Mol Biol Rep (2013) 40:3315–3320 DOI 10.1007/s11033-012-2406-1
Common genetic variation in CYP1B1 is associated with concentrations of T4, FT3 and FT4 in the sera of polycystic ovary syndrome patients Shien Zou • Qing Sang • Huan Wang • Ruizhi Feng • Qiaoli Li • Xinzhi Zhao • Qinghe Xing • Li Jin • Lin He • Lei Wang
Received: 4 September 2012 / Accepted: 18 December 2012 / Published online: 3 January 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract CYP1B1 encodes an estrogen enzyme that oxidizes 17b-estradiol to 4-hydroxyestradiol. The evidence demonstrates there may be a relationship between CYP1B1 and thyroid function. To date, no study has evaluated if genetic polymorphisms that regulate concentrations of serum FT3 and FT4 contribute to Polycyctic Ovary Syndrome (PCOS). To identify polymorphisms in the CYP1B1 locus associated with PCOS, we genotyped three common polymorphisms across the CYP1B1 locus in 226 patients. A test for association of common variants with susceptibility to PCOS was conducted in a large cohort of 609 subjects. The functional polymorphism CYP1B1 L432V (rs1056836) is associated with serum T4 (P = 0.003), serum FT3 (P \ 0.001) and serum FT4 concentrations
Shien Zou, Qing Sang and Huan Wang contribute equally to the paper S. Zou Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China Q. Sang L. Jin L. Wang State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China Q. Sang H. Wang R. Feng Q. Li X. Zhao Q. Xing L. He L. Wang (&) Institutes of Biomedical Science, Fudan University, No. 138 Yixueyuan Road, Shanghai 200032, China e-mail:
[email protected] X. Zhao Q. Xing L. He (&) L. Wang Bio-X Center, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200030, China e-mail:
[email protected]
(P \ 0.001). Our study provides the first evidence that genetic variants in CYP1B1 can be associated with serum T4, FT4 and FT3 levels in PCOS. These findings imply novel pathophysiological links between the CYP1B1 locus and thyroid function in PCOS. Keywords PCOS CYP1B1 Free thyroxin concentration Free triiodothyronine concentration Abbreviations PCOS Polycystic ovary syndrome CYP1B1 Cytochrome P450, subfamily1, polypeptide1 T4 Thyroxin FT4 Free thyroxin T3 Triiodothyronine FT3 Free triiodothyronine SNP Single nucleotide polymorphism LD Linkage disequilibrium TSH Thyroid stimulating hormone
Introduction Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects 5–10 % of the general population and is thought to be one of the leading causes of female subfertility [1]. PCOS is associated with insulin sensitivity and glucose tolerance [2]. In addition, PCOS patients have higher mean Thyroid stimulating hormone (TSH) levels and higher incidences of TSH levels above the upper limit of normal, demonstrating that thyroid dysfunction plays an important role in the phenotypic expression of the disorder [3]. Recently, we have identified a 30 -UTR polymorphism in Gnrhr that is associated with the concentration of serum thyroid stimulation hormone as well as insulin secretion in
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polycystic ovary syndrome patients [4]. Clinically, free thyroxin (FT4) and free triiodothyronine (FT3) are two indexes used to evaluate thyroid function; no study has examined genetic polymorphisms associated with the regulation of concentrations of serum FT3 and FT4 in PCOS. CYP1B1 is a phase I, estrogen enzyme, belonging to a multi-gene super family of monomeric mixed function monooxygenases, that oxidizes 17b-estradiol to 4-hydroxyestradiol [5]. Evidence has shown that estrogen increases thyroid cell proliferation, thyroperoxidase activity and iodide uptake. Iodine/iodide has been reported to alter mRNA expression of several genes involved in the estrogen pathway, including CYP1B1 in MCF7 breast cancer cell line [6, 7]. In addition, an increase in testicular CYP1B1 protein levels has been reported in T3-treated hypophysectomized adult male rats [8]. Altogether, the evidence implies that there may be crosstalk between CYP1B1 expression and thyroid hormone secretion. Compared with normal ovaries, CYP1B1 is down-regulated in PCOS ovaries, implying a relationship between CYP1B1 and the etiology of PCOS [9]. Thus, we explored the role of CYP1B1 genetic variation in PCOS samples that display several endocrine traits, including thyroid function. Using a large cohort (N = 609), we also investigated whether polymorphisms in the CYP1B1 gene are associated with PCOS.
