No evidence of mutations in the P450 aromatase gene in patients with polycystic ovary syndrome

D. Söderlund1, P. Canto1, S. Carranza-Lira2 and J.P. Méndez1,3

1 Research Unit in Developmental Biology, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México, D.F. (06703), México, 2 Gynecologic Endocrinology Service, Hospital de Ginecología y Obstetricia ‘Luis Castelazo Ayala’, Instituto Mexicano del Seguro Social, México, D.F. (01090), México

3 To whom correspondence should be addressed at: Unidad de Investigación Médica en Biología del Desarrollo, Coordinación de Investigación en Salud, Coahuila #5, Colonia Roma, C.P. 06703, Apartado Postal A-047, México, D.F., México. Email: jpmb{at}servidor.unam.mx or Email: teodorost{at}yahoo.com


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Etiology and inheritance pattern in polycystic ovary syndrome (PCOS) remain uncertain. Granulosa cells from follicles of women with PCOS have little, if any, aromatase (encoded by the CYP19 gene) activity; follicles contain low levels of estradiol, P450arom mRNA and aromatase stimulating bioactivity. Mice with targeted disruption of the CYP19 gene present cystic follicles. It has been proposed that chronic exposure to high levels of LH, because of aromatase deficiency, determines the development of ovarian cysts. Herein, we investigated if mutations in the CYP19 gene and/or its ovary promoter are causal in patients with PCOS. METHODS: Twenty-five patients with PCOS and 50 control women were studied. PCR analysis of genomic DNA and complete sequence of all exons of the aromatase gene and its ovary promoter were performed. RESULTS: No heterozygous or homozygous mutant alleles were present in any of the patients studied. CONCLUSIONS: In the population studied, mutations of the P450arom gene or its promoter are not the cause of PCOS. However, these findings do not preclude the possible importance of an aromatase disorder in PCOS etiology. Variations in aromatase complex function could play a role in PCOS etiology, but the determinants of such variations might be located in other genes.

Key words: aromatase deficiency/aromatase gene/mutation/polycystic ovary syndrome/polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Polycystic ovary syndrome (PCOS) is a common disorder present in 6–10% of women at reproductive age (Knochenhauer et al., 1998Go; Asuncion et al., 2000Go). This entity is characterized by chronic anovulation with either oligomenorrhea or amenorrhea and hyperandrogenism (Solomon, 1999Go). The etiology and the possible inheritance pattern remain uncertain.

Biosynthesis of estrogens from androgens is catalyzed by a microsomal enzymatic complex named aromatase, which is composed of a specific cytochrome P450 aromatase (P450arom) and the NADPH cytochrome P450 reductase. Aromatase catalyzes the aromatization of all three major substrates: androstenedione, testosterone and 16-alpha-hydroxy-androstenedione (Simpson et al., 1994Go). P450arom is encoded by the CYP19 gene located on chromosome 15p21.1 (Sparkes et al., 1987Go; Chen et al., 1988Go). This large gene (>123 kb) is composed of nine coding exons (exons II–X) (Simpson et al., 1987Go) and seven tissue-specific promoters associated with the specific 5'-untranslated exons I (Sebastian and Bulun, 2001Go); expression in the ovary uses a proximal promoter II that is regulated primarily by cAMP (Harada et al., 1990Go; Toda et al., 1990Go; Nelson et al., 1993Go) and results in the same translated protein as other tissues (placenta, brain, muscle, bone, etc.); (Simpson et al., 1994, 1997a,bGoSimpson et al.1997a,bGo; Sebastian and Bulun, 2001Go).

