To examine the relationship between genetic polymorphisms of the CYP19A1 gene and obesity-related phenotypes, body mass index (BMI) and waist-to-hip ratio (WHR).
In total, 1241 Chinese women, who were recruited as community controls for a population-based case–control study of breast cancer.
Nineteen haplotype tagging single nucleotide polymorphisms (htSNPs) in four haplotype blocks were genotyped.
Significant associations were observed for WHR at three SNPs that are located in haplotype block 1, including rs2445765, rs1004984 and rs1902584 (P=0.05, 0.04 and 0.01, respectively). Women, particularly premenopausal women, who carried the minor allele at any of these SNPs, had higher WHR than those without it. Of these three SNPs, the strongest association was observed at rs1902584, which is the closest to Promoter I.4, the major promoter for adipose tissue. Haplotype analyses indicated an association between the haplotype TCCAT in block 1 and WHR with a P-value of 0.02.
These results suggested that CYP19A1 genetic polymorphisms may be associated with the risk of obesity among Chinese women, especially among premenopausal women.
The CYP19A1 protein (aromatase) plays a critical role in estrogen biosynthesis and thus affects body fat distribution and regulation.
Obesity has become a worldwide public health problem.1 About 250 million adults, roughly 7% of the world's adult population, are considered obese and two or three times as many may be overweight.2 Body mass index (BMI) and waist-to-hip ratio (WHR) are the commonly used indexes for overall obesity and central abdominal obesity, respectively. Obesity is associated with many chronic conditions, most notably cardiovascular disease, type II diabetes and certain types of cancer.1 Abdominal obesity has been shown to be more closely associated with these chronic diseases through its connection with insulin resistance, hyperinsulinemia, hypertension and hypertriglyceridemia.3 Substantial progress has been made in deciphering the pathogenesis of obesity, and a strong genetic contribution has been implicated.4, 5 Numerous molecular studies have been launched to search for genetic determinants underlying the variations of obesity-related phenotypes, involving more than 600 genes, markers and chromosomal regions.6
The CYP19A1 gene encodes the aromatase, an enzyme that catalyses the conversions of C19 androgens, androstenedione and testosterone, to C18 estrogens, estrone and estradiol, respectively.7, 8, 9 Testosterone inhibits preadipocyte proliferation and differentiation,10, 11 whereas estradiol appears to stimulate them.11 The CYP19A1 gene is expressed in gonadal sites such as the ovaries and testes, and extragonadal sites including adipose tissue. Aromatase-knockout mice progressively accumulate more intra-abdominal adipose tissue than their wild-type littermates,12 which suggests a role of the CYP19A1 gene in obesity pathogenesis. Several whole genome linkage scan studies showed a quantitative trait locus (QTL) for obesity on chromosome 15 q, a region where the CYP19A1 gene is located.13, 14, 15 The CYP19A1 gene is highly polymorphic, and it is conceivable that functional variants of this gene may be related to obesity, particularly central obesity. We evaluated this hypothesis among Chinese women recruited as part of the Shanghai Breast Cancer Study.
Materials and methods
Subjects and data collection
The study sample were recruited as community controls in the Shanghai Breast Cancer Study (SBCS),16 a population-based case–control study among Chinese women. Briefly, for SBCS, cases consisted of permanent Shanghai residents between the ages of 25 and 64 years who were diagnosed with breast cancer between August 1996 and March 1998. Among the 1602 eligible cases, 1459 (91%) participated in the SBCS study. Controls were randomly selected from the general population and frequency matched to case patients by age (5-year intervals) using files from the Shanghai Resident Registry, which contains demographic information for all adult residents of urban Shanghai. The inclusion criteria for controls were identical to those for cases with the exception of a breast cancer diagnosis. Of the 1724 eligible women, 1556 (90%) completed in-person interviews and 1310 controls (84%) provided blood samples. The blood samples were processed the day of collection at the Shanghai Cancer Institute, typically within 6 h of blood draw, and were stored at −70°C. Consent was obtained from all participants, and the study was approved by the institutional review boards of all participating institutions.
A structured questionnaire was used to elicit detailed information on demographic factors, menstrual and reproductive histories, hormone use, dietary habits, prior medical history, physical activity and tobacco and alcohol use. All participants were measured for their current weight, height and circumferences of the waist and hip. The measurements were taken twice according to a standard protocol by trained interviewers who were mostly retired nurses or physicians. A third measurement was taken if any difference between the first two measurements was greater than the tolerance limit (1 kg for weight and 1 cm for height and circumferences). The averages of the two closest measurements were used in the data analyses.
Genetic marker selection
The genetic markers included in the study were chosen using a recent report from the Multiethnic Cohort Study.17 Based on haplotype analyses of 74 single nucleotide polymorphisms (SNPs), Haiman et al.,17 identified four haplotype blocks (blocks 1–4) and 19 haplotype tagging SNPs (htSNPs) for the CYP19A1 gene in Japanese. All of these 19 htSNPs were included in the current study. Among them, five were located in haplotype block 1, five in block 2, three in block 3 and six in block 4.
