Original Paper Received: June 25, 2015 Accepted after revision: October 7, 2015 Published online: November 25, 2015
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
Common Variants in the Sex Hormone-Binding Globulin (SHBG) Gene Influence SHBG Levels in Women with Polycystic Ovary Syndrome Tala M. Abu-Hijleh a Emily Gammoh a Amna S. Al-Busaidi a Zainab H. Malalla a Samira Madan b Naeema Mahmood b Wassim Y. Almawi a
Department of Medical Biochemistry, Arabian Gulf University, b Department of Obstetrics and Gynecology, Salmaniya Medical Complex, Manama, Bahrain
Key Words Haplotypes · Hyper-androgenism · Polycystic ovary syndrome · Sex hormone binding globulin
Abstract Background: Decreased sex hormone-binding globulin (SHBG) levels were associated with polycystic ovary syndrome (PCOS). SHBG polymorphisms associated with reduced SHBG production were tested for their association with PCOS, but with inconclusive results. We tested whether altered SHBG levels and SHBG variants were associated with PCOS. Methods: The study subjects included 242 women with PCOS and 238 control women. SHBG genotyping was done by real-time PCR. Results: Higher minor allele frequency of rs13894, rs858521 and rs727428 was seen in PCOS cases, and significant differences in rs858521 and rs727428 genotypes distribution were seen between PCOS cases and controls. Multivariate regression analysis confirmed the association of only rs727428 with PCOS. Though it was not statistically significant, serum SHBG levels were reduced according to rs727428 genotypes in PCOS cases than in controls. Carriage of rs727428 minor allele was associated with significant increases in free/bioactive testosterone in PCOS cases. Seven-locus (rs9898876-rs13894-rs858521-rs1799941rs6257-rs6259-rs727428) haploview analysis showed increased frequency of GCCGTGA, GTCGTGA and GTCATGG,
© 2015 S. Karger AG, Basel 0250–6807/15/0681–0066$39.50/0 E-Mail [email protected] www.karger.com/anm
and reduced frequency of GTCGTGG haplotypes in PCOS cases than in controls, thus conferring disease susceptibility and protective nature to these haplotypes, respectively. Conclusion: Specific SHBG variants affecting serum SHBG levels and SHBG haplotypes are associated with PCOS, suggesting the role for SHBG as PCOS candidate gene. © 2015 S. Karger AG, Basel
Introduction
Polycystic ovary syndrome (PCOS) is a common multifactorial endocrinopathy, which affects 5–15% of premenopausal women [1, 2]. As stipulated in the Rotterdam criteria, PCOS is characterized by clinical and biochemical hyperandrogenism, in association with anovulation and polycystic ovaries [3]. PCOS is associated with metabolic and reproductive disorders, such as obesity [3], glucose intolerance and insulin resistance (IR) and dyslipidemia [4, 5], and hence an increased risk of arteriosclerosis [6] and type 2 diabetes mellitus (T2DM) [4, 6]. PCOS is multigenic in nature, evidenced by familial clustering of PCOS [7, 8] and by twin studies, which suggested that PCOS pathogenesis is largely (∼70%) due to genetic influences [8, 9]. Sex hormone-binding globulin (SHBG) is a 373-amino acid glycoprotein produced mainly by the liver, which Wassim Y. Almawi, PhD Department of Medical Biochemistry College of Medicine and Medical Sciences, Arabian Gulf University PO Box 22979, Manama (Bahrain) E-Mail wassim @ agu.edu.bh
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
a
Subjects and Methods
Bahraini population. PCOS diagnosis was based on the 2003 Rotterdam criteria, which required 2 of the 3 conditions to be met: (1) anovulation, (2) hyperandrogenism assessed by the presence of hirsutism (modified Ferriman–Gallwey score: >8, acne in the third decade of life, or positive androgenic alopecia) and (3) the presence of polycystic ovary on ultrasound examination. Exclusion criteria included androgen-producing tumors, 21-hydroxylase-deficiency, non-classical adrenal hyperplasia, hyperprolactinemia, active thyroid disease and Cushing’s syndrome. Additional exclusion criteria included extremes of body mass index (BMI; 50 kg/m2), recent/current illness, medications likely to affect carbohydrate metabolism or endocrine parameters for at least 3 months before entering the study. The latter included oral contraceptives and anti-hypertensive, lipid-lowering and anti-inflammatory agents. Demographic data and history of hypertension, diabetes and hypercholesterolemia were taken from all subjects. All participants gave written informed consent, and the local research and ethics committees approved the study. Biochemical Analysis A fasting blood sample was obtained for biochemical and hormonal determinations. Serum samples were analyzed for SHBG by sandwich ELISA (R&D Systems, Minneapolis, Minn., USA); assay sensitivity was 0.01 nmol/ml, and inter-assay and intra-assay precision (CV%) were 5.3 and 4.3%, respectively. Serum luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, testosterone and glucose (ADVIA Centaur, Bayer Vital, Fernwald, Germany) and insulin (IMMULITE 2000, DPC Biermann, Bad Nauheim, Germany) were measured by automated chemiluminescence immunoassays. FT and bioactive testosterone (BT) and free androgen index were determined using Free and Bioavailable Testosterone Calculator (www.issam.ch/freetesto. htm). IR was estimated by the homeostasis model assessment (HOMA-IR), which is defined as fasting serum insulin (μIU/ml) × fasting plasma glucose (mmol/l)/22.5. SHBG Genotyping We selected polymorphisms in SHBG gene with a minor allele frequency (MAF) of >5% in Caucasians, using the SNPbrowser software (version 4.0, Applied Biosystems, Foster City, Calif., USA). SHBG genotyping was performed by the allelic (VIC- and FAM-labeled) discrimination method. Assay-on-demand TaqMan primer pairs for the following SNPs: rs9898876, rs13894, rs858521, rs1799941, rs6257, rs6259 and rs727428 were ordered from Applied Biosystems. The reaction was performed in 6-μl volume on StepOne Plus real-time PCR system, according to manufacturer’s instructions (Applied Biosystems). Replicate blinded quality control samples were included to assess reproducibility of the genotyping procedure; concordance was >99%.
Subjects A total of 389 unrelated self-reported Bahraini Arab women with (n = 242) and without (n = 238) PCOS were recruited from the outpatient endocrinology and obstetrics and gynecology clinics in Manama, Bahrain. Control women comprised eumenorrheic university students and employees, or otherwise healthy volunteers, without clinical evidence of PCOS. Their androgen levels were within the reference range (0.4–3.5 nmol/l) and were studied in the follicular phase of their menstrual cycle. Control women reported no health problems and were recruited from the 5 governorates of Bahrain, and thus were representative of present-day
Statistical Analysis Statistical analysis was performed on SPSS version 22.0 (IBM). Data were expressed as percentages of total (categorical variables) or as mean ± SD (continuous variables). Student’s t test was used in determining differences in means, and Pearson χ2 or Fisher’s exact test was used to assess inter-group significance. SNP genotypes were tested for Hardy–Weinberg equilibrium (HWE) in control women and PCOS cases by Haploview version 4.2 (http://www.broad.mit.edu/mpg/haploview). Genetic Power Calculator (http://pngu.mgh.harvard.edu/∼purcell/cgi-bin/cc2k.cgi)
SHBG Variants and Serum Levels in PCOS
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
67
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
acts by binding circulating steroid sex hormones, including testosterone and estradiol [10, 11], thereby limiting their target tissue bioavailability [10, 12], and is a key regulator of the steroid-signaling system [13]. Increased free testosterone (FT) results in its peripheral conversion to the potent androgen, dihydrotestosterone, and thus leads to induction of hyperandrogenism [14, 15], a hallmark of PCOS [16, 17]. SHBG plays a central role in PCOS pathophysiology, as evidenced by the findings that women with PCOS have increased levels of free circulating androgens and IR, which in turn inhibit the hepatic production and secretion of SHBG [17]. Located on the short arm of chromosome 17 (17p131p12) [18], several genetic variants in the human SHBG gene were reported. Studies documented association between SHBG polymorphisms with altered glucose homeostasis, some of which linked it with altered SHBG levels, while others suggested that it was independent of changes in SHBG levels [16, 19–22]. In view of the link between PCOS and altered glucose homeostasis and IR [4, 23], it was suggested that altered SHBG secretion, stemming from specific SHBG polymorphisms, might contribute to PCOS pathogenesis. For example, studies on predominantly Caucasian populations demonstrated differential association of rs6257, rs6259 (D356N), rs727428 and rs1799941 SHBG variants with decreased SHBG levels and PCOS presence [16, 19, 20, 24]. Other SHBG variants, including rs13894, rs858521 and rs9898876, were also investigated as to their association with altered SHBG levels, but in T2DM and IR [21, 22, 25, 26]. Such a study has yet to be carried out in Middle Eastern women. The aim of the present study was to explore the association of SHBG rs6257, rs6259, rs727428 and rs1799941, in addition to rs13894, rs858521 and rs9898876 SNPs in 242 Bahraini Arab women with PCOS and in 238 control women. This is the first study to examine this association in a Middle Eastern population.
