1 Department of Paediatrics, University of Cambridge, Cambridge CB2 2QQ, 2 Division of Public Health and Primary Health Care, University of Oxford, Oxford OX3 7LF, UK, 3 Endocrine Unit, Hospital Sant Joan de Déu, University of Barcelona, 08950 Barcelona, Spain, 4 Reproductive Medicine Unit, The General Infirmary, Leeds LS2 9NS, UK and 5 Department of Paediatrics, University of Leuven, 3000 Leuven, Belgium
6 To whom correspondence should be addressed at: Department of Paediatrics, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. Email: dbd25{at}cam.ac.uk
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Abstract |
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Key words: genetic association/insulin resistance/polycystic ovarian syndrome/premature pubarche/testosterone
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Introduction |
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Aromatase (EC 1.14.14.1) is a member of the cytochrome P450 family of enzymes (subfamily 19) which catalyses the conversion of C19 androgens to aromatic C18 estrogens. It is induced by FSH and is present in a number of different tissues including adrenals, muscle, placenta, skin, adipose and nervous tissue. Reduced aromatase activity may lead to the development of PCOS, since PCOS has been observed in patients with aromatase deficiency caused by rare loss-of-function mutations (Harada et al., 1992; Ito et al., 1993
; Belgorosky et al., 2003
) and antral follicles taken from PCOS women exhibited no aromatase activity (Takayama et al., 1996
).
The human aromatase gene is 130 kb long. Its 10 exons (the final nine of which are coding) are located within 30 kb of each other (Bulun et al., 2003
), and the 93 kb 5'-flanking region is thought to have a regulatory role. In this study we explored the association between common variation in aromatase and hyperandrogenism in two separate populations of girls and young women, using a haplotype-tag single nucleotide polymorphism (htSNP) genotyping approach.
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Materials and methods |
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Eighty-four of these subjects were subsequently also studied post-menarche in the follicular phase (days 38) of the menstrual cycle, with assessment of biochemical and clinical features of ovarian hyperandrogenism, including hirsutism score, the severity of any acne lesions, serum testosterone, androstenedione, DHEAS and sex hormone-binding globulin (SHBG). They also underwent a 1.75 g/kg body weight (maximum 75 g) 120 min oral glucose tolerance test (Ibáñez et al., 1998). Serum 17-hydroxyprogesterone (OHP) levels in response to leuprolide acetate, a GnRH agonist (Procrin, 500 µg sc; Abbott, Spain) were measured, and biochemical ovarian hyperandrogenism was defined as a peak 17-OHP >160 ng/dl (Ibáñez et al., 1994
).
Healthy controls (n=71) were either short, normal Barcelona-Spanish girls (heights between the 10th and 25th percentiles, i.e. within the familial range and well above what is considered pathological) attending a clinic for concerns about growth (none were subsequently found to have a pathological cause) or were subjects undergoing minor non-endocrine-related surgical procedures such as those on the ear, nose or throat. None of the controls had a history of PP or PCOS or showed any symptoms or signs of androgen excess. The study protocol was approved by the Institutional Review Board of Barcelona University Hospital of Sant Joan de Déu. Informed consent was obtained from parents and/or study subjects, including permission for the collection and genotyping of DNA samples. Birthweight and gestational age data were obtained from hospital records or from the girls' paediatricians, and transformed into SD scores (Ibáñez et al., 1998).