Genomic DNA was extracted from buffy coats using a Puregene DNA purification kit (Gentra Systems, Minneapolis, MN, USA) following the manufacturer's protocol. SNP rs1004984 was genotyped using a high throughput Masscode assay at BioServe Biotechnologies, Ltd (Laurel, MD, USA). SNP rs700519 was genotyped by using polymerase chain reaction–restriction-fragment length polymorphism and confirmed by direct sequencing using BigDye Terminator Chemistry on an ABI 3700 (ABI). Genotyping of the other 17 SNPs was performed by running the 5′ nuclease Taqman allelic discrimination assay using an ABI 7900 (ABI, Applied Biosystems, Foster City, CA, USA). Details concerning assays, primers, probes and procedures are available upon request.
The laboratory staff was blind to the identity of the subjects. Quality control samples were included in the genotyping assays. Each 96-well plate of genomic DNA contained multiple quality controls, including one water, two samples of CEPH 1347-02, two known study duplicates and two blinded study duplicates. The agreement of the genotypes determined for the blinded quality control samples was 98.7%.
The χ2-test was performed to examine Hardy–Weinberg equilibrium (HWE) for each of the 19 SNPs. SNPs rs12907866 and rs700519 deviated significantly from HWE and they were excluded from the association analyses and haplotype reconstruction. Strong linkage disequilibrium (LD) was observed among the SNPs within each haplotype block (Nobuhiko et al., unpublished). Haplotypes were reconstructed based on SNPs within each block using program PHASE.18 The SNPs used for haplotype reconstruction were arranged according to their chromosomal locations in the order of rs2446405 – rs2445765 – rs2470144 – rs1004984 – rs1902584 for block 1, hCV1664178 – rs12900137 – rs730154 – rs936306 – rs1902586 for block 2, rs749292 – rs6493494 – rs1008805 for block 3 and rs727479 – rs2414096 – rs10046 – rs4646 for block 4.
The relationships between the polymorphisms with BMI and WHR were tested using analysis of covariance (ANCOVA), with age and physical activity adjusted as covariates. All statistical tests were based on two-sided probabilities using SAS, version 9.0 (SAS Institute Inc., Cary, NC, USA).
Among the 1310 eligible women who provided blood, genotypes were available for the CYP19A1 genetic polymorphisms among 1241 (95%), who were included in this study. Descriptive characteristics of these participants are shown in Table 1. The mean age of the study subjects was 49.2±8.7 years (mean±s.d.). Only 2.6% and 3.4% of the women had ever smoked or consumed alcohol on a regular basis, respectively. Postmenopausal women had significantly higher WHR and BMI, 0.815 and 24.2, than premenopausal women, 0.791 and 22.7, respectively. More postmenopausal women participated in regular physical activity than premenopausal women, 42.4 vs 16.6%. There was no significant difference between subjects included in this study and all the control subjects included in the Shanghai Breast Cancer Study with regard to the distribution of major demographic factors (data not shown).
Genotype and allele distributions and detailed information for the genetic markers are summarized in Table 2. Table 3 showed the results of association analyses. Significant associations between WHR and genotypes were observed at three SNPs that are located in haplotype block 1, that is, rs2445765, rs1004984 and rs1902584 (P=0.05, 0.04 and 0.01, respectively). Women with the minor allele at any of these SNPs tended to have higher WHR than those without it. Haplotype analyses indicated an association between haplotype TCCAT in block 1 and WHR with a P-value of 0.02 (Figure 1). Women having two copies of the haplotype TCCAT had the highest WHR 0.814 and those without this haplotype had the lowest WHR (0.797). Statistically significant associations for WHR were also observed at SNPs rs6493494 (in block 3) and rs10046 (in block 4) with P-values of 0.04 and 0.007, respectively. No association was observed between WHR and the other genetic markers. We did not find any significant association for BMI at any genetic marker (Table 3 and Figure 1).
Analyses stratified by menopause status indicated that the associations of genotypes with obesity were mainly evident in premenopausal women. Among premenopausal women, for the five studied SNPs located in haplotype block 1, significant associations with WHR were observed at three SNPs, that is, rs2445765, rs1004984 and rs1902584 and borderline associations were observed at the other two SNPs, rs2446405 and rs2470144 (Table 4). Haplotype analyses confirmed the above associations with a P-value of 0.005 (Figure 1). Women having two copies of the haplotype TCCAT had the highest WHR (0.822) and those without this haplotype had the lowest (0.788). Associations for WHR were also observed at rs6493494 (in block 3), and rs10046 (in block 4) with P-values of 0.04 and 0.03, respectively. For BMI, significant associations were observed at rs2445765, rs2414096 and rs10046 among premenopausal women (P=0.008, 0.02 and 0.03, respectively). Women carrying minor alleles of rs2445765 and rs10046 tended to have higher BMI, while minor allele of rs2414096 was associated with lower BMI. Haplotype analyses indicated a suggestive association between haplotype TCCAT in block 1 and BMI with a P-value of 0.09 (Figure 1). We did not find any association between any genetic markers with BMI and WHR among postmenopausal women. However, after correction for multiple testing, none of the above associations reach statistically significant.
