RESEARCH ARTICLE
Single nucleotide polymorphisms of INS, INSR, IRS1, IRS2, PPAR-G and CAPN10 genes in the pathogenesis
of polycystic ovary syndrome
Maheswari Thangavelu1, Usha Rani Godla2, Solomon F.D. Paul1, Maddaly Ravi1*
1
Faculty of Biomedical Sciences, Sri Ramachandra University, Porur, Chennai, India.
2
Department of Obstetrics and Gynecology, Sri Ramachandra Medical Centre, Porur, Chennai, India.
*Corresponding Author:
Maddaly Ravi PhD, MNASc
Associate Professor
Department of Human Genetics, Faculty of Biomedical Sciences, Technology and Research,
Sri RamachandraUniversity, Porur,
Chennai – 600116. India.
E-mail address: [email protected]
Running Title: Role of single nucleotide polymorphisms in PCOS
Key words: Anovulation, Hyperandrogenism, Infertility, Polycystic ovary syndrome, Single nucleotide
polymorphism, Real time polymerase chain reaction.
Single nucleotide polymorphisms of INS, INSR, IRS1, IRS2, PPAR-G and CAPN10 genes in the pathogenesis
of polycystic ovary syndrome
Abstract
Purpose
Polycystic ovary syndrome (PCOS) is the most common and a complex female endocrine disorder. It is the leading
cause of female infertility. We aimed to investigate the association of single nucleotide polymorphisms of INS,
INSR, IRS1, IRS2, PPAR-G and CAPN10 gene in the pathogenesis of PCOS.
Method
A Hospital-based, observational case–control study was carried out in 169 PCOS and 169 control women in the
Southern region of India. Genotype was carried out by Real-time polymerase chain reaction. A chi squared test was
performed and the genotypes were verified to comply with the Hardy – Weinberg equilibrium. Odds ratio and 95%
confidence interval were calculated to assess the relative risk.
Results
Comparison of clinical characteristics of women with PCOS and controls reveal an increase in Body mass index
(BMI), luteinizing hormone/ follicle stimulating hormone (LH/FSH) ratio, glucose levels, insulin, testosterone,
hirsutism and antral follicular count in PCOS women. The variant rs1801278 (p=0.002; OR- 2.88; 95%CI- 1.43,
5.80) show an association with PCOS. In the genotypic (p=0.0002) and allelic model (p=0.000) significance
persisted even after Bonferroni correction. The genotypes of SNP’s strongly influence BMI, LH, LH/FSH ratio,
ovarian volume and antral follicular count in PCOS women.
Conclusion
The study results were suggestive of a positive association between Gly972Arg of IRS1 and PCOS in the South
Indian population while INS, INS2, PPAR-G and CAPN10 failed to show any association in link with PCOS in our
studied population. Further studies focusing the role of IRS1 are warranted to delineate its implication towards
PCOS.
Key words
Anovulation, Hyperandrogenism, Infertility, Polycystic ovary syndrome, Single nucleotide polymorphism, Real time
polymerase chain reaction.
Introduction
Polycystic Ovary Syndrome (PCOS), the most common and highly complex endocrinopathy affects the female
population of reproducing age at an increasing prevalence of about 4-8% [Azziz et al. 2004]. PCOS, the leading
cause of oligo-anovulatory infertility is characterized by insulin resistance, hyperinsulinemia (found in 50–70 % of
women diagnosed) [Franca et al. 2010; Navaratnarajah et al. 2008] and many other metabolic abnormalities that
including obesity and hyperandrogenism, which has resulted in increased risk of diabetes mellitus, dyslipidemia,
atherosclerosis and also endometrial carcinoma [Carmina et al. 2006; Giallauria et al. 2008; Orio et al. 2006].