Oxford population ptudy
A total of 224 young women volunteers who considered themselves normal were recruited by invitation from general practice surgeries and by advertisement (requesting healthy volunteers to enrol for a study of women's health) in Oxford universities (Michelmore et al., 1999, 2001
). All volunteers were aged between 18 and 25 (mean 21.5) years and 97% of them were Caucasian in ethnic origin (the remainder being Indian or Afro-Caribbean). In these young women the prevalence of polycystic ovaries on transabdominal ultrasound was higher than that reported in other population-based studies (Polson et al., 1988
; Tayob et al., 1990
), but no direct evidence of selection bias was detected (Michelmore et al., 2001
). A total of 115 women who were recruited in the original survey were excluded from this study as they were using oral contraceptives (the only exclusion criterion). Blood samples for hormonal analyses were collected in the fasting state during the early follicular phase of the menstrual cycle. Ovaries were scanned by ultrasound for polycystic appearance. A symptom score for PCOS was calculated on the basis of number of features from: menstrual irregularity, acne, hirsutism, testosterone >3 nmol/l, and LH > 10 IU/l (Michelmore et al., 1999
, 2001
). Around one third of the volunteers were shown to have polycystic ovaries, and of these 7580% had symptoms of PCOS (Michelmore et al., 1999
, 2001
). Ethical approval for the study was granted by the Central Oxford Research Ethics Committee and informed consent was obtained from all subjects.
Assays
For the Barcelona cases and controls, serum hormone concentrations [insulin intra- and inter-assay coefficients of variation (CV) were 4.7 and 7.2% respectively, testosterone 9.2 and 10.4%, androstenedione 6 and 11.9%, DHEAS 5.3 and 3.9%, SHBG 3.0% and 4.4%] were measured by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) kits as previously described (Ibáñez et al., 1993, 1998
, 2000
). Glucose was measured using a glucose oxidase method. For the Oxford population study, plasma hormone concentrations (including testosterone inter-assay CV <10%, estradiol inter-assay CV < 3%, androstenedione inter-assay CV <7%, 17-hydroxyprogesterone inter-assay CV <4%, SHBG inter-assay CV <5%, LH inter-assay CV <2%, FSH inter-assay CV <3%, insulin CV: intra-assay 8.3% and inter-assay 12.2%, and c-peptide intra-assay CV 3.4% and inter-assay CV 10.0%) were measured by radioimmunoassay or ELISA as previously described (Michelmore et al., 1999
, 2001
).
Aromatase genotyping
Linkage disequilibrium (LD) maps of aromatase single nucleotide polymorphisms (SNP) have been constructed to define htSNP that allow high predictability of haplotypes (Haiman et al., 2003). Four blocks of strong LD were found, the largest of which covered a 50 kb region containing the entire aromatase coding region and part of the 3'-untranslated region. The haplotypes in this region could be inferred from eight htSNP genotypes (Haiman et al., 2003
). Close inspection, however, revealed that the increased diversity of aromatase in African-Americans alone accounts for four of the htSNP genotypes necessary to define this region (Haiman et al., 2003
). In races such as Caucasians and Latinas, haplotypes in this LD-block could be inferred from just four htSNP genotypes, so we adopted this strategy for constructing aromatase coding-region haplotypes in our populations. For convenience, SNP genotypes are reported according to the nomenclature of Haiman et al. (2003)
, and genotyping was performed on genomic DNA that had been extracted from leukocytes (Ibáñez et al., 1993
; Michelmore et al., 2001
). Replicate genotyping on the same DNA gave <1% inconsistent calls throughout.
SNP44 genotyping
SNP 44 (Haiman et al., 2003) (hCV8234947, rs12907866) was genotyped using PCR amplification and detection of SNP alleles by restriction fragment length polymorphism (RFLP) analyses. DNA (10 ng) was amplified (20 µl total volume) along with 1 x reaction buffer, 200 µmol/l each dNTP (Promega Ltd, UK), 1 mmol/l magnesium, 12 pmol each primer (forward: 5'-GCATTGCAGGTAGAGAGAATGAGAAAC-3'; reverse: 5'-CCTGCTCCATGTTCTCAAGACAGATATA-3'), 10% (v/v) glycerol and 0.5 IU Biotaq DNA Polymerase (Bioline, UK). The mix was heated to 94°C for 5 min, followed by 20 cycles of 94°C (45 s), 59°C (45 s, dropping 0.5°C per cycle) and 72°C (45 s). After this the samples underwent 15 cycles of 94°C (45 s), 49°C (45 s) and 72°C (45 s), prior to a final incubation at 72°C for 10 min. Ten microlitres of the resulting PCR product was incubated at 65°C for 16 h along with 5 IU of TaqI (New England Biolabs, UK). Separating the resulting products by agarose gel electrophoresis produced a 425 bp band for the A allele and 258 and 167 bp bands for the G allele.