Table 1. Baseline and endocrine parameters of women with PCOS and control women
Parameter
Cases1
Controls1
p value2
Age, years BMI, kg/m2 Menarche, years Luteinizing hormone, IU/l Follicle stimulating hormone, IU/l Thyroid stimulating hormone, μIU/ml Fasting insulin HOMA-IR Total testosterone, nmol/l FT, pmol/l Bioavailable testosterone, pmol/l Free androgen index SHBG, nmol/l High-density lipoprotein cholesterol, mmol/l Triglycerides, mmol/l
28.7±6.1 29.1±6.7 12.4±1.4 5.8 (0.8–56.3) 5.3 (0.5–44.9) 2.8±2.2 11.4 (1.6–118.7) 4.3±2.7 1.5±1.0 28.9±19.4 655.1±445.4 7.7±4.1 33.8±18.8 1.3±0.5 1.4±0.9
27.5±7.0 26.2±5.3 12.6±1.4 5.4 (0.4–66.6) 5.2 (0.4–18.6) 1.8±1.1 6.4 (1.7–31.8) 1.9±0.8 1.4±1.0 20.9±16.4 455.6±288.9 3.2±2.2 67.1±46.1 1.5±0.5 1.1±0.7
0.072 5.0 × 10–6 0.266 0.266 0.824 0.004 2.7 × 10–4 1.4 × 10–4 0.469 0.003 2.2 × 10–4 0.003 1.0 × 10–6 0.383 0.068
1
A total of 242 PCOS cases and 238 control women were included. t test (variables with normal distribution), Mann–Whitney U test (variables that were not normally distributed). Values are mean ± SD and percentage of total within each group/subgroup. 2 Student’s
Results
Study Subjects The clinical and biochemical characteristics of study subjects are presented in table 1. Age at examination, menarche, along with fasting serum HDL cholesterol and triglycerides, as well as follicle stimulating hormone, luteinizing hormone and total testosterone were compara68
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
ble between women with PCOS and control women. Significant differences in mean BMI (p < 0.001), FT (p = 0.003) and BT (p < 0.001) and free androgen index (p = 0.003), as well as in fasting insulin (p < 0.001) and HOMAIR (p < 0.001), were seen between PCOS cases and control women. In addition, significantly lower serum levels of SHBG (p < 0.001) and higher levels of thyroid stimulating hormone (p = 0.006; though within reference range) were seen between women with PCOS and control women. Accordingly, these were selected as the covariates that were controlled for in later analysis. SHBG SNPs Analyses The 7 tested SHBG variants were in HWE in control women (p > 0.05), while deviation from HWE was noted for rs9898876, rs13894 and rs6259 in women with PCOS (table 2). Of the tested SHBG SNPs, higher MAF of rs13894 (p = 0.011; χ2 = 6.445) and rs727428 (p = 0.002; χ2 = 9.735), and lower MAF of rs858521 (p = 0.015; χ2 = 5.973) were seen in women with PCOS compared to control women; MAF of the remaining SNPs were comparable between PCOS cases and control women (table 2). The calculated ORs were 1.467, 1.582 and 0.697 for rs13894, rs727428, and rs858521, respectively, after controlling for key covariates. Significant difference in rs858521 (p = 0.020) and rs727428 (p = 0.002) genotype distribution was seen beAbu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
was used in calculating the power for detecting an association between SHBG variants and PCOS. The parameters used were 242 women with PCOS and 238 control women, genotypic relative risk for heterozygote (1/2) and minor allele homozygous (2/2), and the MAF for PCOS cases and controls for the 7 tested SNPs, and assuming a 14.0% population prevalence of PCOS (unpublished Bahrain Ministry of Health statistics). Assuming these parameters, we calculated the overall power (71.2%) as the average power of the 7 tested SNPs. All analyses were conducted under additive genetic effect, as it is a conservative model, using SNPStats (bioinfo.iconcologia.net/ snpstats/). This assumes that the risk conferred by an allele increases r-fold for heterozygotes and r2-fold for homozygotes. Linkage disequilibrium analysis was performed by Haploview 4.2, and haplotype reconstruction was performed by the expectation maximization method (Haploview 4.2). Logistic regression analysis was performed to determine the odds ratios and 95% confidence intervals associated with the risk of PCOS, taking control women as the reference group. Statistical significance was set at values of p < 0.05.
Table 2. SHBG SNPs analyzed
SNP
rs9898876 rs13894 rs858521 rs1799941 rs6257 rs6259 rs727428
Location1
7623644 7626584 7626829 7630105 7630399 7633209 7634474
Alleles
G:T C:T C:G G:A T:C G:A G:A
Cases
Controls
HWE
MAF
HWE
MAF
0.043 0.022 0.120 1.000 0.025 1.000 0.745
0.126 0.401 0.386 0.182 0.186 0.042 0.055
0.480 0.063 0.420 0.437 0.165 1.000 0.110
0.107 0.321 0.479 0.183 0.183 0.057 0.435
Power2 χ2
p value
86.7 68.9 74.8 79.0 56.8 62.2 69.9
0.508 0.011 0.015 0.999 0.727 0.292 0.002
0.439 6.445 5.973 0.000 0.122 1.113 9.735
aOR (95% CI)3
1.467 (1.091–1.973) 0.697 (0.521–0.931)
1.582 (1.185–2.112)
1
Location on chromosome based on dbSNP build 125. Study power (%) based on case–control statistics (B vs. b). 3 Covariates that were adjusted for were BMI, HOMA-IR, SHBG and FT. 2
Table 3. Genotype frequencies of SHBG polymorphisms
SNP
Genotype
PCOS cases
Controls
p value
OR (95% CI)
p value
aOR (95% CI)1
rs9898876
G/G G/T T/T
189 (78.1)2 45 (18.6) 8 (3.3)
191 (80.3) 43 (18.1) 4 (1.7)
0.46
1.00 (reference) 1.06 (0.63–1.78) 2.37 (0.58–9.65)
0.22
1.00 (reference) 1.10 (0.50–2.40) 7.31 (0.64–83.37)
rs13894
C/C C/T T/T
97 (40.1) 96 (39.7) 49 (20.2)
117 (49.2) 89 (37.4) 32 (13.4)
0.07
1.00 (reference) 1.33 (0.86–2.07) 1.93 (1.08–3.44)
0.14
1.00 (reference) 1.05 (0.54–2.03) 2.20 (0.95–5.09)
rs858521
C/C C/G G/G
98 (40.5) 101 (41.7) 43 (17.8)
77 (32.4) 94 (39.5) 67 (28.1)
0.05
1.00 (reference) 0.86 (0.55–1.37) 0.52 (0.30–0.90)
0.27
1.00 (reference) 1.34 (0.68–2.64) 0.69 (0.31–1.54)
rs1799941
G/G G/A A/A
162 (66.9) 72 (29.8) 8 (3.3)
167 (70.2) 55 (23.1) 16 (6.7)
0.17
1.00 (reference) 1.32 (0.83–2.09) 0.53 (0.20–1.43)
0.56
1.00 (reference) 1.44 (0.74–2.81) 1.17 (0.20–7.00)
rs6257
T/T T/C C/C
165 (68.2) 64 (26.4) 13 (5.4)
165 (69.3) 59 (24.8) 14 (5.9)
0.93
1.00 (reference) 0.92 (0.58–1.46) 1.06 (0.45–2.53)
0.75
1.00 (reference) 0.79 (0.40–1.54) 1.14 (0.27–4.81)
rs6259
G/G G/A
222 (91.7) 20 (8.3)
211 (88.7) 27 (11.3)
0.34
1.00 (reference) 0.72 (0.36–1.43)
0.34
1.00 (reference) 0.63 (0.24–1.65)
rs727428
G/G G/A A/A
58 (23.7) 101 (41.9) 83 (34.4)
69 (29.0) 131 (55.0) 38 (16.0)
2.0 × 10–4
1.00 (reference) 0.93 (0.57–1.53) 2.65 (1.48–4.76)
0.002
1.00 (reference) 0.94 (0.46–1.91) 3.91 (1.50–10.18)
1 Covariates
that were controlled for were BMI, HOMA-IR, SHBG and FT. of subjects (percent total).
tween women with PCOS and control women (table 3); the genotype distribution of the remaining SNPs was comparable between cases and controls. Binary logistic regression confirmed this association, which included PCOS or control status as an independent variable and
the SHBG SNPs as independent variables. Taking homozygous wild-type genotype as reference (OR 1.00), increased PCOS risk was seen with rs727428 minor-allele homozygous genotype (OR 2.65, 95% CI 1.48–4.76), while reduced PCOS risk was associated with rs858521
SHBG Variants and Serum Levels in PCOS
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
69
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
2 Number
Table 4. SHBG values for women with PCOS according to SHBG SNP genotype1
SNP
1/1
1/2
3/3
p value2
rs9898876 rs13894 rs858521 rs1799941 rs6257 rs6259 rs727428
49.7±34.3 48.6±39.1 46.1±32.4 47.5±40.0 50.0±43.9 48.1±41.1 71.4±50.6
48.0±36.2 53.9±41.9 48.8±31.8 50.4±44.1 49.5±36.8 58.4±41.8 51.1±40.9
63.9±31.5 40.6±32.9 53.9±34.4 70.6±48.5 45.8±32.3 – 32.0±23.6
0.589 0.100 0.540 0.173 0.785 0.222 0.008
1
Genotypes were coded as 1/1 (homozygous major allele), 1/2 (heterozygous) and 2/2 (homozygous minor allele). 2 One-way analysis of variance (2-tailed). Values are mean SHBG ± SD (nmol/l).
Table 5. Multivariate analyses for association between serum SHBG and rs727428 genotype, BMI, testosterone and IR in women with PCOS
SNP
G/G
G/A
A/A
p value1
BMI Fasting insulin HOMA-IR Total testosterone FT BT
28.1±5.8 15.4±12.0 3.1±2.3 1.42±1.07 23.8±16.1 0.56±0.39
29.4±6.5 16.8±11.2 4.0±2.6 1.43±1.03 30.3±21.1 0.65±0.49
28.2±5.8 17.5±10.9 4.7±2.8 1.57±0.88 36.6±22.3 0.81±0.50
0.396 0.292 0.422 0.826 0.039 0.015
1 One-way analysis of variance (2-tailed). Values are mean ± SD.
Influence of rs727428 on SHBG Serum Levels and PCOS Features We analyzed the influence of rs727428 genotypes on SHBG serum levels, insulin status, lipid profile and testosterone levels. Carriage of rs727428 minor allele was not influenced by menarche or BMI, and did not affect IR (HOMA-IR) or serum lipids (table 5). Compared with major-allele homozygous (G/G) genotype carriers, there was a progressive increase in FT (p = 0.039) and BT (p = 0.015) in rs727428 genotypes in heterozygous (G/A), and more so in minor-allele homozygous (A/A) genotype carriers (table 5). 70
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
Haploview Analysis Haploview analysis revealed limited linkage disequilibrium (LD) between the 7 tested SHBG variants (fig. 1). Extensive diversity in the haplotype assignment was seen, with the majority of haplotypes (81.1%) captured by 11 haplotypes, which were thus designated as ‘common’. Taking GCGGTGG haplotype as common (OR 1.00), there was positive association of GCCGTGA, GTCGTGA and GTCATGG haplotypes, and negative association of GTCGTGG haplotype with PCOS (table 6). The association was confirmed by multivariate regression analysis after controlling for BMI, HOMA-IR, SHBG and FT.