Furthermore, women with PCOS have expressed several interrelated features including chronic anovulation,
polycystic ovaries and oligomenorrhea which are coupled with anomalous androgen and insulin-related parameters
irrespective of other standard reproductive factors [Adams et al. 2004]. Various studies have also suggested that
women with PCOS are at a higher risk for gestational diabetes, miscarriages, preeclampsia and preterm labor
[Boomsma et al. 2006]. The genetic basis of the disease is still not clearly understood owing to the difficulties in
determining the inheritability of PCOS. The genes that regulate insulin secretion and action, ovarian and adrenal
steroidogenesis are considered as candidate genes which determine the expression of several integral phenotypes of
PCOS. Insulin gene thought to play a functionally central role in insulin secretion and/or action and also in the
signaling pathways. The insulin gene to the 5’ flanking region has variable number of tandem repeats and has shown
to influence transcriptional activity of gene in vitro [Paquette et al. 1998]. The insulin receptor gene (INSR) is a
hetradimeric complex containing 22 exons. Exons 17-21 encode tyrosine kinase domain which is essential for
insulin signal transduction. Any polymorphisms in INSR gene can introduce changes in insulin receptor function and
may predispose to the development of PCOS [Siegel et al. 2002]. Insulin receptor substrate1(IRS1) and 2 (IRS2)
involved in the insulin signaling by activating phosphatidylinositol-3- kinase, reported to be associated with insulin
resistance, type2 DM and PCOS [Dilek et al. 2005]. Peroxisome proliferator receptor gamma (PPAR-G) is a nuclear
receptor and plays a critical role in carbohydrate, lipid metabolism and adipocyte differentiation by regulating
multiple genes [Sanchez et al. 2002]. The calpain 10 (CAPN10) gene is calcium dependent and is expressed at
mRNA and protein levels by several tissue types. This gene was recognized as type2DM susceptibility loci and is
allied to proinsulin processing, insulin secretion and insulin resistance [Horikawa et al. 2000; Baier et al. 2000]. In
the present study we tried to investigate the role of single nucleotide polymorphisms of INS, INSR, IRS1, IRS2,
PPAR-G and CAPN10 genes in Indian population which were previously studied in different populations with
consideration of utmost link towards PCOS. Polymorphism of the above said genes in link with PCOS has been
reported by several studies in different populations but very few studies have been reported in the Indian population.
INS-VNTR in link with PCOS is studied for the first time in Indian population. The SNP rs1805097 of IRS2 and
rs2975766, rs7607759 of CAPN10 gene in relation towards PCOS is also studied for the first time in Indian
population. This study will shed light on the polymorphic pattern in the selected population and its association
towards the syndrome.
Materials and methods
Study subjects
The study was conducted on altogether 338 women, comprising 169 cases (PCOS) and 169 controls. PCOS subjects
were selected based on observation of oligoamenorrhea/ anovulation, clinical or biochemical evidence of
hyperandrogenism and/or polycystic ovaries on ultrasonography (The Rotterdam criteria, 2003) and normal,
unaffected, age-matched fertile women with regular menstrual cycles (interval of 28-35 days) and with normal
ovaries from the same geographical region were included in the study as controls. Women with galactorhea,
hyperthyroidism, any systemic disease that affects their reproductive physiology, or any medication which interferes
with the normal function of the hypothalamic-pituitary-gonadal axis were excluded from the study. Subject’s age
group was in the range from ≥ 20yrs to ≤40yrs. The study was approved by the institutional ethical committee of Sri
Ramachandra University. A written informed consent was collected from all the subjects enrolled in the study.
Subject’s history and other anthropometric assessments were carried out.
Biochemical and Hormonal analysis
Blood Samples were taken after an eight hour fast through vein puncture from the subjects for carrying out
biochemical and hormonal analysis on day 2 (D2) or day 3 (D3) of their follicular phase. Tests were conducted
using Siemens-ADVIA Centaur Automated System and were assayed by chemiluminescent immuno assay (CLIA).
Hirsutism was calculated using the modified Ferriman-Gallwey scoring method (Ferriman& Gallwey, 1961). IR was
assessed using the homeostatic model assessment (HOMA-IR), calculated as (fasting Insulin× fasting glucose)/22.5
[Li et al. 2014]. PCO was confirmed by ultrasound assessments by means of a transvaginal ultrasonography with a
transvaginal probe of curved array 5.0 -2.0 MHz (for ovary) with a frequency of 5.9MHz using diagnostic
ultrasound system, Sonoscape Co., Ltd, China; with 12 or more follicles in each ovary, measuring 2-9 mm in
diameter and/or increased ovarian volume (10 cm3).