SNP50 genotyping
SNP 50 (Haiman et al., 2003) (rs2414096) was genotyped using mismatch primer PCRRFLP (Hao et al., 2002
). DNA (10 ng) was amplified (20 µl total volume) along with 1 x reaction buffer, 200 µmol/l each dNTP, 1.5 mmol/l magnesium, 12 pmol of each primer (forward: 5'-TTGTTACCCTCAAAAAAGCGACC-3'; reverse: 5'-CTCAAACTCCAATCTAGAGGTTCAAAG-3'), 5% (v/v) dimethylsulphoxide and 0.5 IU Biotaq DNA Polymerase. The mix was heated to 94°C for 5 min, followed by 20 cycles of 94°C (45 s), 55°C (45 s, dropping 0.5°C per cycle) and 72°C (20 s). After this the samples underwent 15 cycles of 94°C (45 s), 45°C (45 s) and 72°C (20 s), prior to a final incubation at 72°C for 10 min. Of the resulting PCR product, 10 µl was incubated at 60°C for 16 h along with 2.5 IU of BsiEI (New England Biolabs). This produced a 124 bp band for the A allele and 102 and 22 bp bands for the G allele.
SNP60 genotyping
SNP 60 (Siegelmann-Danieli and Buetow, 1999; Haiman et al., 2003
) (int 7_14A, IVS7+26, G-A) was genotyped using tetra-primer amplification refractory mutation system (ARMS) PCR (Ye et al., 2001
). DNA (30 ng) was amplified (10 µl total volume) along with 1 x reaction buffer, 200 µmol/l each dNTP, 2.5 mmol/l magnesium, 10 pmol each inner primer (forward: 5'-CACAGTCATAACATATGTGGCATTGA-3'; reverse: 5'-GAGGTACTGACCTGAACTAACTGTAAGTC-3') and 1pmol each outer primer (forward: 5'-TGGGATTACAGAACTGCTAGAGAAAGTA-3'; reverse: 5'-TAGACAATATTTTGACTTTTTTTCCAGC-3'), 10% (v/v) glycerol and 0.5 IU Biotaq DNA Polymerase. The mix was heated to 94°C for 5 min, followed by 20 cycles of 94°C (45 s), 57°C (45 s, dropping 0.5°C per cycle) and 72°C (45 s). After this the samples underwent 15 cycles of 94°C (45 s), 47°C (45 s) and 72°C (45 s), prior to a final incubation at 72°C for 10 min. This produced a 194 bp band for A allele and a 135 bp band for the G allele (with a full PCR product band at 274 bp).
SNP64 genotyping
SNP 64 (Haiman et al., 2003) (rs4646) was also genotyped by ARMS PCR. The cycling conditions were similar to those for SNP 60, except that the annealing temperatures were 7°C higher throughout. The reaction mix was the same as for SNP 60 with the exception of the inner primer sequences (forward: 5'-GGGTTGTCACCAAGCTAGGTGCTACTT-3prime; reverse: 5prime;-CTGGTGTGAACAGGAGCAGATGGCC-3prime) and outer primer sequences (forward: 5prime;-ACCCCAAGAAACTCAGACAGGTGTCTG-3prime; reverse: 5prime;-TTGTTAATGAAGGCCTATCCTTCTCAAAGCACA-3'). This produced a 223 bp band for G allele and a 176 bp band for the T allele (with a full PCR product band at 346 bp).