Discussion
Previous studies investigated the role of SHBG in PCOS pathophysiology, but with inconclusive findings. In the present study, we assessed the association of SHBG Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
minor-allele homozygous genotype (OR 0.52, 95% CI 0.30–0.90; table 4), thus assigning PCOS susceptibility and protective nature to these genotypes, respectively. Multivariate regression analysis, confirmed the positive association of only rs727428 minor-allele homozygous genotype (adjusted OR 3.91, 95% CI 1.50–10.18; table 4).
rs6259
rs727428
3
4
5
6
7
Block 1 (10 kb) 2
1
3
0 71
56 2
70
84 73
9 89
25 77
0 91
55 11
11 41
2 95
Fig. 1. Haploview graph of SHBG SNPs analyzed. The positions of
the 7 SNPs used (build 37.3) are indicated along with the basic gene structure and displayed above the Haploview output. The relative LD between specific pair of SHBG SNPs is indicated by the color scheme, which represents the LD relationships. This is based on D’ values (normalized linkage disequilibrium measure or D) multiplied by 100; D’ is calculated as D divided by the theoretical maximum for the observed allele frequencies. Values approaching zero indicate absence of LD, and those approaching 100 indicate complete LD. The squares colored red represent varying degrees of LD 2 scores; darker shades indicating stronger LD.
71
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
Color version available online
rs6257
rs13894
rs1799941
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
rs858521
SHBG Variants and Serum Levels in PCOS
rs9898876
polymorphisms with altered serum SHBG levels and the presence of PCOS. Four of the tested SNPs (rs6259, rs6257, rs1799941 and rs727428) were previously studied in relation to their association with PCOS, but with variable results [19, 20, 27]. The remaining 3 SNPs (rs989887, rs13894, rs858521) were also investigated, but in relation to their effects on SHBG levels and cancer and osteoporosis [21, 22, 25, 26, 28]. Our study demonstrated a significant link between rs727428, but not the other studied SNPs, and PCOS and hyperandrogenism in Bahraini women. To our knowledge, this is the first study to evaluate the association of SHBG polymorphism and changes in SHBG levels in the Arab-speaking community. All subjects included were self-reported Bahraini Arab women and were consecutively enrolled, so as to minimize the possibility of type II errors. PCOS diagnosis was based on the 2003 Rotterdam criteria, as compared with other studies in which the National Institute of Health
diagnostic criteria were used [16, 19]. As some of SHBG SNPs studied, notably rs1799941, rs6257 and rs6259, were previously linked with T2DM [29, 30], we excluded women with T2DM or IR in order to limit the possible contribution of altered glucose homeostasis. Our study population differed from those included in earlier studies with regards to mean BMI and testosterone. For example, mean BMI was higher than that reported in the study by Wickham et al. [20], but comparable to that of the Mediterranean women in the study by Martínez-García et al. [16], while both FT and total testosterone levels were lower than it was in the subjects of the study by MartínezGarcía et al. [16]. Accordingly, BMI, HOMA-IR, SHBG and FT were the main covariates that were controlled for in subsequent analysis. SHBG levels inversely correlated with age (r = –0.138; p = 0.018), FT (r = –0.481; p = 1.0 × 10–7), HOMA-IR (r = –0.196; p = 0.001), but not with BMI, among women with PCOS. SHBG is a sensitive biomarker of IR and the metabolic syndrome, and the low SHBG serum levels seen in women with PCOS linked with IR (HOMA-IR) is the result of inhibition of hepatic SHBG production, as shown elsewhere [31, 32]. SHBG levels have also been shown to correlate with both fat mass [31, 33] and BMI [33, 34], and visceral adiposity positively correlated with SHBG levels [35]. In our hands, there was lack of correlation between SHBG levels and BMI (p = 0.289; r2 = 0.014), which persisted after controlling for HOMA-IR (p = 0.552; r2 = 0.004), suggesting lack of contribution of obesity to altered SHBG secretion. Population-based studies documented strong association between altered testosterone and low SHBG levels and PCOS [36, 37]. Recent studies on Turkish [38], Mediterranean [16], European [19] and US [20] women indicated that SHBG variants rs6257 [16, 19], rs6259 [19, 20, 38], rs727428 [16, 19, 20] and rs1799941 [16, 20] were associated with PCOS. In our hands, rs13894 was significantly associated with PCOS among Bahraini women, evidenced by the increased MAF and homozygous minor allele-carriers in women with PCOS. Carriage of rs727428 minor allele was significantly linked with increased PCOS risk, with an OR of 1.58 recorded for heterozygous (G/A) and more for homozygous minor allele-carriers (A/A), indicating a ‘dose–response’ like relationship. In contrast, rs858521 was negatively associated with PCOS, thus assigning a protective nature to this SNP. In our hands, rs6259 was not linked with altered PCOS risk, in agreement with the study of Bendlová et al. [39], but in contrast to a study on Mediterranean women, which showed that carriage of rs6259 minor allele was associated with re-
Table 6. Distribution of SHBG haplotype in PCOS cases and control women
Haplotype1
Frequency2
PCOS cases
Controls
χ2
p value
OR (95% CI)
Adjusted OR (95% CI)3
GCGGTGG GCCGTGA GCGGCGA GCCATGG GTGGTGG GTCGTGA GCCGTGG GTCATGG GTGGCGA TCCGTGA GTCGTGG
0.123 0.122 0.087 0.080 0.076 0.070 0.067 0.057 0.050 0.044 0.035
0.104 0.155 0.088 0.077 0.076 0.095 0.052 0.075 0.062 0.040 0.026
0.141 0.091 0.086 0.083 0.077 0.048 0.081 0.041 0.040 0.047 0.043
2.349 7.282 0.013 0.107 0.002 6.265 2.434 4.045 1.838 0.234 1.708
0.125 0.007 0.908 7.44 0.963 0.009 0.119 0.042 0.175 0.628 0.191
1.00 (reference) 1.83 (1.18–2.85)
1.00 (reference) 2.08 (1.07–4.04)
2.26 (1.20–3.81)
7.80 (1.15–52.93)
1.89 (1.02–3.51)
2.62 (1.08–6.98)
0.49 (0.14–1.72)
0.12 (0.02–0.77)
Bold and underlined indicates minor allele. haplotype constructed from rs9898876, rs13894, rs858521, rs1799941, rs6257, rs6259, rs727428 alleles. 2 Haplotype frequency. 3 Covariates that were controlled for were BMI, HOMA-IR, SHBG and FT. 1 SHBG
72
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
than the women included here, thus prompting the speculation of contribution of advancing age [40] and altered sex hormone levels [30, 41] to SHBG circulating levels. Mechanistically, increased SHBG production, stemming from genetic variations in SHBG gene, or from nongenetic factors, may influence the levels of inactive protein-bound, as well as free bioactive SHBG levels. Given the role of SHBG in regulating sex hormone levels [10, 11], these SNPs could result in altered testosterone levels [25, 26], and thus PCOS-associated hyperandrogenemia. To test this notion, we examined the impact of carriage of rs727428 minor allele on SHBG serum levels and its correlation with BMI, IR and total, BT and FT pools. Carriage of rs727428 minor allele was associated with significant reduction in SHBG serum levels, in partial agreement with 2 recent studies, which documented the association of rs727428 with reduced SHBG serum levels, but not with PCOS [19, 20], but in apparent disagreement with the study by Martínez-García et al. [16], which reported on the lack of correlation between rs727428 and SHBG serum levels. The lack of replication of such findings may be related to the phenotypic heterogeneity of PCOS, differences in the studied population (age, BMI, menopausal status), and experimental design differences, notably with regard to PCOS definition, sample size and SHBG concentration measurement assays. In conclusion, we confirmed an association between PCOS and the SHBG variants rs13894, rs727428 and rs858521 SNPs, of which only rs727428 was associated with altered circulating levels of SHBG. The strength of Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
duced risk of PCOS [16]. This discrepancy could be explained by a difference in the racial/ethnic background and in selection of study subjects. Of the SNPs associated with PCOS in this study (rs13894, rs858521 and rs727428), only rs727428 remained associated with altered SHBG circulating levels and PCOS, after adjusting for BMI, HOMA-IR and FT, with a calculated adjusted OR of 3.91. This was in agreement with the study by Martínez-García et al. [16], which reported a strong association between rs727428 and PCOS susceptibility, but with a lower OR of 1.29. This suggested a causal role for SHBG rs727428 in SHBG expression and the pathogenesis of PCOS. Differences in study designs and ethnic/racial backgrounds may explain our findings and earlier studies. Neither rs6257 nor rs6259 were significantly associated with altered SHBG levels or PCOS, in agreement with the studies by Wickham et al. [20] and Bendlová et al. [39], but in disagreement with the study by Ding et al. [29], which reported on significantly higher and lower SHBG levels in postmenopausal women associated with rs6259 and rs6257, respectively. Furthermore, it was reported that rs6257 (and rs1799941), but not rs6259, was associated with altered SHBG levels in age- and weightadjusted analysis of postmenopausal women [21]. While differences in the sensitivity of SHBG assays, and the racial/ethnic origin of the participants, may partially explain these discrepancies, it should be noted that the studies by both Ding et al. [29] and Riancho et al. [21] used postmenopausal women, who were significantly older
our study is that cases and controls were ethnically matched, hence minimizing the problems of differences in genetic background, and that potential covariates were controlled for in the analysis. However, our study also has shortcomings, namely the relatively small sample size due to the Bahraini population of 731,000 (http:// population-of.com/en/Bahrain/), which in turn affected the power required to detect effects of SHBG variants on SHBG levels. Another limitation is the retrospective case–control study design, which prompted the speculation of cause–effect relationship, and that it was limited to Bahraini Arabs, thus necessitating parallel studies on other ethnic groups. Follow-up studies on additional SHBG variants, and populations of related and distant
ethnic origin are needed to confirm (or alternatively rule out) whether tested and other novel variants in SHBG contribute to the pathophysiology of PCOS.
Acknowledgments The authors wish to thank Dr. Mona Arekat for her helpful suggestions. The study was supported by grants from the Islamic Bank for Development and Arabian Gulf University R&EC.
