respectively. The assays employed for serum hormone measurements have been reported previously (17).
Nonclassical congenital adrenal hyperplasia and hyperprolactinemia were ruled out in all of the patients by ACTH-stimulated serum 17-hydroxyprogesterone levels below 30 nmol/L (18) and basal serum PRL concentrations below 24 µg/L.
Nine of the 19 patients, who had oligomenorrhea defined by menstrual cycles longer than 36 days (19) fulfilled endocrine criteria for the diagnosis of PCOS (2).
When the screening of the 19 hirsute patients described above detected any variant, the analysis was extended to a larger group of hyperandrogenemic hirsute patients with or without menstrual dysfunction (17) and to a group of 50 healthy women who did not have hirsutism, acne, alopecia, or menstrual disturbances (20). Data from these patients and controls regarding aspects of the pathophysiology of hirsutism and hyperandrogenism different from those studied here have been previously reported (17, 20).
The hospital ethics committee approved the study, and informed consent was obtained from every patient and control.
Molecular genetic analysis
Genomic DNA was extracted from peripheral leukocytes following the manufacturer’s instructions (Wizard Genomic DNA Purification Kit, Promega Corp., Madison, WI). A broad screening was performed in the 19 patients for mutations in DAX-1, SF-1, and StAR genes, and in the coding regions of CYP11A. We have not evaluated the variable tandem repeat polymorphism in the promoter of CYP11A, as our previous study in a large series of hirsute patients showed no association either with hirsutism or hyperandrogenism (7).
DNA was used to amplify different portions of the genes described above. PCR amplifications were performed using an automated thermocycler (GeneAmp PCR System 2400, PE Applied Biosystems, Foster City, CA).
The primers used for PCR amplification of each exon and intron-exon boundaries of CYP11A and SF-1 genes and for amplification of the 5'-regulatory regions of SF-1 gene (from -246 to +55 in exon 1), DAX-1 (position -194 to +98 in exon 1), and StAR gene (position -950 to -753, which contains a putative SF-1-binding domain), were designed using OLIGO 4.0 Primer Analysis Software (Molecular Biology Insights, Inc., Plymouth, MN). Primer sequences and PCR conditions are available from the authors on request.
The primers used for the amplification of the two exons of DAX-1 and for the promoter and seven exons of the StAR gene have been previously described by others (21, 22).
Heteroduplex analysis (23) was used to detect variants, which were identified by direct sequencing of PCR products using an ABI 310 automated sequencer (PE Applied Biosystems). Digestion by SphI or MspI endonuclease restriction enzymes was used for the screening of mutations that cause changes in target sites for these enzymes. Mutations were confirmed by direct sequencing.
Variants present in more than 2% of the study population and in similar proportions in patients and healthy controls were considered common polymorphisms (24).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. We identified several novel variants
in the SF-1 and StAR genes and in CYP11A. Analysis of SF-1
gene in the 19 hirsute patients initially screened detected 2 different
heterozygous mutations. Two patients presented a GC change in exon
4, resulting in a Gly146Ala missense mutation.
This mutation creates a restriction site for SphI, and
digestion by this enzyme was used for further testing. The presence of
the Gly146Ala mutation in 2 of 48 hyperandrogenic
patients and 2 of 50 controls suggested that this variant is a
polymorphism (24).
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C change in exon 6,
resulting in an Arg365Pro missense mutation.
Using digestion by MspI, which is prevented by the
Arg365Pro mutation, the latter was not found in
any other patient or control.
No mutations were found in the promoter region of DAX-1 or in
coding regions of the DAX-1 and StAR gene. On the contrary, a CT bp
change at position -33 from the transcription start site of the StAR
gene was identified in 1 of the 19 patients initially screened. This
mutation results in a target site for MspI, and digestion by
this restriction enzyme was used to evaluate another 29 patients and 43
controls. A total of 3 patients and 3 controls presented this variant,
which therefore was considered a polymorphism (24).
Finally, analysis of CYP11A-coding regions identified a
GA change in exon 3, resulting in a Val179Ile
missense mutation. The mutation was found in 1 of 29 patients and was
not present in 50 nonaffected individuals. No other variants were
identified in other coding regions of CYP11A.
None of the affected patients or controls had more than one of the variants described above.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Furthermore, using promoter constructs containing successive truncations of the 5'-flanking sequence of the human CYP17 gene, these researchers have recently shown that the region between -235/-109 bp of the transcription site is critical to the increased CYP17 messenger ribonucleic acid expression and activity in PCOS thecal cells (25). Cotransfection with SF-1 was required for the detection of basal and forskolin-stimulated CYP17 promoter activity, in agreement with the presence of SF-1 response elements in that region (25).