Statistical analyses
All genotype frequencies were assessed for consistency with HardyWeinberg equilibrium (the Barcelona samples being assessed separately for cases and controls due to the possibility of natural selection occurring for these loci in the cases). Statistical analyses were performed using SPSS for windows (version 10.0.7) (SPSS Inc., USA). In the Barcelona study there was 80% statistical power to be able to detect a difference of 0.4 SD of a quantitative variable at the quoted SNP 50 allele frequencies. For the Barcelona SNP genotypes there was 80% statistical power to detect a difference between minor allele frequencies of 0.5 and 0.3. In the Oxford population study there was 80% statistical power to be able to detect a difference of 0.6 SD of a quantitative variable at the quoted SNP 50 allele frequencies. Aromatase coding-region haplotypes were reconstructed from the four SNP genotypes using Phase (version II) software (Stephens et al., 2001). For both studies, haplotypes were reconstructed using data from the complete set of samples (i.e. in Barcelona both the cases and controls combined and in the Oxford population the full group including those taking oral contraceptives). Haplotypes were expressed in terms of aromatase alleles in the order SNP 44, 50, 60 then 64. Hormone concentrations and quantitative phenotypic data were compared across aromatase genotypes and haplotypes by one-way ANOVA. Where appropriate, adjustment was made for insulin sensitivity [calculated as homeostatic model assessment (HOMA) (Levy et al., 1998
) from fasting plasma insulin and glucose concentrations], age (at presentation or assessment), puberty stage (when analysing puberty-related hormone concentrations), body mass index (BMI) or birthweight. In the Barcelona study, frequencies of aromatase haplotypes were compared using the
2 goodness-of-fit test. Frequencies of individual haplotypes were compared (presence against absence in cases and controls) by the
2-test.
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Results |
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None of the other genotypes and haplotypes were associated with clinical or biochemical features of hyperandrogenism in this group.
Oxford population study
All aromatase SNP genotypes in this cross-sectional study were consistent with HardyWeinberg equilibrium. The SNP 50 genotype was associated with PCOS symptom scores (P=0.008 with dominant model; Table IV), but the aromatase coding-region haplotype was not associated with any clinical or biochemical feature of hyperandrogenism (all P>0.05; SNP genotype and haplotype prevalences are shown in Table IITable II). This association with SNP 50 was independent of BMI, insulin sensitivity and ultrasound diagnosis of polycystic ovaries, all of which were independently associated with the PCOS symptom score (all P<0.03). The SNP 50 genotype was also associated with variation in circulating testosterone concentrations (P=0.02 with dominant model; Table IV). There was no detectable association between this genotype and other features of PCOS or with insulin sensitivity (P=0.7), even after adjusting for BMI (P=0.6). Variation in estradiol quartile was also associated with the SNP 50 genotype (P=0.014 with additive model; Table IV) but it was not related to the estradiol to testosterone ratio (P=0.4).
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Discussion |
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The aetiology of PP, ovarian hyperandrogenism and PCOS has been linked to insulin resistance and obesity (Dunaif, 1997). Our findings were independent of insulin sensitivity and BMI. Genotype distributions between PP girls and healthy controls were different, with the SNP 50 A allele (which was more prevalent in PP girls) being associated with increased testosterone concentrations in both the Barcelona PP casecontrol study and the Oxford population study (where it was also associated with increased PCOS symptom scores). Aromatase coding-region haplotypes in both our Barcelona controls and Oxford young women showed similar prevalences to those of Haiman et al. (2003)
. Variation in them was also shown to be associated with alterations in risk for PP, itself a risk factor for PCOS (Ibáñez et al., 1993
, 2000
). In particular, comparing haplotypes differing in the SNP 50 genotype, one variant was associated with a 2-fold decreased risk for having PP. In 84 post-pubertal subjects, reduced insulin sensitivity and aromatase coding region haplotypes were independently associated with functional ovarian hyperandrogenism risk. The lack of confirmation of the haplotype effects in the Oxford population study may well have been due to a lack of sufficient statistical power, with the four SNP genotyped being resolved into nine different haplotypes in that group of only 109 young women.