Disclosure Statement None of the authors have any conflict of interest to declare.
References
SHBG Variants and Serum Levels in PCOS
10
11
12
13
14
15
16
17
mic approaches to the study of polycystic ovary syndrome. Mol Cell Endocrinol 2013; 370: 65–77. Botwood N, Hamilton-Fairley D, Kiddy D, Robinson S, Franks S: Sex hormone-binding globulin and female reproductive function. J Steroid Biochem Mol Biol 1995;53:529–531. Hammond GL, Wu TS, Simard M: Evolving utility of sex hormone-binding globulin measurements in clinical medicine. Curr Opin Endocrinol Diabetes Obes 2012;19:183–189. Travison TG, Zhuang WV, Lunetta KL, Karasik D, Bhasin S, Kiel DP, Coviello AD, Murabito JM: The heritability of circulating testosterone, oestradiol, oestrone and sex hormone binding globulin concentrations in men: the Framingham heart study. Clin Endocrinol (Oxf) 2014;80:277–282. Kahn SM, Hryb DJ, Nakhla AM, Romas NA, Rosner W: Sex hormone-binding globulin is synthesized in target cells. J Endocrinol 2002; 175:113–120. Hogeveen KN, Cousin P, Pugeat M, Dewailly D, Soudan B, Hammond GL: Human sex hormone-binding globulin variants associated with hyperandrogenism and ovarian dysfunction. J Clin Invest 2002;109:973–981. Rosner W, Hryb DJ, Khan MS, Nakhla AM, Romas NA: Sex hormone-binding globulin: anatomy and physiology of a new regulatory system. J Steroid Biochem Mol Biol 1991; 40: 813–820. Martínez-García MÁ, Gambineri A, Alpañés M, Sanchón R, Pasquali R, Escobar-Morreale HF: Common variants in the sex hormonebinding globulin gene (SHBG) and polycystic ovary syndrome (PCOS) in Mediterranean women. Hum Reprod 2012;27:3569–3576. O’Reilly MW, Taylor AE, Crabtree NJ, Hughes BA, Capper F, Crowley RK, Stewart PM, Tomlinson JW, Arlt W: Hyperandrogenemia predicts metabolic phenotype in polycystic ovary syndrome: the utility of serum
18
19
20
21
22
23
24
25
androstenedione. J Clin Endocrinol Metab 2014;99:1027–1036. Berube D, Seralini GE, Gagne R, Hammond GL: Localization of the human sex hormonebinding globulin gene (SHBG) to the short arm of chromosome 17 (17p12–p13). Cytogenet Cell Genet 1990;54:65–67. Pau C, Saxena R, Welt CK: Evaluating reported candidate gene associations with polycystic ovary syndrome. Fertil Steril 2013; 99: 1774–1778. Wickham EP 3rd, Ewens KG, Legro RS, Dunaif A, Nestler JE, Strauss JF 3rd: Polymorphisms in the SHBG gene influence serum SHBG levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2011; 96:E719–E727. Riancho JA, Valero C, Zarrabeitia MT, García-Unzueta MT, Amado JA, GonzálezMacías J: Genetic polymorphisms are associated with serum levels of sex hormone binding globulin in postmenopausal women. BMC Med Genet 2008;9:112. Sunbul M, Eren F, Nacar C, Agirbasli M: Sex hormone binding globulin gene polymorphisms and metabolic syndrome in postmenopausal Turkish women. Cardiol J 2013; 20:287–293. DeUgarte CM, Bartolucci AA, Azziz R: Prevalence of insulin resistance in the polycystic ovary syndrome using the homeostasis model assessment. Fertil Steril 2005;83:1454–1460. Ferk P, Teran N, Gersak K: The (TAAAA)n microsatellite polymorphism in the SHBG gene influences serum SHBG levels in women with polycystic ovary syndrome. Hum Reprod 2007;22:1031–1036. Napoli N, Varadharajan A, Rini GB, Del Fiacco R, Yarramaneni J, Mumm S, Villareal DT, Armamento-Villareal R: Effects of polymorphisms of the sex hormone-binding globulin (SHBG) gene on free estradiol and bone mineral density. Bone 2009;45:1169–1174.
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
73
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
1 Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO: The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745–2749. 2 Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R: Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078–3082. 3 Legro RS, Arslanian SA, Ehrmann DA, Hoeger KM, Murad MH, Pasquali R, Welt CK; Endocrine Society: Diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2013;98:4565–4592. 4 Moran LJ, Misso ML, Wild RA, Norman RJ: Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review and metaanalysis. Hum Reprod Update 2010; 16: 347– 363. 5 Vélez LM, Motta AB: Association between polycystic ovary syndrome and metabolic syndrome. Curr Med Chem 2014; 21: 3999– 4012. 6 Huang G, Coviello A: Clinical update on screening, diagnosis and management of metabolic disorders and cardiovascular risk factors associated with polycystic ovary syndrome. Curr Opin Endocrinol Diabetes Obes 2012;19:512–519. 7 Deligeoroglou E, Kouskouti C, Christopoulos P: The role of genes in the polycystic ovary syndrome: predisposition and mechanisms. Gynecol Endocrinol 2009;25:603–609. 8 Vink JM, Sadrzadeh S, Lambalk CB, Boomsma DI: Heritability of polycystic ovary syndrome in a Dutch twin-family study. J Clin Endocrinol Metab 2006;91:2100–2104. 9 Insenser M, Montes-Nieto R, Murri M, Escobar-Morreale HF: Proteomic and metabolo-
74
31 Nestler JE, Powers LP, Matt DW, Steingold KA, Plymate SR, Rittmaster RS, Clore JN, Blackard WG: A direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome. J Clin Endocrinol Metab 1991;72:83–89. 32 Pugeat M, Nader N, Hogeveen K, Raverot G, Déchaud H, Grenot C: Sex hormone-binding globulin gene expression in the liver: drugs and the metabolic syndrome. Mol Cell Endocrinol 2010;316:53–59. 33 Kim JW, Kim DY: Effects of aerobic exercise training on serum sex hormone binding globulin, body fat index, and metabolic syndrome factors in obese postmenopausal women. Metab Syndr Relat Disord 2012;10:452–457. 34 Maggio M, Lauretani F, Basaria S, Ceda GP, Bandinelli S, Metter EJ, Bos AJ, Ruggiero C, Ceresini G, Paolisso G, et al: Sex hormone binding globulin levels across the adult lifespan in women – the role of body mass index and fasting insulin. J Endocrinol Invest 2008; 31:597–601. 35 Lim U, Turner SD, Franke AA, Cooney RV, Wilkens LR, Ernst T, Albright CL, Novotny R, Chang L, Kolonel LN, et al: Predicting total, abdominal, visceral and hepatic adiposity with circulating biomarkers in Caucasian and Japanese American women. PLoS One 2012;7:e43502. 36 Baldani DP, Skrgatic L, Cerne JZ, Oguic SK, Gersak BM, Gersak K: Association between serum levels and pentanucleotide polymorphism in the sex hormone binding globulin gene and cardiovascular risk factors in females with polycystic ovary syndrome. Mol Med Rep 2015;11:3941–3947.
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
37 Papalou O, Livadas S, Karachalios A, Tolia N, Kokkoris P, Tripolitakis K, Diamanti-Kandarakis E: White blood cells levels and PCOS: direct and indirect relationship with obesity and insulin resistance, but not with hyperandogenemia. Hormones (Athens) 2015;14:91– 100. 38 Hacıhanefioğlu B, Aybey B, Hakan Özön Y, Berkil H, Karşıdağ K: Association of anthropometric, androgenic and insulin-related features with polymorphisms in exon 8 of SHBG gene in women with polycystic ovary syndrome. Gynecol Endocrinol 2013; 29: 361– 364. 39 Bendlová B, Zavadilová J, Vanková M, Vejrazková D, Lukásová P, Vcelák J, Hill M, Cibula D, Vondra K, Stárka L, et al: Role of D327N sex hormone-binding globulin gene polymorphism in the pathogenesis of polycystic ovary syndrome. J Steroid Biochem Mol Biol 2007;104:68–74. 40 Lecomte P, Lecureuil N, Lecureuil M, Osorio Salazar C, Valat C: Age modulates effects of thyroid dysfunction on sex hormone binding globulin (SHBG) levels. Exp Clin Endocrinol Diabetes 1995;103:339–342. 41 Rannevik G, Jeppsson S, Johnell O, Bjerre B, Laurell-Borulf Y, Svanberg L: A longitudinal study of the perimenopausal transition: altered profiles of steroid and pituitary hormones, SHBG and bone mineral density. Maturitas 2008;61:67–77.
Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
26 Thompson DJ, Healey CS, Baynes C, Kalmyrzaev B, Ahmed S, Dowsett M, Folkerd E, Luben RN, Cox D, Ballinger D, et al: Identification of common variants in the SHBG gene affecting sex hormone-binding globulin levels and breast cancer risk in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2008;17:3490–3498. 27 Garcia-Closas M, Brinton LA, Lissowska J, Richesson D, Sherman ME, Szeszenia-Dabrowska N, Peplonska B, Welch R, Yeager M, Zatonski W, et al: Ovarian cancer risk and common variation in the sex hormone-binding globulin gene: a population-based casecontrol study. BMC Cancer 2007;7:60. 28 Xu WH, Zheng W, Cai Q, Cheng JR, Cai H, Xiang YB, Shu XO: The Asp(327)Asn polymorphism in the sex hormone-binding globulin gene modifies the association of soy food and tea intake with endometrial cancer risk. Nutr Cancer 2008;60:736–743. 29 Ding EL, Song Y, Manson JE, Hunter DJ, Lee CC, Rifai N, Buring JE, Gaziano JM, Liu S: Sex hormone-binding globulin and risk of type 2 diabetes in women and men. N Engl J Med 2009;361:1152–1163. 30 Goto A, Chen BH, Song Y, Cauley J, Cummings SR, Farhat GN, Gunter M, Van Horn L, Howard BV, Jackson R, et al: Age, body mass, usage of exogenous estrogen, and lifestyle factors in relation to circulating sex hormone-binding globulin concentrations in postmenopausal women. Clin Chem 2014;60: 174–185.