Molecular analysis
DNA was isolated from peripheral blood using modified salting-out method. Genotyping of single nucleotide
polymorphisms (SNP’s) was carried out with Real-time polymerase chain reaction technology (Taqman® SNP
Genotyping Assay; Applied Biosystems, Carlsbad, California, USA). Each reaction mixture had a final volume of
5μl [2X Taqman Genotyping Master Mix -2.50μl, 20X Taqman Drug Metabolism Genotyping assay mix – 0.25μl,
Genomic DNA diluted in distilled water – 2.25μl (3-20ng of genomic DNA)]. Thermal Cycle reaction with initial
denaturation of 950 for 10mins followed by 40 cycles of denaturation 95 0 for 15 secs, Annealing/Extension 60o for 1
min was carried out in a 384 well optical plate on a 7900- HT Fast Real Time PCR machine. The Taqman Drug
Metabolism Genotyping assay mix contains the primers and fluorescent probes. The alleles were labeled with VIC®
-dye and FAM™ dye.
Statistical analysis
The continuous variables were expressed as mean ± standard deviation. All statistical analysis was performed using
the SPSS statistical software version 9.0. Hardy–Weinberg equilibrium (HWE) tests were performed by comparison
of observed and expected genotype frequencies using chi-squared goodness-of-fit test. The genotype and allele
frequencies of each polymorphism were compared between subjects with PCOS and the controls by the chi-square
test. The odds ratio and 95% confidence interval were calculated using wild type genotypes or alleles as the
reference group. A p-value < 0.05 was considered to be statistically significant. Significant values were further
confirmed by multiple testing using Bonferroni correction to address the multiple comparisons problem. Pair-wise
linkage disequilibrium was computed as both D' and r2 for the PPAR-G and CAPN10 genes using Haploview v. 4.1.
SNP-SNP interactions among variants of PPAR-G gene were assessed by non-parametric Multifactor
Dimensionality Reduction (MDR) analysis using software version 3.0.2.
Results
The anthropometric and clinical parameters between cases and controls are measured and presented in Table1. The
mean age of POS and control women were 26.92 and 27.52. BMI between PCOS and control women were more or
less similar with mean 25.14 and 24.18 respectively. The mean LH level in controls was 4.68 and a two fold
increase in mean value was observed in PCOS women (9.12). However, a decrease in the FSH levels were observed
in PCOS (6.18) compared to controls (7.11). Also the mean LH/FSH ratio was increased among PCOS women
(1.62) compared to controls (0.71). A highly significant difference was observed in the mean testosterone levels of
PCOS women (71.65) compared to their controls (28.23). Correspondingly, the mean hirsutism scores of PCOS
women (4.65) were higher than the controls (1.83). The mean insulin levels and HOMA-IR levels in PCOS and
controls were 13.77, 10.76 and 3.08, 2.28 respectively. Glucose levels were elevated in PCOS women. The
unilateral right and left ovarian volume in PCOS and control were 10.33, 9.26 and 4.79, 4.33 and the unilateral right
and left AFC in PCOS and control were 10.43, 10.27 and 6.07, 5.78 respectively.
All the genotype frequencies were distributed according to the Hardy-Weinberg equilibrium (HWE) in both case and
control subjects except for rs1801278 of IRS1 gene (cases only). In the rs689 variant of INS, T alleles were more
compared to the A alleles in both cases and controls. The minor allele frequency was14% in control and 12% in the
PCOS case groups (Table 2). The genotypic distribution between PCOS and control subjects did not show any
significant association. In the INSR gene (rs1799817), the frequency of T allele was found to be more (66.27%) than
the C allele (33.73%) in both cases and controls (Table 3). No significant association was observed at the dominant
model or allelic model. SNP rs1801278 of IRS1 gene (Table 4), the minor allele frequency among cases and controls
were 53% and 43%. Significant difference was observed between PCOS and control subjects in genotypes (GA0.002, AA-0.002), dominant model (0.001) and the allelic model (<0.001). This significance was maintained after
Bonferroni correction for multiple testing (GA-0.0002; AA-0.0002; GA+AA- 0.0001). The frequency of A allele
(53.25%) was more compared to the G allele (46.75%) in PCOS women and the frequency of G allele (57.40%) was
more compared to A allele (42.60%) in control women. Polymorphism of rs1805097 of IRS2 gene is depicted in
Table 5. The frequency of homozygous GG was 49.11% and 49.70 % in PCOS and control while the homozygous
AA was much lesser in both PCOS and control with 8.88% and 11.83%. No significant difference was seen in the
genotypic, dominant and the allelic model for the variant rs1805097. Table 6 explains two variants rs1801282 and
rs3856806 of PPAR-G gene. The genotype distribution frequency for rs1801282 among cases and controls for
homozygous GG were higher 76.33% and 73.96% compared to the homozygous CC which was 2.37% and 1.78%.