Since 1992, 10 well documented cases of congenital aromatase deficiency have been reported in nine different families. This syndrome is caused by mutations in the CYP19 gene which lead to excessive circulating androgens in the fetus, associated with maternal virilization (Harada et al., 1992Go; Conte et al., 1994Go; Morishima et al., 1995Go; Portrat-Doyen et al., 1996Go; Carani et al., 1997Go; Müllis et al., 1997Go; Ludwig et al., 1998Go; Deladoëy et al., 1999Go; Hermann et al., 2002Go). Prenatal exposure of the female fetus to adrenal androgens yields ambiguous external genitalia. Later in life, aromatase deficiency is responsible for sexual infantilism, primary amenorrhea, tall stature, virilization at puberty, delayed skeletal maturation and osteopenia (Harada et al., 1992Go; Bulun, 1996Go; Müllis et al., 1997Go; Ludwig et al., 1998Go; Deladoëy et al., 1999Go; Belgorosky et al., 2003Go).

It has been demonstrated that granulosa cells obtained from medium-sized follicles of women with PCOS have little, if any, aromatase activity (Erikson et al., 1979Go). Likewise, Jakimiuk et al. (1998)Go determined that all PCOS follicles contained low levels of estradiol (E2), P450arom mRNA and aromatase stimulating bioactivity when compared to control follicles. This indicates that E2 production is low in PCOS follicles because there is insufficient aromatase stimulating bioactivity to increase P450arom mRNA expression. In ArKO mice (Fisher et al., 1998Go; Britt et al., 2000Go), an experimental model with targeted disruption of the CYP19 gene, large hemorrhagic cystic follicles are observed. It has been proposed that chronic exposure to abnormally high levels of LH, due to the aromatase deficiency, determines the development of ovarian cysts in these rodents (Risma et al., 1995, 1997GoRisma et al., 1997Go).

On these bases, the purpose of the present study was to determine if mutations in the CYP19 gene and/or its ovary promoter are present and causal in patients with PCOS.


    Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We studied 25 PCOS patients. All women had a Mexican–mestizo ethnic origin and their age ranged from 16 to 32 years. All the patients fulfilled the Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group (2004)Go criteria for PCOS (two out of three) including: (i) oligo-and/or anovulation, (ii) clinical and/or biochemical signs of hyperandrogenism, (iii) polycystic ovaries; as well as exclusion of other causes of oligomenorrhea and hyperandrogenism. In all cases hirsutism was present [score ≥ 6 of the Ferriman and Gallwey modified scale (1961)Go] and there was at least a first degree relative with confirmed PCOS. A pelvic ultrasound demonstrated presence of 12 or more follicles in each ovary measuring 2–9 mm in diameter, and/or increased ovarian volume (>10 ml) (Table I). Likewise, the presence of congenital adrenal hyperplasia, Cushing syndrome, hyperprolactinemia or hypothyroidism was ruled out. The control group included 50 age-matched women. All control subjects had normal menstrual cycles in the past 12 months, no clinical evidence of hyperandrogenism, and were not taking anovulatories.


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Table I. Clinical findings and diagnostic features in 25 polycystic ovary syndrome (PCOS) patients

 
Informed consent was obtained from the patients before participating. The study was approved by the Institute's human research committee.

Methods
Genomic DNA was prepared from peripheral blood leukocytes by standard techniques according to Sambrook and Russell (2001)Go. Blood was collected in tubes containing EDTA and was transferred and red blood cell lysis buffer (20 mM Tris–Cl pH 7.6) was added and the capped tubes were inverted in order to mix the contents. The solution was incubated at room temperature for 10 min. Afterwards, the tubes were centrifuged at maximum speed for 20 s at room temperature and the supernatant fluid was removed. Ice-cold cell lysis buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and proteinase K (20 mg/ml) were added and incubated for 15 min at 37 °C. Afterwards, 4 mg/ml DNase-free RNase was added and incubated for 15 min at 37 °C. Potassium acetate solution (5 M potassium acetate, glacial acetic acid and H2O) was added and the contents of the tube were mixed by vortexing. The precipitated protein/SDS complex was centrifugated at maximum speed for 3 min at 4 °C. The supernatant was transferred to a fresh microfuge tube containing isopropanol. The solution was mixed and the DNA precipitate recovered by centrifuging the tube at maximum speed for 1 min at room temperature. The supernatant was removed by aspiration and 70% ethanol was added to the DNA pellet. Centrifugation of the tube at maximum speed for 1 min at room temperature was performed. The supernatant was removed by aspiration and the DNA pellet was dried in air for 15 min; finally, the DNA pellet was redissolved in TE (pH 7.6).