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
Common Variants in the Sex Hormone-Binding Globulin (SHBG) Gene Influence SHBG Levels in Women with Polycystic Ovary Syndrome Tala M. Abu-Hijleh a Emily Gammoh a Amna S. Al-Busaidi a Zainab H. Malalla a Samira Madan b Naeema Mahmood b Wassim Y. Almawi a
Department of Medical Biochemistry, Arabian Gulf University, b Department of Obstetrics and Gynecology, Salmaniya Medical Complex, Manama, Bahrain
Key Words Haplotypes · Hyper-androgenism · Polycystic ovary syndrome · Sex hormone binding globulin
Abstract Background: Decreased sex hormone-binding globulin (SHBG) levels were associated with polycystic ovary syndrome (PCOS). SHBG polymorphisms associated with reduced SHBG production were tested for their association with PCOS, but with inconclusive results. We tested whether altered SHBG levels and SHBG variants were associated with PCOS. Methods: The study subjects included 242 women with PCOS and 238 control women. SHBG genotyping was done by real-time PCR. Results: Higher minor allele frequency of rs13894, rs858521 and rs727428 was seen in PCOS cases, and significant differences in rs858521 and rs727428 genotypes distribution were seen between PCOS cases and controls. Multivariate regression analysis confirmed the association of only rs727428 with PCOS. Though it was not statistically significant, serum SHBG levels were reduced according to rs727428 genotypes in PCOS cases than in controls. Carriage of rs727428 minor allele was associated with significant increases in free/bioactive testosterone in PCOS cases. Seven-locus (rs9898876-rs13894-rs858521-rs1799941rs6257-rs6259-rs727428) haploview analysis showed increased frequency of GCCGTGA, GTCGTGA and GTCATGG,
© 2015 S. Karger AG, Basel 0250–6807/15/0681–0066$39.50/0 E-Mail [email protected] www.karger.com/anm
and reduced frequency of GTCGTGG haplotypes in PCOS cases than in controls, thus conferring disease susceptibility and protective nature to these haplotypes, respectively. Conclusion: Specific SHBG variants affecting serum SHBG levels and SHBG haplotypes are associated with PCOS, suggesting the role for SHBG as PCOS candidate gene. © 2015 S. Karger AG, Basel
Introduction
Polycystic ovary syndrome (PCOS) is a common multifactorial endocrinopathy, which affects 5–15% of premenopausal women [1, 2]. As stipulated in the Rotterdam criteria, PCOS is characterized by clinical and biochemical hyperandrogenism, in association with anovulation and polycystic ovaries [3]. PCOS is associated with metabolic and reproductive disorders, such as obesity [3], glucose intolerance and insulin resistance (IR) and dyslipidemia [4, 5], and hence an increased risk of arteriosclerosis [6] and type 2 diabetes mellitus (T2DM) [4, 6]. PCOS is multigenic in nature, evidenced by familial clustering of PCOS [7, 8] and by twin studies, which suggested that PCOS pathogenesis is largely (∼70%) due to genetic influences [8, 9]. Sex hormone-binding globulin (SHBG) is a 373-amino acid glycoprotein produced mainly by the liver, which Wassim Y. Almawi, PhD Department of Medical Biochemistry College of Medicine and Medical Sciences, Arabian Gulf University PO Box 22979, Manama (Bahrain) E-Mail wassim @ agu.edu.bh
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
a
Subjects and Methods
Bahraini population. PCOS diagnosis was based on the 2003 Rotterdam criteria, which required 2 of the 3 conditions to be met: (1) anovulation, (2) hyperandrogenism assessed by the presence of hirsutism (modified Ferriman–Gallwey score: >8, acne in the third decade of life, or positive androgenic alopecia) and (3) the presence of polycystic ovary on ultrasound examination. Exclusion criteria included androgen-producing tumors, 21-hydroxylase-deficiency, non-classical adrenal hyperplasia, hyperprolactinemia, active thyroid disease and Cushing’s syndrome. Additional exclusion criteria included extremes of body mass index (BMI; 50 kg/m2), recent/current illness, medications likely to affect carbohydrate metabolism or endocrine parameters for at least 3 months before entering the study. The latter included oral contraceptives and anti-hypertensive, lipid-lowering and anti-inflammatory agents. Demographic data and history of hypertension, diabetes and hypercholesterolemia were taken from all subjects. All participants gave written informed consent, and the local research and ethics committees approved the study. Biochemical Analysis A fasting blood sample was obtained for biochemical and hormonal determinations. Serum samples were analyzed for SHBG by sandwich ELISA (R&D Systems, Minneapolis, Minn., USA); assay sensitivity was 0.01 nmol/ml, and inter-assay and intra-assay precision (CV%) were 5.3 and 4.3%, respectively. Serum luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, testosterone and glucose (ADVIA Centaur, Bayer Vital, Fernwald, Germany) and insulin (IMMULITE 2000, DPC Biermann, Bad Nauheim, Germany) were measured by automated chemiluminescence immunoassays. FT and bioactive testosterone (BT) and free androgen index were determined using Free and Bioavailable Testosterone Calculator (www.issam.ch/freetesto. htm). IR was estimated by the homeostasis model assessment (HOMA-IR), which is defined as fasting serum insulin (μIU/ml) × fasting plasma glucose (mmol/l)/22.5. SHBG Genotyping We selected polymorphisms in SHBG gene with a minor allele frequency (MAF) of >5% in Caucasians, using the SNPbrowser software (version 4.0, Applied Biosystems, Foster City, Calif., USA). SHBG genotyping was performed by the allelic (VIC- and FAM-labeled) discrimination method. Assay-on-demand TaqMan primer pairs for the following SNPs: rs9898876, rs13894, rs858521, rs1799941, rs6257, rs6259 and rs727428 were ordered from Applied Biosystems. The reaction was performed in 6-μl volume on StepOne Plus real-time PCR system, according to manufacturer’s instructions (Applied Biosystems). Replicate blinded quality control samples were included to assess reproducibility of the genotyping procedure; concordance was >99%.
Subjects A total of 389 unrelated self-reported Bahraini Arab women with (n = 242) and without (n = 238) PCOS were recruited from the outpatient endocrinology and obstetrics and gynecology clinics in Manama, Bahrain. Control women comprised eumenorrheic university students and employees, or otherwise healthy volunteers, without clinical evidence of PCOS. Their androgen levels were within the reference range (0.4–3.5 nmol/l) and were studied in the follicular phase of their menstrual cycle. Control women reported no health problems and were recruited from the 5 governorates of Bahrain, and thus were representative of present-day
Statistical Analysis Statistical analysis was performed on SPSS version 22.0 (IBM). Data were expressed as percentages of total (categorical variables) or as mean ± SD (continuous variables). Student’s t test was used in determining differences in means, and Pearson χ2 or Fisher’s exact test was used to assess inter-group significance. SNP genotypes were tested for Hardy–Weinberg equilibrium (HWE) in control women and PCOS cases by Haploview version 4.2 (http://www.broad.mit.edu/mpg/haploview). Genetic Power Calculator (http://pngu.mgh.harvard.edu/∼purcell/cgi-bin/cc2k.cgi)
SHBG Variants and Serum Levels in PCOS
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
67
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
acts by binding circulating steroid sex hormones, including testosterone and estradiol [10, 11], thereby limiting their target tissue bioavailability [10, 12], and is a key regulator of the steroid-signaling system [13]. Increased free testosterone (FT) results in its peripheral conversion to the potent androgen, dihydrotestosterone, and thus leads to induction of hyperandrogenism [14, 15], a hallmark of PCOS [16, 17]. SHBG plays a central role in PCOS pathophysiology, as evidenced by the findings that women with PCOS have increased levels of free circulating androgens and IR, which in turn inhibit the hepatic production and secretion of SHBG [17]. Located on the short arm of chromosome 17 (17p131p12) [18], several genetic variants in the human SHBG gene were reported. Studies documented association between SHBG polymorphisms with altered glucose homeostasis, some of which linked it with altered SHBG levels, while others suggested that it was independent of changes in SHBG levels [16, 19–22]. In view of the link between PCOS and altered glucose homeostasis and IR [4, 23], it was suggested that altered SHBG secretion, stemming from specific SHBG polymorphisms, might contribute to PCOS pathogenesis. For example, studies on predominantly Caucasian populations demonstrated differential association of rs6257, rs6259 (D356N), rs727428 and rs1799941 SHBG variants with decreased SHBG levels and PCOS presence [16, 19, 20, 24]. Other SHBG variants, including rs13894, rs858521 and rs9898876, were also investigated as to their association with altered SHBG levels, but in T2DM and IR [21, 22, 25, 26]. Such a study has yet to be carried out in Middle Eastern women. The aim of the present study was to explore the association of SHBG rs6257, rs6259, rs727428 and rs1799941, in addition to rs13894, rs858521 and rs9898876 SNPs in 242 Bahraini Arab women with PCOS and in 238 control women. This is the first study to examine this association in a Middle Eastern population.