The genotype distribution of rs3856806 for cases and controls for genotypes CC, CT and TT are as follows; 72.78%,
23.67%, 3.55% and 69.23%, 28.99%, 1.78%. The minor allele frequency for cases and control subjects were more
or less similar for both the variants. The variants rs7607759 and rs2975766 of CAPN10 gene are illustrated in Table
7. No significant difference was observed in the genotype distribution, dominant model and allelic model between
cases and controls for both the SNP’s but increased risk was observed in GG genotype (p= 0.558; OR 1.37; CI 0.473.98) of rs7607759 and genotypes GA (p= 0.121; OR 1.83; CI 0.84-3.98), AA (p= 0.531; OR 2.12; CI 0.19-23.65),
dominant model (p= 0.099; OR 1.85; CI 0.88-3.91) and allelic model (p= 0.086; OR 1.82; CI 0.91-3.66) of
rs2975766. However neither of these findings remains significant. The Clinical and hormonal variables were
compared with the genotypes of all the SNP’s and are presented in Figure 8-15. The genotypes of rs689 showed
increase in Waist/hip ratio, LH, LH/FSH and right ovarian volume in PCOS women. Post-hoc test was done to find
out which specific groups differed, and it revealed that the TT/TA combination differed significantly. The
rs1799817 of INSR gene, increase in LH (p-0.049) was observed in PCOS women with CC/CT combination. In
rs1801278 of IRS1 gene and rs3856806 of PPAR-G gene the phenotypic variables did not show any significant
difference in the PCOS group but unilateral left antral follicular count was significantly more in the control groups
with AA,GA combinations (p-0.036) in rs1801278 and CC,TT combinations (p-0.020) in rs3856806 showing risk.
In IRS2 gene, an increase in BMI was seen in PCOS women (p-0.024) and the combination genotypes between AA,
GA and AA, GG groups contributed to the difference. The SNP rs1801282 of PPAR-G, the homozygous GG
genotype displayed decrease in levels of Waist/hip ratio, LH, LH/FSH, right ovarian volume and right and left antral
follicular count compared to the CC and CG genotype. In variant rs7607759 of CAPN10 gene, genotypes did not
show any changes in the phenotype except for hirsutism grades in control women (p- 0.028). The genotypes
rs2975766 revealed relation towards LH and LH/FSH ratio in PCOS women and waist/hip ratio among control
subjects. Pairwise linkage disequilibrium between rs1801282 and rs3856806 of PPAR-G (D′=0.289 and r2=0.22)
and rs7607759 and rs2975766 of CAPN10 (D′=0.211 and r2=0.87) was not strong and significant. Haplotype
analysis show no haplotypes between the SNP’s with an estimated frequency more than 5%. SNP-SNP interactions
were analyzed by MDR. The prediction accuracy of the model was estimated by tenfold cross-validation. The best
model chosen had the highest cross-validation consistency (CVC) of 10/10 and a testing accuracy (TA) of 0.526.
The best model consisted of 2 PPAR-G gene variants (rs1801282 and rs3856806) and is shown in Table 16 and the
combinations of genotype interaction of the two SNPs are depicted in Figure 1.
Discussion
Several biochemical and hormonal variables were checked to find out the difference in PCOS and control women.