Therefore, variants in the genes encoding the enzymes involved in androgen synthesis and in those of their regulatory factors might be related to the up-regulation of steroidogenic enzymes found in hyperandrogenism and hirsutism. As these factors are present in both the adrenal and the ovary, their abnormalities might explain not only the increased synthesis of androgens in the ovary, but also the frequent finding of adrenal hyperandrogenism in hirsute and PCOS patients (26).
The first and critical step in ovarian and adrenal steroidogenesis is the conversion of cholesterol into pregnenolone. This step is mediated by the actions of StAR and P450scc. The genes encoding both proteins are influenced by several factors, including DAX-1 and SF-1, which are believed to play a crucial role in the development and function of the adrenal and gonads. The interaction between DAX-1 and SF-1 is poorly understood. DAX-1 is a potent repressor of StAR gene expression, blocking steroidogenesis (12) and also repressing the transcriptional activity of SF-1 (27). SF-1 is generally considered to be a transcriptional activator mediated by recruiting coactivators; however, binding of DAX-1 to SF-1 turns SF-1 into transcriptional repressor, because DAX-1 is able to recruit corepressors (27). The influence of DAX-1 and SF-1 is not limited to the initial steps of adrenal and ovarian steroidogenesis; it also modulates the activity of enzymes such as P450c17 and 3ß-hydroxysteroid dehydrogenase (28, 29).
In the present study we screened the genes encoding these factors and enzymes in women presenting with hirsutism and increased serum androgen levels. The novel variants found in our patients were not frequent enough to explain the hyperandrogenism of these women.
The G146A variant in SF-1 gene and the CT change at -33
of the StAR protein gene can be considered polymorphisms using the
standard definition, as more than 2% of our population is heterozygous
for that locus (24) The small proportion of patients
affected and the similar proportion of controls carrying the variant
suggest that these are common polymorphisms (30) and that
the contribution of these variants to the pathogenesis of
hyperandrogenism, if any, is minor.
On the contrary, the variants found in exon 6 of the SF-1 gene and in exon 3 of CYP11A were not present in controls. Comparison of the human and murine CYP11A sequences revealed that mice present Ile at the equivalent position, suggesting that the Val179Ile missense mutation found in one patient probably has no important consequences for the function of this enzyme. Moreover, Val and Ile are similar amino acids in terms of composition, polarity, and molecular volume, properties that correlate with the evolutionary exchangeability of protein residues (31, 32).
The Arg365Pro mutation found in one patient may have a relevant impact on the function of SF-1, as an Arg is present at equivalent positions of the murine, rat, and bovine SF-1 genes, and the introduction of a proline can affect the secondary structure of the SF-1 molecule.
Further work is needed to properly describe the Arg365Pro missense mutation in exon 6 of SF-1. Examination of parental genotypes is needed to evaluate the Mendelian inheritance of this variant or the possibility of a de novo mutation. Moreover, functional studies using transcription assays after mutagenesis in appropriate vectors will be required to evaluate the impact of the Arg365Pro missense mutation on SF-1 activity in different cell systems.
In conclusion, variants in DAX-1, SF-1, and StAR genes and in coding regions of CYP11A are seldom found in women with hyperandrogenic hirsutism. Moreover, the variants identified in our present study are either equally frequent in patients and controls or are rare or conservative changes and thus not likely to be significant contributors to hyperandrogenism. Therefore, these genes do not appear to play a major role in the pathogenesis of hirsutism and hyperandrogenism.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received May 15, 2000.
Revised July 20, 2000.
Revised October 4, 2000.
Accepted January 4, 2001.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase
(CYP17) gene in hyperandrogenic states. Clin Endocrinol (Oxf). 46:131–134.[CrossRef][Medline]
-hydroxylase and steroidogenic acute regulatory
protein gene promoters in normal and polycystic ovary syndrome theca
cells. J Clin Endocrinol Metab. 85:2304–2311.This article has been cited by other articles:
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  I. A. Pikuleva CHOLESTEROL-METABOLIZING CYTOCHROMES P450 Drug Metab. Dispos., April 1, 2006; 34(4): 513 - 520. [Abstract] [Full Text] [PDF]
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 H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]
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 W. Zheng, Y.-T. Gao, X.-O. Shu, W. Wen, Q. Cai, Q. Dai, and J. R. Smith Population-Based Case-Control Study of CYP11A Gene Polymorphism and Breast Cancer Risk Cancer Epidemiol. Biomarkers Prev., May 1, 2004; 13(5): 709 - 714. [Abstract] [Full Text] [PDF]
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 B. Peral, J. L. San Millan, R. Castello, P. Moghetti, and H. F. Escobar-Morreale The Methionine 196 Arginine Polymorphism in Exon 6 of the TNF Receptor 2 Gene (TNFRSF1B) Is Associated with the Polycystic Ovary Syndrome and Hyperandrogenism J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3977 - 3983. [Abstract] [Full Text] [PDF]
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