Table 1. Baseline and endocrine parameters of women with PCOS and control women
Parameter
Cases1
Controls1
p value2
Age, years BMI, kg/m2 Menarche, years Luteinizing hormone, IU/l Follicle stimulating hormone, IU/l Thyroid stimulating hormone, μIU/ml Fasting insulin HOMA-IR Total testosterone, nmol/l FT, pmol/l Bioavailable testosterone, pmol/l Free androgen index SHBG, nmol/l High-density lipoprotein cholesterol, mmol/l Triglycerides, mmol/l
28.7±6.1 29.1±6.7 12.4±1.4 5.8 (0.8–56.3) 5.3 (0.5–44.9) 2.8±2.2 11.4 (1.6–118.7) 4.3±2.7 1.5±1.0 28.9±19.4 655.1±445.4 7.7±4.1 33.8±18.8 1.3±0.5 1.4±0.9
27.5±7.0 26.2±5.3 12.6±1.4 5.4 (0.4–66.6) 5.2 (0.4–18.6) 1.8±1.1 6.4 (1.7–31.8) 1.9±0.8 1.4±1.0 20.9±16.4 455.6±288.9 3.2±2.2 67.1±46.1 1.5±0.5 1.1±0.7
0.072 5.0 × 10–6 0.266 0.266 0.824 0.004 2.7 × 10–4 1.4 × 10–4 0.469 0.003 2.2 × 10–4 0.003 1.0 × 10–6 0.383 0.068
1
A total of 242 PCOS cases and 238 control women were included. t test (variables with normal distribution), Mann–Whitney U test (variables that were not normally distributed). Values are mean ± SD and percentage of total within each group/subgroup. 2 Student’s
Results
Study Subjects The clinical and biochemical characteristics of study subjects are presented in table 1. Age at examination, menarche, along with fasting serum HDL cholesterol and triglycerides, as well as follicle stimulating hormone, luteinizing hormone and total testosterone were compara68
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
ble between women with PCOS and control women. Significant differences in mean BMI (p < 0.001), FT (p = 0.003) and BT (p < 0.001) and free androgen index (p = 0.003), as well as in fasting insulin (p < 0.001) and HOMAIR (p < 0.001), were seen between PCOS cases and control women. In addition, significantly lower serum levels of SHBG (p < 0.001) and higher levels of thyroid stimulating hormone (p = 0.006; though within reference range) were seen between women with PCOS and control women. Accordingly, these were selected as the covariates that were controlled for in later analysis. SHBG SNPs Analyses The 7 tested SHBG variants were in HWE in control women (p > 0.05), while deviation from HWE was noted for rs9898876, rs13894 and rs6259 in women with PCOS (table 2). Of the tested SHBG SNPs, higher MAF of rs13894 (p = 0.011; χ2 = 6.445) and rs727428 (p = 0.002; χ2 = 9.735), and lower MAF of rs858521 (p = 0.015; χ2 = 5.973) were seen in women with PCOS compared to control women; MAF of the remaining SNPs were comparable between PCOS cases and control women (table 2). The calculated ORs were 1.467, 1.582 and 0.697 for rs13894, rs727428, and rs858521, respectively, after controlling for key covariates. Significant difference in rs858521 (p = 0.020) and rs727428 (p = 0.002) genotype distribution was seen beAbu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
was used in calculating the power for detecting an association between SHBG variants and PCOS. The parameters used were 242 women with PCOS and 238 control women, genotypic relative risk for heterozygote (1/2) and minor allele homozygous (2/2), and the MAF for PCOS cases and controls for the 7 tested SNPs, and assuming a 14.0% population prevalence of PCOS (unpublished Bahrain Ministry of Health statistics). Assuming these parameters, we calculated the overall power (71.2%) as the average power of the 7 tested SNPs. All analyses were conducted under additive genetic effect, as it is a conservative model, using SNPStats (bioinfo.iconcologia.net/ snpstats/). This assumes that the risk conferred by an allele increases r-fold for heterozygotes and r2-fold for homozygotes. Linkage disequilibrium analysis was performed by Haploview 4.2, and haplotype reconstruction was performed by the expectation maximization method (Haploview 4.2). Logistic regression analysis was performed to determine the odds ratios and 95% confidence intervals associated with the risk of PCOS, taking control women as the reference group. Statistical significance was set at values of p < 0.05.
Table 2. SHBG SNPs analyzed
SNP
rs9898876 rs13894 rs858521 rs1799941 rs6257 rs6259 rs727428
Location1
7623644 7626584 7626829 7630105 7630399 7633209 7634474
Alleles
G:T C:T C:G G:A T:C G:A G:A
Cases
Controls
HWE
MAF
HWE
MAF
0.043 0.022 0.120 1.000 0.025 1.000 0.745
0.126 0.401 0.386 0.182 0.186 0.042 0.055
0.480 0.063 0.420 0.437 0.165 1.000 0.110
0.107 0.321 0.479 0.183 0.183 0.057 0.435
Power2 χ2
p value
86.7 68.9 74.8 79.0 56.8 62.2 69.9
0.508 0.011 0.015 0.999 0.727 0.292 0.002
0.439 6.445 5.973 0.000 0.122 1.113 9.735
aOR (95% CI)3
1.467 (1.091–1.973) 0.697 (0.521–0.931)
1.582 (1.185–2.112)
1
Location on chromosome based on dbSNP build 125. Study power (%) based on case–control statistics (B vs. b). 3 Covariates that were adjusted for were BMI, HOMA-IR, SHBG and FT. 2
Table 3. Genotype frequencies of SHBG polymorphisms
SNP
Genotype
PCOS cases
Controls
p value
OR (95% CI)
p value
aOR (95% CI)1
rs9898876
G/G G/T T/T
189 (78.1)2 45 (18.6) 8 (3.3)
191 (80.3) 43 (18.1) 4 (1.7)
0.46
1.00 (reference) 1.06 (0.63–1.78) 2.37 (0.58–9.65)
0.22
1.00 (reference) 1.10 (0.50–2.40) 7.31 (0.64–83.37)
rs13894
C/C C/T T/T
97 (40.1) 96 (39.7) 49 (20.2)
117 (49.2) 89 (37.4) 32 (13.4)
0.07
1.00 (reference) 1.33 (0.86–2.07) 1.93 (1.08–3.44)
0.14
1.00 (reference) 1.05 (0.54–2.03) 2.20 (0.95–5.09)
rs858521
C/C C/G G/G
98 (40.5) 101 (41.7) 43 (17.8)
77 (32.4) 94 (39.5) 67 (28.1)
0.05
1.00 (reference) 0.86 (0.55–1.37) 0.52 (0.30–0.90)
0.27
1.00 (reference) 1.34 (0.68–2.64) 0.69 (0.31–1.54)
rs1799941
G/G G/A A/A
162 (66.9) 72 (29.8) 8 (3.3)
167 (70.2) 55 (23.1) 16 (6.7)
0.17
1.00 (reference) 1.32 (0.83–2.09) 0.53 (0.20–1.43)
0.56
1.00 (reference) 1.44 (0.74–2.81) 1.17 (0.20–7.00)
rs6257
T/T T/C C/C
165 (68.2) 64 (26.4) 13 (5.4)
165 (69.3) 59 (24.8) 14 (5.9)
0.93
1.00 (reference) 0.92 (0.58–1.46) 1.06 (0.45–2.53)
0.75
1.00 (reference) 0.79 (0.40–1.54) 1.14 (0.27–4.81)
rs6259
G/G G/A
222 (91.7) 20 (8.3)
211 (88.7) 27 (11.3)
0.34
1.00 (reference) 0.72 (0.36–1.43)
0.34
1.00 (reference) 0.63 (0.24–1.65)
rs727428
G/G G/A A/A
58 (23.7) 101 (41.9) 83 (34.4)
69 (29.0) 131 (55.0) 38 (16.0)
2.0 × 10–4
1.00 (reference) 0.93 (0.57–1.53) 2.65 (1.48–4.76)
0.002
1.00 (reference) 0.94 (0.46–1.91) 3.91 (1.50–10.18)
1 Covariates
that were controlled for were BMI, HOMA-IR, SHBG and FT. of subjects (percent total).
tween women with PCOS and control women (table 3); the genotype distribution of the remaining SNPs was comparable between cases and controls. Binary logistic regression confirmed this association, which included PCOS or control status as an independent variable and
the SHBG SNPs as independent variables. Taking homozygous wild-type genotype as reference (OR 1.00), increased PCOS risk was seen with rs727428 minor-allele homozygous genotype (OR 2.65, 95% CI 1.48–4.76), while reduced PCOS risk was associated with rs858521
SHBG Variants and Serum Levels in PCOS
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
69
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
2 Number
Table 4. SHBG values for women with PCOS according to SHBG SNP genotype1
SNP
1/1
1/2
3/3
p value2
rs9898876 rs13894 rs858521 rs1799941 rs6257 rs6259 rs727428
49.7±34.3 48.6±39.1 46.1±32.4 47.5±40.0 50.0±43.9 48.1±41.1 71.4±50.6
48.0±36.2 53.9±41.9 48.8±31.8 50.4±44.1 49.5±36.8 58.4±41.8 51.1±40.9
63.9±31.5 40.6±32.9 53.9±34.4 70.6±48.5 45.8±32.3 – 32.0±23.6
0.589 0.100 0.540 0.173 0.785 0.222 0.008
1
Genotypes were coded as 1/1 (homozygous major allele), 1/2 (heterozygous) and 2/2 (homozygous minor allele). 2 One-way analysis of variance (2-tailed). Values are mean SHBG ± SD (nmol/l).
Table 5. Multivariate analyses for association between serum SHBG and rs727428 genotype, BMI, testosterone and IR in women with PCOS
SNP
G/G
G/A
A/A
p value1
BMI Fasting insulin HOMA-IR Total testosterone FT BT
28.1±5.8 15.4±12.0 3.1±2.3 1.42±1.07 23.8±16.1 0.56±0.39
29.4±6.5 16.8±11.2 4.0±2.6 1.43±1.03 30.3±21.1 0.65±0.49
28.2±5.8 17.5±10.9 4.7±2.8 1.57±0.88 36.6±22.3 0.81±0.50
0.396 0.292 0.422 0.826 0.039 0.015
1 One-way analysis of variance (2-tailed). Values are mean ± SD.
Influence of rs727428 on SHBG Serum Levels and PCOS Features We analyzed the influence of rs727428 genotypes on SHBG serum levels, insulin status, lipid profile and testosterone levels. Carriage of rs727428 minor allele was not influenced by menarche or BMI, and did not affect IR (HOMA-IR) or serum lipids (table 5). Compared with major-allele homozygous (G/G) genotype carriers, there was a progressive increase in FT (p = 0.039) and BT (p = 0.015) in rs727428 genotypes in heterozygous (G/A), and more so in minor-allele homozygous (A/A) genotype carriers (table 5). 70
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
Haploview Analysis Haploview analysis revealed limited linkage disequilibrium (LD) between the 7 tested SHBG variants (fig. 1). Extensive diversity in the haplotype assignment was seen, with the majority of haplotypes (81.1%) captured by 11 haplotypes, which were thus designated as ‘common’. Taking GCGGTGG haplotype as common (OR 1.00), there was positive association of GCCGTGA, GTCGTGA and GTCATGG haplotypes, and negative association of GTCGTGG haplotype with PCOS (table 6). The association was confirmed by multivariate regression analysis after controlling for BMI, HOMA-IR, SHBG and FT.