Methods
Mol Biol Rep (2013) 40:3315–3320
Serum thyroid stimulating hormone, T3, T4, FT3 and FT4 were measured using ELISA (VIDAS Reproduction Hormones kit and thyroid function kit, Biomerieux Corp, France). We recruited 383 healthy, nondiabetic women volunteers (mean age (SEM): 29.7(0.3)) with regular menstrual cycles to serve as a control group. None of the controls were diagnosed with hyperandrogenism or any other endocrine disorders related to PCOS. All patients and controls were of Han Chinese descedant and were recruited from the same area. The Fudan University Ethics Review Committee approved the study, and informed consent was obtained from all participants. Genotyping Genomic DNA was extracted from peripheral blood using standard DNA extraction methods. The three SNPs (rs2551188, rs1056836, rs9341266), from the HapMap project database (http://www.hapmap.org), were selected to cover an 8.5 kb region of CYP1B1. The SNPs are all tag SNPs, which will provide enough genetic information in the later association study. The three SNPs were genotyped using Taqman allelic discrimination assays (Assay-on-Demand or Assay-on-design): Assay ID C_15895262_10 was used to genotype for Rs2551188; Assay ID C_3099976_30 was used to genotype for Rs1056836; and, Rs9341266 was determined by Assay-on-demand.
Subjects
Statistical analysis
A total of 226 PCOS patients (mean age (SEM): 27.3(0.2)) were recruited from the outpatient clinic of the Xi’an fourth hospital and the Shaanxi hospital for women and children. Recruitment was based on the revised Rotterdam diagnostic criteria [10]. Women who met at least two of the following three criteria were defined as PCOS cases: [1] oligo-ovulation and/or anovulation; [2] clinical and/or biochemical signs of hyperandrogenism; and [3] and polycystic ovaries. Other causes of hyperandrogenism or menstrual disorder were excluded. None of the patients had taken hormonal medication, including oral contraceptives, for at least 3 months before starting the study. Standardized initial screening was performed on a random cycle day between 09:00 and 11:00 h. Fasting blood withdrawal was performed on the third day of follicular phase between 09:00 and 11:00 h. Serum levels of luteinizing hormone, follicle stimulating hormone and prolactin were measured by ELISA (Human LH, FSH, Prolactin kit, Fenghua Corp., China). Testosterone was measured by RIA (Coat-A-Count free testosterone kit; Diagnostic Products Corp., LA, CA). Estradiol and progesterone were measured by a radioimmunoassay protocol as previously described [11].
Quantitative variables were expressed in our analysis as the mean ± standard error mean (SEM). The Hardy–Weinberg equilibrium test was performed using the Chi-squared test run on Plink (http://pngu.mgh.harvard.edu/*purcell/plink/). Genotypes and allele frequencies for the case and control groups were compared using the chi-squared test. Pairwise linkage disequilibrium was tested using Haploview [12]. Haplotype frequencies were estimated by SHEsis [13]. Differences in haplotype frequency distributions between the PCOS group and the control group were assessed using the CLUMP program. Biochemical differences between two continuous variables were estimated using the Mann–Whitney U test or t test, as appropriate. One-way analysis of covariance (ANOVA) was used to analyze the associations between genotype and clinical features in the PCOS group. Clinical features of interest were chosen as dependent variables, with BMI acting as the covariate. P \ 0.05 was considered to be statistically significant. Unless otherwise stated, analyses were performed using Statistical Package for Social Sciences, SPSS, version 11.5 (SPSS Inc., Chicago, IL).