Not much difference was observed in the mean values of body mass index in PCOS (25.14) and controls (24.18) in
our population but waist/hip ratio was observed to be significantly high in PCOS women. Waist/hip ratio could
perhaps be a better indicator risk than BMI. LH levels were higher in PCOS women and lower in control and FSH
levels were higher in control and lower in PCOS women. A decrease in FSH levels disrupts follicular growth. Also,
a significant difference was observed in the Testosterone levels of PCOS women compared to that of controls,
supporting with the hypothesis that an increase in testosterone levels leads to suppression of normal menstruation
and ovulation. The augmented GnRH pulse frequency is hugely associated with hyperandrogenism and increased
ovarian volume. An increase in testosterone levels above a normal range leads to an unusual increase in hair growth
(hirsutism). An increased conversion of testosterone to dihydrotestosterone via the enzyme 5 alpha reductase within
the pilosebaceous unit may be one of the root causes for hirsutism. As testosterone increases, a decline in the serum
prolactin levels and FSH levels were observed which possibly lead to the suppression of normal menstruation and
ovulation. A significant difference in the Glucose levels, fasting insulin levels and HOMA-IR index were found
between PCOS and control women with an elevation in PCOS women. We also found that the unilateral antral
follicular count and ovarian volume of the right ovary showed a marginally higher mean value than that of the left in
women with PCOS.
The variable number tandem repeats positioned at -23bp in the 5’ flanking region of INS gene is considered to be a
susceptible loci and acts as a surrogate marker. The rs689 polymorphism has shown association with PCOS by the
mutant class III alleles also presenting with increased serum insulin levels and BMI in PCOS women [Waterworth et
al. 1997]. The frequency of class III allele (12%) reported in our population was much lower compared to the
Japanese, European, Korean and Han Chinese which was 97%, 30%, 94% and 93.5% accordingly. The homozygous
class I alleles in our study were higher in both PCOS and control. This variant rs689 of INS gene was not associated
with PCOS in the present study. But the heterozygote TA and homozygous AA genotypes showed an increase in
waist/hip ratio (p=0.020), increase in luteinizing hormone levels (p=0.020) and right ovarian volume (p=0.042) in
PCOS women. Alterations in insulin action and β- cell function may lead to augmented GnRH pulse frequency
which increases LH secretion [Li et al. 2014] and is hugely associated with hyperandrogenism and increased ovarian
volume [Kralovicova 2006]. Similar to our results were studies on anovulatory women with PCOS selected based on
NIH criteria in Czech women reporting a negative association with odds ratio 1.44(0.50-4.18; p-0.59) [Vancova et
al. 2002]; Another study with 216 PCOS and 192 non PCOS women in Han Chinese population reported no
significant difference between cases and control groups either in allele (p-0.996) or genotype (p-0.802) frequencies
[Xu et al. 2009]; also a family based association study reported no excess transmission of alleles of rs689 VNTR
polymorphism, regardless of parent of origin [Powell et al. 2005].
The rs1799817 polymorphism of INSR gene shows a negative association towards PCOS women in our population.
But the genotypes influenced the phenotypic expression. Women with TT genotype of His1058His showed an
increase in insulin, endometrial thickness, ovarian volume and right antral follicular count and women with CC
genotype show an increase in testosterone, hirsutism grade and pre- prandial glucose in PCOS women. Insulin
resistance might up-regulate testosterone production in theca cells, LH secretion in pituitary which may perhaps
disturb follicular maturation and lead to PCOS [Le 2006]. The rs1799817 polymorphism influenced an increase in
LH levels (p=0.049) in PCOS women. The frequencies of CC genotypes (PCOS-11.24%, control-13.02%) were
much lower in our population. While CC genotype frequency in other population reported (PCOS-43.9%, control52.8%); (PCOS-34%, control-27.66%) [Mukherjee et al. 2009; Bagheri et al. 2015]. Lack of association of
His1058His was also stated in other studies as reported by Bagheri et al, Tehrani et al and Urbanek et al. Two
GWAS studies in 2011 and 2012 by Chen et al. and Shi et al, reported an association of rs1799817 of INSR gene in
PCOS women. Increase in frequency of uncommon T allele in lean PCOS those with body mass index < 27 kg/m2)
compared with lean controls were reported [Siegel et al. 2002]. Yet another study told genetic variation in exon 17
of INSR is associated with insulin resistance and hyperandrogenemia among lean Indian women with polycystic
ovary syndrome but not obese women. The difference in phenotypic expression and association of exon17
polymorphism towards PCOS in other populations could be due to the frequency of distribution of genotypes TT,
CT and CC across population where the effect of genotype influences suppression or increased expression of
phenotypic change.