Discussion
Previous studies investigated the role of SHBG in PCOS pathophysiology, but with inconclusive findings. In the present study, we assessed the association of SHBG Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
minor-allele homozygous genotype (OR 0.52, 95% CI 0.30–0.90; table 4), thus assigning PCOS susceptibility and protective nature to these genotypes, respectively. Multivariate regression analysis, confirmed the positive association of only rs727428 minor-allele homozygous genotype (adjusted OR 3.91, 95% CI 1.50–10.18; table 4).
rs6259
rs727428
3
4
5
6
7
Block 1 (10 kb) 2
1
3
0 71
56 2
70
84 73
9 89
25 77
0 91
55 11
11 41
2 95
Fig. 1. Haploview graph of SHBG SNPs analyzed. The positions of
the 7 SNPs used (build 37.3) are indicated along with the basic gene structure and displayed above the Haploview output. The relative LD between specific pair of SHBG SNPs is indicated by the color scheme, which represents the LD relationships. This is based on D’ values (normalized linkage disequilibrium measure or D) multiplied by 100; D’ is calculated as D divided by the theoretical maximum for the observed allele frequencies. Values approaching zero indicate absence of LD, and those approaching 100 indicate complete LD. The squares colored red represent varying degrees of LD 2 scores; darker shades indicating stronger LD.
71
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
Color version available online
rs6257
rs13894
rs1799941
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
rs858521
SHBG Variants and Serum Levels in PCOS
rs9898876
polymorphisms with altered serum SHBG levels and the presence of PCOS. Four of the tested SNPs (rs6259, rs6257, rs1799941 and rs727428) were previously studied in relation to their association with PCOS, but with variable results [19, 20, 27]. The remaining 3 SNPs (rs989887, rs13894, rs858521) were also investigated, but in relation to their effects on SHBG levels and cancer and osteoporosis [21, 22, 25, 26, 28]. Our study demonstrated a significant link between rs727428, but not the other studied SNPs, and PCOS and hyperandrogenism in Bahraini women. To our knowledge, this is the first study to evaluate the association of SHBG polymorphism and changes in SHBG levels in the Arab-speaking community. All subjects included were self-reported Bahraini Arab women and were consecutively enrolled, so as to minimize the possibility of type II errors. PCOS diagnosis was based on the 2003 Rotterdam criteria, as compared with other studies in which the National Institute of Health
diagnostic criteria were used [16, 19]. As some of SHBG SNPs studied, notably rs1799941, rs6257 and rs6259, were previously linked with T2DM [29, 30], we excluded women with T2DM or IR in order to limit the possible contribution of altered glucose homeostasis. Our study population differed from those included in earlier studies with regards to mean BMI and testosterone. For example, mean BMI was higher than that reported in the study by Wickham et al. [20], but comparable to that of the Mediterranean women in the study by Martínez-García et al. [16], while both FT and total testosterone levels were lower than it was in the subjects of the study by MartínezGarcía et al. [16]. Accordingly, BMI, HOMA-IR, SHBG and FT were the main covariates that were controlled for in subsequent analysis. SHBG levels inversely correlated with age (r = –0.138; p = 0.018), FT (r = –0.481; p = 1.0 × 10–7), HOMA-IR (r = –0.196; p = 0.001), but not with BMI, among women with PCOS. SHBG is a sensitive biomarker of IR and the metabolic syndrome, and the low SHBG serum levels seen in women with PCOS linked with IR (HOMA-IR) is the result of inhibition of hepatic SHBG production, as shown elsewhere [31, 32]. SHBG levels have also been shown to correlate with both fat mass [31, 33] and BMI [33, 34], and visceral adiposity positively correlated with SHBG levels [35]. In our hands, there was lack of correlation between SHBG levels and BMI (p = 0.289; r2 = 0.014), which persisted after controlling for HOMA-IR (p = 0.552; r2 = 0.004), suggesting lack of contribution of obesity to altered SHBG secretion. Population-based studies documented strong association between altered testosterone and low SHBG levels and PCOS [36, 37]. Recent studies on Turkish [38], Mediterranean [16], European [19] and US [20] women indicated that SHBG variants rs6257 [16, 19], rs6259 [19, 20, 38], rs727428 [16, 19, 20] and rs1799941 [16, 20] were associated with PCOS. In our hands, rs13894 was significantly associated with PCOS among Bahraini women, evidenced by the increased MAF and homozygous minor allele-carriers in women with PCOS. Carriage of rs727428 minor allele was significantly linked with increased PCOS risk, with an OR of 1.58 recorded for heterozygous (G/A) and more for homozygous minor allele-carriers (A/A), indicating a ‘dose–response’ like relationship. In contrast, rs858521 was negatively associated with PCOS, thus assigning a protective nature to this SNP. In our hands, rs6259 was not linked with altered PCOS risk, in agreement with the study of Bendlová et al. [39], but in contrast to a study on Mediterranean women, which showed that carriage of rs6259 minor allele was associated with re-
Table 6. Distribution of SHBG haplotype in PCOS cases and control women
Haplotype1
Frequency2
PCOS cases
Controls
χ2
p value
OR (95% CI)
Adjusted OR (95% CI)3
GCGGTGG GCCGTGA GCGGCGA GCCATGG GTGGTGG GTCGTGA GCCGTGG GTCATGG GTGGCGA TCCGTGA GTCGTGG
0.123 0.122 0.087 0.080 0.076 0.070 0.067 0.057 0.050 0.044 0.035
0.104 0.155 0.088 0.077 0.076 0.095 0.052 0.075 0.062 0.040 0.026
0.141 0.091 0.086 0.083 0.077 0.048 0.081 0.041 0.040 0.047 0.043
2.349 7.282 0.013 0.107 0.002 6.265 2.434 4.045 1.838 0.234 1.708
0.125 0.007 0.908 7.44 0.963 0.009 0.119 0.042 0.175 0.628 0.191
1.00 (reference) 1.83 (1.18–2.85)
1.00 (reference) 2.08 (1.07–4.04)
2.26 (1.20–3.81)
7.80 (1.15–52.93)
1.89 (1.02–3.51)
2.62 (1.08–6.98)
0.49 (0.14–1.72)
0.12 (0.02–0.77)
Bold and underlined indicates minor allele. haplotype constructed from rs9898876, rs13894, rs858521, rs1799941, rs6257, rs6259, rs727428 alleles. 2 Haplotype frequency. 3 Covariates that were controlled for were BMI, HOMA-IR, SHBG and FT. 1 SHBG
72
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
than the women included here, thus prompting the speculation of contribution of advancing age [40] and altered sex hormone levels [30, 41] to SHBG circulating levels. Mechanistically, increased SHBG production, stemming from genetic variations in SHBG gene, or from nongenetic factors, may influence the levels of inactive protein-bound, as well as free bioactive SHBG levels. Given the role of SHBG in regulating sex hormone levels [10, 11], these SNPs could result in altered testosterone levels [25, 26], and thus PCOS-associated hyperandrogenemia. To test this notion, we examined the impact of carriage of rs727428 minor allele on SHBG serum levels and its correlation with BMI, IR and total, BT and FT pools. Carriage of rs727428 minor allele was associated with significant reduction in SHBG serum levels, in partial agreement with 2 recent studies, which documented the association of rs727428 with reduced SHBG serum levels, but not with PCOS [19, 20], but in apparent disagreement with the study by Martínez-García et al. [16], which reported on the lack of correlation between rs727428 and SHBG serum levels. The lack of replication of such findings may be related to the phenotypic heterogeneity of PCOS, differences in the studied population (age, BMI, menopausal status), and experimental design differences, notably with regard to PCOS definition, sample size and SHBG concentration measurement assays. In conclusion, we confirmed an association between PCOS and the SHBG variants rs13894, rs727428 and rs858521 SNPs, of which only rs727428 was associated with altered circulating levels of SHBG. The strength of Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
duced risk of PCOS [16]. This discrepancy could be explained by a difference in the racial/ethnic background and in selection of study subjects. Of the SNPs associated with PCOS in this study (rs13894, rs858521 and rs727428), only rs727428 remained associated with altered SHBG circulating levels and PCOS, after adjusting for BMI, HOMA-IR and FT, with a calculated adjusted OR of 3.91. This was in agreement with the study by Martínez-García et al. [16], which reported a strong association between rs727428 and PCOS susceptibility, but with a lower OR of 1.29. This suggested a causal role for SHBG rs727428 in SHBG expression and the pathogenesis of PCOS. Differences in study designs and ethnic/racial backgrounds may explain our findings and earlier studies. Neither rs6257 nor rs6259 were significantly associated with altered SHBG levels or PCOS, in agreement with the studies by Wickham et al. [20] and Bendlová et al. [39], but in disagreement with the study by Ding et al. [29], which reported on significantly higher and lower SHBG levels in postmenopausal women associated with rs6259 and rs6257, respectively. Furthermore, it was reported that rs6257 (and rs1799941), but not rs6259, was associated with altered SHBG levels in age- and weightadjusted analysis of postmenopausal women [21]. While differences in the sensitivity of SHBG assays, and the racial/ethnic origin of the participants, may partially explain these discrepancies, it should be noted that the studies by both Ding et al. [29] and Riancho et al. [21] used postmenopausal women, who were significantly older
our study is that cases and controls were ethnically matched, hence minimizing the problems of differences in genetic background, and that potential covariates were controlled for in the analysis. However, our study also has shortcomings, namely the relatively small sample size due to the Bahraini population of 731,000 (http:// population-of.com/en/Bahrain/), which in turn affected the power required to detect effects of SHBG variants on SHBG levels. Another limitation is the retrospective case–control study design, which prompted the speculation of cause–effect relationship, and that it was limited to Bahraini Arabs, thus necessitating parallel studies on other ethnic groups. Follow-up studies on additional SHBG variants, and populations of related and distant
ethnic origin are needed to confirm (or alternatively rule out) whether tested and other novel variants in SHBG contribute to the pathophysiology of PCOS.
Acknowledgments The authors wish to thank Dr. Mona Arekat for her helpful suggestions. The study was supported by grants from the Islamic Bank for Development and Arabian Gulf University R&EC.
Disclosure Statement None of the authors have any conflict of interest to declare.