In this large scale, population-based study, we found that genetic polymorphisms in the CYP19A1 gene were associated with obesity-related phenotypes among Chinese women, especially the polymorphisms located in haplotype block 1 among premenopausal women. These results are not unexpected, given the important role of the CYP19A1 gene in estrogen metabolism and in preadipocyte proliferation and differentiation.
The CYP19A1 protein, aromatase, may indirectly affect body fat distribution and regulation by modulating the ratio of androgens to estrogens in adipose tissue. Estrogen stimulates preadipocyte proliferation and differentiation through the α- and β-estrogen receptors, which have been found in subcutaneous and intra-abdominal fat cells.11 Conversely, testosterone may inhibit preadipocyte proliferation and differentiation and stimulate catecholamine-mediated lipolysis through the upregulation of β-adrenoreceptors.19 The total amount of estrogen synthesized by adipose tissue may be small, but the concentrations achieved in local tissue are probably high and thus exert significant biological influence locally.20 Such a source of estrogen may play an important, but as of yet largely unrecognized, physiological and pathophysiological role.20 CYP19A1 gene knockout mice showed not only elevated androgens but also abdominal obesity and insulin resistance,12 clearly indicating involvement of aromatase in the development of abdominal obesity. It is not surprising then that the observed associations of CYP19A1 polymorphisms with obesity in this study.
The CYP19A1 gene spans about 123 kb with a coding region of 9 exons (about 30 kb, exons II–X) and a 93 kb regulatory region (exon I). The regulatory region contains at least 10 distinct promoters that regulate in a tissue- or signaling pathway-specific manner.21 Among the 10 promoters, promoters I.3, I.4 and II are expressed in adipose tissue with promoter I.4 being the major one. Promoter I.4 is regulated by class I cytokines such as interleukin (IL)-6 and IL-11, as well as by tumor necrosis factor alpha (TNF-α).20 Downstream of the transcriptional start site for promoter I.4, there is a specificity protein-1 (Sp1) binding site.22 Upstream of the transcriptional start site of promoter I.4, there is a gamma interferon activating sequence (GAS) element, downstream of which is a glucocorticoid response element (GRE).22 All of these three elements are required for expression from promoter I.4.
The SNPs located in haplotype block 1 are close to promoter I.4, therefore they may have effects in regulating the transcription and affecting the activity of the aromatase in adipose tissue. Among the five study SNPs in haplotype block 1, rs1902584 has the shortest distance to the three response elements, 493 bp upstream of GAS, 646 bp upstream of GRE and 915 bp upstream of Sp1.22 Interestingly, the strongest association in our study was observed at this SNP, rs1902584. In premenopausal women, the majority of circulating estrogens are synthesized by granulosa ovarian cells. In postmenopausal women, however, estrogen is produced primarily through the conversion of androgens to estrogens catalyzed by aromatase in the adipose tissue. In our study, we found that the association of the CYP19A1 gene with obesity was restricted to premenopausal women. We have no special explanation for this specific association. It appears unlikely that circulating estrogens could contribute to the development of central obesity, since premenopausal women, on the average, have a much higher circulating estrogen level than postmenopausal women.
Signal of association was also observed for the SNPs located in haplotype block 3 and 4. Block 4 SNPs were located in the coding region and 3′ UTR, which may have functional significance by affecting mRNA stability or regulation of translation termination.23 These SNPs may also be in strong LD with other functional variants elsewhere in the block. Block 3 SNPs were between the promoter I.f and I.2, which were expressed in brain and placenta, respectively. We have no special explanation for the association of block 3 SNPs with obesity.
Among our Chinese premenopausal women, some SNPs were associated with WHR but not with BMI, while some SNPs were associated with BMI but not with WHR. A plausible explanation for this is as follows. WHR indicates abdominal obesity, while BMI suggests whole body obesity. Studies have shown that aromatase activity may differ across body sites. Aromatase activity is 6–30 times higher in fat taken from the upper thigh, buttocks and flank vs the abdomen.24, 25 It is also possible that aromatase regulation varies across fat deposit locations.26 Thus, the effect of CYP19A1 polymorphisms on obesity-related phenotypes may differ across the body.
In summary, we found that genetic polymorphisms of the CYP19A1 gene were related to obesity-related phenotypes among Chinese women, especially among premenopausal women. If these results are not by chance and can be confirmed in further studies, they might indicate that the CYP19A1 gene may modify the risk of obesity through estrogen biosynthesis.
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We wish to thank Drs Qi Dai and Fan Jin and Ms Jia-Rong Cheng for their contributions in coordinating data and specimen collection in Shanghai, Ms Qing Wang and Ms Regina Courtney for technical assistance in genotyping assays and Ms Allison Reed for technical assistance in the preparation of this manuscript. This study was supported by National Cancer Institute USPHS RO1CA64277 and RO1CA90899.
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Cite this article
Long, J., Shu, X., Cai, Q. et al. CYP19A1 genetic polymorphisms may be associated with obesity-related phenotypes in Chinese women. Int J Obes 31, 418–423 (2007) doi:10.1038/sj.ijo.0803439
- genetic polymorphism
- body mass index
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