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GG genotype showed significantly higher serum T4, FT4 and FT3 concentrations compared to patients with GC and CC genotypes (Fig. 2a, b, c).
Results CYP1B1 common genetic variant rs1056836 association with serum T4, FT3 and FT4 in PCOS
Association of CYP1B1 polymorphisms with PCOS SNPs position and CYP1B1 gene structure were shown in Fig. 1. We tested whether three common genetic variants across the CYP1B1 locus were associated with several clinical metabolic traits in 226 PCOS patients. Only rs1056836 was found to be associated with serum T4, serum FT3 and serum FT4. Clinical and endocrine/metabolic characteristics of the 226 patients by rs1056836 genotype are presented in Table 1. Rs1056836 was found to be associated with serum T4 concentration (P = 0.003, Mann–Whitney U test, Fig. 2a), serum FT3 concentration (P \ 0.001, Mann–Whitney U test, Fig. 2b), and serum FT4 concentration (P \ 0.001, Mann–Whitney U test, Fig. 2c). In addition, patients with
The three common genetic variants were further scored in 609 individuals (226 PCOS patients and 383 healthy controls). Clinically characteristic PCOS cases and controls are shown in Table 2. Genotype and allele frequencies for the three polymorphisms in the PCOS cases did not differ from those in the healthy controls (Table 3). The genotypes of the three polymorphisms were in Hardy–Weinberg equilibrium in both cases and controls (data not shown). We also analyzed linkage disequilibrium for each pair of the SNPs in the PCOS and control subjects (Table 4). A haplotype analysis was performed among the three SNPs. None of the haplotypes were associated with PCOS (data not shown).
Discussion In this study, we investigated if common genetic variation in the CYP1B1 locus was associated with thyroid function of PCOS and etiology of PCOS. In a study of PCOS
Fig. 1 The Structure of CYP1B1 gene and location of the SNPs in CYP1B1 gene. The Position of SNPs are indicated by vertical lines Table 1 Clinical and metabolic characteristics of PCOS women (n = 226) by CYP1B1 rs1056836 (G/C) genotype
The data represent the mean ± SEM; the P value was determined using one-way ANOVA analysis (when data is in normal distribution) or the Mann–Whitney U test (when data is not in normal distribution); the values with P \ 0.01 are in bold LH luteinizing hormone, FSH follicle stimulating hormone, PRL prolactin, E2 estradiol, P progesterone, T testosterone, TSH thyroid stimulating hormone, T3 triiodothyonine, T4 thyroxin, FT3 free triiodothyonine, FT4 free thyroxin, GLU glucose, INS insulin
GG N BMI
CG 4
19.91 ± 0.89
CC 56
22.40 ± 0.49
P
166 22.44 ± 0.26
0.344
LH (miu/ml)
4.31 ± 1.55
5.37 ± 0.64
5.12 ± 0.34
0.872
FSH (miu/ml)
6.64 ± 0.49
5.80 ± 0.23
6.44 ± 0.30
0.499
PRL (ng/ml)
19.15 ± 4.65
24.85 ± 2.32
25.02 ± 1.23
0.754
E2 (pg/ml)
45.22 ± 6.34
49.57 ± 4.38
50.03 ± 3.24
0.968
P (ng)
1.04 ± 0.09
0.87 ± 0.04
1.70 ± 0.34
0.396
T (ng/ml) TSH (miu/ml)
0.36 ± 0.08 2.72 ± 0.84
0.50 ± 0.04 3.22 ± 0.26
0.45 ± 0.03 3.57 ± 0.19
0.477 0.519
T3 (nmol/l)
2.43 ± 0.54
1.71 ± 0.06
2.35 ± 0.70
0.870
T4 (nmol/l)
119.53 ± 36.14
88.20 ± 2.63
88.23 ± 1.26
0.003
FT3 (pmol/l)
7.46 ± 2.72
4.47 ± 0.10
4.59 ± 0.09