For each PCR amplification, genomic DNA (0.3 µg) in the presence of 0.2 mM dNTP, 2 U Taq DNA polymerase (Ampli Taq, Perkin-Elmer Corp., Branchburg, NJ) and 400 nM of specific set of P450arom primers was used. All coding exons of the P450arom gene, with the flanking intron sequences and the 5' untranslated exon (ovary promoter) were amplified by PCR, using oligonucleotide primers previously described (Müllis et al., 1997Go; Hermann et al., 2002Go). We analyzed 139 bases of the 5'-promoter region of the human aromatase gene, which included the transcriptional initiation site, the consensus sequences of a ‘TATA-box’ and a ‘CAAT-box’, besides the complete sequence of the exon 1 (Harada et al., 1990Go). The sequence of the primers of the promoter were described previously by Müllis et al. (1997)Go and were: forward primer 5'-AGGAAGAAGAATCTGGACAG-3' (nucleotide position –362 to –381 according to the sequence described in GenBank: X55983) and reverse primer 5'-CTCCAACTCCAGTTCCAACAC-3' (nucleotide position 669 to 689 according to the sequence described in GenBank: X55983). Thirty cycles of PCR amplifications were performed in a Thermal Cycler (PE Applied Biosystems, Foster City, CA). Except for the last, all cycles were 30 s at 98 °C, 30 s at 56 °C, and 3 min at 73 °C. In the last cycle the annealing temperature was at 53 °C. After amplification, PCR products were electrophoresed on 1.2% agarose gels and stained with ethidium bromide to verify the correct size of the expected fragments.

PCR products from the P450arom gene and from the ovarian promoter were purified by QIAEX II (Qiagen, Hilden, Germany). DNA sequences of both the sense and antisense strands (300 nmol DNA template/reaction) of each PCR product were determined by cycle sequencing on an automated DNA sequencer ABI 377 (PE Applied Biosystems) using the DNA Sequencing Kit BigDye Terminator Cycle Sequencing Ready Reaction (Perkin-Elmer Corp.). PCR conditions for cycle sequencing were performed following the protocol supplied by the manufacturer. In each case both strands were sequenced and compared.

Sequence variations are described in relation to a reference sequence for which the accession numbers from GenBank are M28420, M32245 and X55983. Each sequence variation was confirmed in three independent PCR amplifications and sequencings.


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
In all individuals studied, no gross deletions or insertions were detected after PCR amplification of the nine exons of the P450arom gene and after amplification of the ovary promoter. Direct sequencing of all exons of the P450arom gene demonstrated that no heterozygous or homozygous mutant alleles were present in any of the patients studied. After sequencing the ovary promoter of the P450arom gene a C->T polymorphism was detected at position –41. This base change was observed in a heterozygotic fashion in one patient and in two controls; whilst it was detected in a homozygotic fashion in three patients and in two controls (Figure 1).



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Figure 1. Part of the sequence of the ovary promoter of the P450arom gene. The homozygous base change (C->T) at position –41 (*) presented by three patients and two controls is depicted in the middle panel. In the right panel, both C and T can be observed at position –41 (*), demonstrating the heterozygous state presented by one patient and two controls.

 

    Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Due to its high prevalence and because it constitutes the most common cause of anovulatory infertility and hirsutism, several attempts have been made in order to determine the presence of causal mutations or recurrent polymorphisms in various genes that intervene on the synthesis of androgenic precursors (Carey et al., 1994Go; Franks, 1995Go; Gharani et al., 1997Go) or in genes that influence gonadotropin secretion or other mechanisms related to gonadotropin or androgen secretion or action (Ciaraldi et al., 1992Go; Kiddy et al., 1992Go; Dunaif et al., 1995Go). Likewise, Urbanek et al. (1999)Go studied, by linkage analysis, 37 candidate genes (including CYP19), finding only evidence for linkage with follistatin. In none of these studies has a particular molecular disorder been found as the precise cause of PCOS (Legro, 1999Go). Although this syndrome is a familial disorder, tending to aggregate in families with a high number of sisters and mothers affected, the genetic basis of its origin remains controversial (Franks, 1995Go); although a study of 150 patients in 10 families of women with PCOS suggested an autosomal dominant mode of inheritance (Carey et al., 1993Go).