References
SHBG Variants and Serum Levels in PCOS
10
11
12
13
14
15
16
17
mic approaches to the study of polycystic ovary syndrome. Mol Cell Endocrinol 2013; 370: 65–77. Botwood N, Hamilton-Fairley D, Kiddy D, Robinson S, Franks S: Sex hormone-binding globulin and female reproductive function. J Steroid Biochem Mol Biol 1995;53:529–531. Hammond GL, Wu TS, Simard M: Evolving utility of sex hormone-binding globulin measurements in clinical medicine. Curr Opin Endocrinol Diabetes Obes 2012;19:183–189. Travison TG, Zhuang WV, Lunetta KL, Karasik D, Bhasin S, Kiel DP, Coviello AD, Murabito JM: The heritability of circulating testosterone, oestradiol, oestrone and sex hormone binding globulin concentrations in men: the Framingham heart study. Clin Endocrinol (Oxf) 2014;80:277–282. Kahn SM, Hryb DJ, Nakhla AM, Romas NA, Rosner W: Sex hormone-binding globulin is synthesized in target cells. J Endocrinol 2002; 175:113–120. Hogeveen KN, Cousin P, Pugeat M, Dewailly D, Soudan B, Hammond GL: Human sex hormone-binding globulin variants associated with hyperandrogenism and ovarian dysfunction. J Clin Invest 2002;109:973–981. Rosner W, Hryb DJ, Khan MS, Nakhla AM, Romas NA: Sex hormone-binding globulin: anatomy and physiology of a new regulatory system. J Steroid Biochem Mol Biol 1991; 40: 813–820. Martínez-García MÁ, Gambineri A, Alpañés M, Sanchón R, Pasquali R, Escobar-Morreale HF: Common variants in the sex hormonebinding globulin gene (SHBG) and polycystic ovary syndrome (PCOS) in Mediterranean women. Hum Reprod 2012;27:3569–3576. O’Reilly MW, Taylor AE, Crabtree NJ, Hughes BA, Capper F, Crowley RK, Stewart PM, Tomlinson JW, Arlt W: Hyperandrogenemia predicts metabolic phenotype in polycystic ovary syndrome: the utility of serum
18
19
20
21
22
23
24
25
androstenedione. J Clin Endocrinol Metab 2014;99:1027–1036. Berube D, Seralini GE, Gagne R, Hammond GL: Localization of the human sex hormonebinding globulin gene (SHBG) to the short arm of chromosome 17 (17p12–p13). Cytogenet Cell Genet 1990;54:65–67. Pau C, Saxena R, Welt CK: Evaluating reported candidate gene associations with polycystic ovary syndrome. Fertil Steril 2013; 99: 1774–1778. Wickham EP 3rd, Ewens KG, Legro RS, Dunaif A, Nestler JE, Strauss JF 3rd: Polymorphisms in the SHBG gene influence serum SHBG levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2011; 96:E719–E727. Riancho JA, Valero C, Zarrabeitia MT, García-Unzueta MT, Amado JA, GonzálezMacías J: Genetic polymorphisms are associated with serum levels of sex hormone binding globulin in postmenopausal women. BMC Med Genet 2008;9:112. Sunbul M, Eren F, Nacar C, Agirbasli M: Sex hormone binding globulin gene polymorphisms and metabolic syndrome in postmenopausal Turkish women. Cardiol J 2013; 20:287–293. DeUgarte CM, Bartolucci AA, Azziz R: Prevalence of insulin resistance in the polycystic ovary syndrome using the homeostasis model assessment. Fertil Steril 2005;83:1454–1460. Ferk P, Teran N, Gersak K: The (TAAAA)n microsatellite polymorphism in the SHBG gene influences serum SHBG levels in women with polycystic ovary syndrome. Hum Reprod 2007;22:1031–1036. Napoli N, Varadharajan A, Rini GB, Del Fiacco R, Yarramaneni J, Mumm S, Villareal DT, Armamento-Villareal R: Effects of polymorphisms of the sex hormone-binding globulin (SHBG) gene on free estradiol and bone mineral density. Bone 2009;45:1169–1174.
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
73
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
1 Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO: The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745–2749. 2 Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R: Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078–3082. 3 Legro RS, Arslanian SA, Ehrmann DA, Hoeger KM, Murad MH, Pasquali R, Welt CK; Endocrine Society: Diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2013;98:4565–4592. 4 Moran LJ, Misso ML, Wild RA, Norman RJ: Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review and metaanalysis. Hum Reprod Update 2010; 16: 347– 363. 5 Vélez LM, Motta AB: Association between polycystic ovary syndrome and metabolic syndrome. Curr Med Chem 2014; 21: 3999– 4012. 6 Huang G, Coviello A: Clinical update on screening, diagnosis and management of metabolic disorders and cardiovascular risk factors associated with polycystic ovary syndrome. Curr Opin Endocrinol Diabetes Obes 2012;19:512–519. 7 Deligeoroglou E, Kouskouti C, Christopoulos P: The role of genes in the polycystic ovary syndrome: predisposition and mechanisms. Gynecol Endocrinol 2009;25:603–609. 8 Vink JM, Sadrzadeh S, Lambalk CB, Boomsma DI: Heritability of polycystic ovary syndrome in a Dutch twin-family study. J Clin Endocrinol Metab 2006;91:2100–2104. 9 Insenser M, Montes-Nieto R, Murri M, Escobar-Morreale HF: Proteomic and metabolo-
74
31 Nestler JE, Powers LP, Matt DW, Steingold KA, Plymate SR, Rittmaster RS, Clore JN, Blackard WG: A direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome. J Clin Endocrinol Metab 1991;72:83–89. 32 Pugeat M, Nader N, Hogeveen K, Raverot G, Déchaud H, Grenot C: Sex hormone-binding globulin gene expression in the liver: drugs and the metabolic syndrome. Mol Cell Endocrinol 2010;316:53–59. 33 Kim JW, Kim DY: Effects of aerobic exercise training on serum sex hormone binding globulin, body fat index, and metabolic syndrome factors in obese postmenopausal women. Metab Syndr Relat Disord 2012;10:452–457. 34 Maggio M, Lauretani F, Basaria S, Ceda GP, Bandinelli S, Metter EJ, Bos AJ, Ruggiero C, Ceresini G, Paolisso G, et al: Sex hormone binding globulin levels across the adult lifespan in women – the role of body mass index and fasting insulin. J Endocrinol Invest 2008; 31:597–601. 35 Lim U, Turner SD, Franke AA, Cooney RV, Wilkens LR, Ernst T, Albright CL, Novotny R, Chang L, Kolonel LN, et al: Predicting total, abdominal, visceral and hepatic adiposity with circulating biomarkers in Caucasian and Japanese American women. PLoS One 2012;7:e43502. 36 Baldani DP, Skrgatic L, Cerne JZ, Oguic SK, Gersak BM, Gersak K: Association between serum levels and pentanucleotide polymorphism in the sex hormone binding globulin gene and cardiovascular risk factors in females with polycystic ovary syndrome. Mol Med Rep 2015;11:3941–3947.
Ann Nutr Metab 2016;68:66–74 DOI: 10.1159/000441570
37 Papalou O, Livadas S, Karachalios A, Tolia N, Kokkoris P, Tripolitakis K, Diamanti-Kandarakis E: White blood cells levels and PCOS: direct and indirect relationship with obesity and insulin resistance, but not with hyperandogenemia. Hormones (Athens) 2015;14:91– 100. 38 Hacıhanefioğlu B, Aybey B, Hakan Özön Y, Berkil H, Karşıdağ K: Association of anthropometric, androgenic and insulin-related features with polymorphisms in exon 8 of SHBG gene in women with polycystic ovary syndrome. Gynecol Endocrinol 2013; 29: 361– 364. 39 Bendlová B, Zavadilová J, Vanková M, Vejrazková D, Lukásová P, Vcelák J, Hill M, Cibula D, Vondra K, Stárka L, et al: Role of D327N sex hormone-binding globulin gene polymorphism in the pathogenesis of polycystic ovary syndrome. J Steroid Biochem Mol Biol 2007;104:68–74. 40 Lecomte P, Lecureuil N, Lecureuil M, Osorio Salazar C, Valat C: Age modulates effects of thyroid dysfunction on sex hormone binding globulin (SHBG) levels. Exp Clin Endocrinol Diabetes 1995;103:339–342. 41 Rannevik G, Jeppsson S, Johnell O, Bjerre B, Laurell-Borulf Y, Svanberg L: A longitudinal study of the perimenopausal transition: altered profiles of steroid and pituitary hormones, SHBG and bone mineral density. Maturitas 2008;61:67–77.
Abu-Hijleh/Gammoh/Al-Busaidi/Malalla/ Madan/Mahmood/Almawi
Downloaded by: University of Hong Kong 198.143.53.1 - 1/19/2016 9:50:56 PM
26 Thompson DJ, Healey CS, Baynes C, Kalmyrzaev B, Ahmed S, Dowsett M, Folkerd E, Luben RN, Cox D, Ballinger D, et al: Identification of common variants in the SHBG gene affecting sex hormone-binding globulin levels and breast cancer risk in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2008;17:3490–3498. 27 Garcia-Closas M, Brinton LA, Lissowska J, Richesson D, Sherman ME, Szeszenia-Dabrowska N, Peplonska B, Welch R, Yeager M, Zatonski W, et al: Ovarian cancer risk and common variation in the sex hormone-binding globulin gene: a population-based casecontrol study. BMC Cancer 2007;7:60. 28 Xu WH, Zheng W, Cai Q, Cheng JR, Cai H, Xiang YB, Shu XO: The Asp(327)Asn polymorphism in the sex hormone-binding globulin gene modifies the association of soy food and tea intake with endometrial cancer risk. Nutr Cancer 2008;60:736–743. 29 Ding EL, Song Y, Manson JE, Hunter DJ, Lee CC, Rifai N, Buring JE, Gaziano JM, Liu S: Sex hormone-binding globulin and risk of type 2 diabetes in women and men. N Engl J Med 2009;361:1152–1163. 30 Goto A, Chen BH, Song Y, Cauley J, Cummings SR, Farhat GN, Gunter M, Van Horn L, Howard BV, Jackson R, et al: Age, body mass, usage of exogenous estrogen, and lifestyle factors in relation to circulating sex hormone-binding globulin concentrations in postmenopausal women. Clin Chem 2014;60: 174–185.