The significant findings in this study appear to relate to the SNP 50 genotypes. The question therefore arises as to whether this SNPS is causing these associations directly or whether it is just in LD with another causal SNPS. Clearly there are many SNPS in this aromatase block that are in strong LD (Haiman et al., 2003), hence the relatively small number of SNP that need to be typed to be used as haplotype tags. Indeed one SNP, only 667 base pairs away from SNP 50 [SNP 51 in Haiman et al. (2003)
] in exon 3 of the gene, is in strong LD with SNP 50 (Haiman et al., 2003
) and has already been shown to be associated with breast cancer risk in women (Siegelmann-Danieli and Buetow, 1999
) and with circulating estradiol concentrations (Somner et al., 2004
). However, in those studies the issue of causality has not been addressed either. The DNA sequence around SNP 50, when the G allele is present, is a consensus branchpoint sequence for pre-RNA processing, albeit further from the 3' splice site than branchpoints are usually found (Buvoli et al., 1997
). When the A allele is present this branchpoint sequence disappears, so if it were physiologically active there may be an alteration in the efficiency of aromatase pre-RNA processing and ultimately aromatase expression. This suggestion remains speculative at present, however.
While our samples sizes may be relatively small for genetic association studies, the detailed phenotype assessments in both populations would increase the power to identify true positive associations. The consistent associations in two very separate populations further increases the validity of the findings and reduces the likelihood of population stratification bias, although family-based transmission studies would be needed to confirm this. The Barcelona control girls were not recruited from a completely unselected population; however, they had no evidence of endocrine or any other pathology. Similarly, while it is possible that the Oxford volunteers may have over-selected themselves for the presence of PCOS features, no selection bias was detected (Michelmore et al., 2001), and as in the Barcelona control girls, the genotype and haplotype frequencies were similar to those observed in other populations (Haiman et al., 2003
). Our associations were largely with hyperandrogenism, and future casecontrol studies would be needed to confirm the relevance of these findings to actual PCOS risk. Such studies would need to be much larger, in view of the widely heterogeneous pathogenesis of PCOS.
Previously described rare loss-of-function mutations in aromatase have demonstrated that variation in this estrogen biosynthesis pathway can indeed lead to PCOS in females (Harada et al., 1992; Ito et al., 1993
; Conte et al., 1994
; Morishima et al., 1995
; Mullis et al., 1997
; Belgorosky et al., 2003
). So far the aromatase mutations described have been in exons 5 (Belgorosky et al., 2003
), 9 (Belgorosky et al., 2003
; Morishima et al., 1995
; Mullis et al., 1997
) and 10 (Ito et al., 1993
; Conte et al., 1994
), and in the boundary between exons and introns 3 (Mullis et al., 1997
) and 6 (Harada et al., 1992
). Our findings which relate to SNP 50, which is also in the coding-region, suggest that more subtle, common variations in aromatase are also associated with variation in risk for PCOS. Overall PCOS risk may involve oligogenic contributions (Franks et al., 2001
) [as well as environmental components (Crosignani and Nicolosi, 2001
)] including that made by aromatase, in addition to other genes such as those of the androgen receptor (Ibáñez et al., 2003
), insulin receptor (Tucci et al., 2001
), cholesterol side-chain cleavage enzyme (Gharani et al., 1997
), follistatin (Urbanek et al., 1999
) and the insulin VNTR (Michelmore et al., 2001
). Alternatively, as PCOS is such a heterogeneous disorder, different genetic variants could associate with PCOS risk in different populations (Sanders et al., 2002
).
In summary, this is the first association study that has found a link between genetic variation in the aromatase gene and androgen excess in females. This was confirmed in two independent populations, one a population survey and the other a casecontrol study. The fact that these two study groups, which gave very similar associations with aromatase genotypes, were established by different selection criteria gives these findings extra credence. Previously both a linkage (Gharani et al., 1997) and an association (Urbanek et al., 1999
) study failed to find a relationship between aromatase and PCOS, but these studies were performed without the power associated with the use of htSNPS and therefore some of the haplotypic variation in the gene may have been missed.
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Acknowledgements |
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Submitted on December 22, 2004; resubmitted on January 24, 2005; accepted on March 4, 2005.