In PCOS, the endocrine hallmarks are hyperandrogenemia and in a lesser extent LH hypersecretion. It seems, however, that the abnormality in gonadotropin secretion is a result of the excessive androgen secretion by the ovary. The increased activity of various steroidogenic enzymes (17{alpha}-hydroxylase/17–20 lyase, 3{beta}-hydroxysteroid dehydrogenase and 17{beta}-hydroxysteroid hydrogenase) in theca cells from women with PCOS, suggests that the increase in steroidogenic activity is intrinsic, probably genetic (Nelson et al., 1999Go). Eventually, hyperstimulation of the ovaries by the increase of LH and FSH levels is the consequence of the inability of the ovary to aromatize androgens to estrogens (Ludwig et al., 1998Go), which are required to restrain gonadotropin secretion (Belgorosky et al., 2003Go). Likewise, in aromatase deficiency an amplification of FSH signaling might occur in the presence of high intraovarian androgens and this could be involved in the subsequent development of ovarian follicular cysts (Simpson et al., 1987Go).

Estrogen biosynthesis is catalyzed by the aromatase complex being the P450arom encoded by the CYP19 gene. Likewise, biochemical studies have demonstrated that follicles of women with PCOS are abnormal and targeted disruption of the CYP19 gene in mice (ArKO) induces ovaries containing numerous follicles with abundant granulosa cells and evidences antrum formation that appears arrested before ovulation. No corpora lutea is present and the stroma is hyperplastic, indicative of overstimulation by LH (Fisher et al., 1998Go). The ArKO mouse is infertile as a consequence of disrupted folliculogenesis and failure to ovulate. Testosterone concentrations are markedly elevated and FSH and LH assays show that LH levels are elevated 2- to 10-fold in the ArKO females compared with wild type (Fisher et al., 1998Go). On these bases, we studied the CYP19 gene and its ovary promoter, which is a plausible candidate gene for PCOS because of its function, to determine if PCOS was caused by mutations in this gene, due to the fact that to date there are no data identifying a direct genetic cause for PCOS.

In our patients, however, we did not identify any gross mutations in the P450arom gene or the ovarian promoter of this gene. The only sequence variation we detected, comparing to the original sequence description made by Harada et al. (1990)Go, was a C to T change at position –41 of the ovary promoter. This base variation was described by Toda et al. (1990)Go, when reporting the characterization of the human aromatase P-450 gene. This sequence change which is located between the TATA box (–27 to –22) and the CAAT box (–83 to –79) was found in a heterozygotic fashion in 1/25 patients and in 2/50 controls. It was also found in a homozygotic form in three patients and two controls.

This study demonstrates that at least in the population studied (small number of cases and one specific ethnic group), mutations of the P450arom gene or its ovarian promoter are not the cause of PCOS. However, with our present data, the possibility of the existence of causative mutations in the untranslated regions of exons 2 and 10 and within introns cannot be completely ruled out.

Although we have shown that variation in the P450arom gene is unlikely to be responsible of PCOS, these findings do not preclude the possible importance of an aromatase disorder in the etiology of this syndrome. Unknown cis or trans elements could be involved in the regulation of gene expression. Likewise, variations in the function of the aromatase complex could play a role in PCOS etiology, but the determinants of such variations might be located in other genes different from the P450arom gene.


    Acknowledgements
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Leonor Enciso from the Unidad de Instrumentos, Coordinación de Investigación en Salud, Instituto Mexicano del Seguro Social, for her technical assistance. This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México. Grant number: G29790M.


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 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 24, 2004; resubmitted on November 19, 2004;



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