The rs1801278 of IRS1 gene, show 21 % PCOS subjects with Glycine to Arginine substitution whereas; in control
15% had this substitution. The Gly972Arg polymorphism towards susceptibility of PCOS reveal that the
heterozygous Gly/Arg genotype may increase the risk of developing PCOS by 2.30 times (p=0.002) and having
Arg/Arg genotype which is the mutant in our population might increase a 2.88 times risk in the development of
PCOS (P=0.002). These findings remained significant even after Bonferroni correction for multiple comparisons
(p=0.0002). The dominant model (Gly/Arg+Arg/Arg) did contribute to a relative risk of 2.42 (P=0.001), Bonferroni
corrected value (p=0.0001). And the allele Arg showed a risk of 1.94 in developing PCOS (p-<0.001), Bonferroni
corrected value (p=0.0001). But Gly972Arg polymorphism did not show a change in the phenotypes except for the
left antral follicular count in control subjects (p=0.036). Consistent to our results, a study from the Japanese
population reported significantly more IRS-1 972Arg carriers among PCOS women compared to healthy controls
(10.6% vs. 4.8%, p=0.029), and had a significantly increased risk of developing PCOS (odds ratio: 3.31, 95%
confidence interval: 1.49–7.35). They also found that the IRS1 polymorphism was not associated with its phenotypes
such as BMI, insulin resistance or androgen levels in PCOS women [Baba et al. 2007]. Also, women carrying IRS-1
Gly/Arg had an increased risk of PCOS (OR = 2.49, 95% C.I. 1.16-5.37, p = 0.019) [Lin et al. 2014]. Two recent
meta- analysis on PCOS women reported that Gly/Arg and Gly/Gly genotype is significantly associated with risk of
developing PCOS (odds ratio 3.31; 1.49-7.35) which is chiefly mediated by higher levels of fasting insulin [Ruan et
al. 2012]. The IRS-1 variant allele occurs significantly more frequently among PCOS patients than among healthy
women [Sir-Petermann et al. 2001]. On the other hand Spanish, Croatian, Chilean, Taiwanese, Caucasian, Slovak
and Iranian population did not find any association of Gly972Arg towards PCOS. However a study analyzing 250
PCOS and 299 controls reported IRS-1 polymorphic alleles having a similar distribution between cases and controls,
and seems to have a probable protective role against development of specific PCOS sub-phenotypes [Dasgupta et al.
2012].
In rs1805097 of IRS2, we report that Asp was the minor allele in our taken population and the Caucasians and the
African Americans also presented Asp as the minor allele [Millan et al. 2004]. Nevertheless Lin et al. reported Gly
as the minor allele and association of Asp/Asp genotype with higher fasting insulin and HOMA index [Goodarzi et
al. 2005]. While Ehrmann and his team reported association of Gly/Gly with higher 2h OGTT levels. The variant
rs1805097 (Gly1057Asp) of IRS2 gene was not associated with PCOS in the studied population but this
polymorphism may possibly have an effect in weight gain in PCOS women. The non- association was confirmed in
Spanish and Greek population as well. IRS1 and IRS2 polymorphisms influence glucose homeostasis and could
influence obesity, regardless of women presented with PCOS [Taylor 1997]. Levels of both IRS-1 and IRS-2 are
increased in theca cells from women with PCOS which leads to proliferation and increased androgen synthesis [Yen
et al., 2004].
The Ala variant of rs1801282 of PPAR-G gene was found to be higher in PCOS and controls. The Ala/Ala variant
tend to decrease waist/hip ratio, lower LH levels, LH/FSH ratio, right OV and AFC in PCOS women in our
population. Ala variant cuts the risk of PCOS with decline in levels of BMI and fasting Insulin [Xu et al. 2009].
Polymorphism of rs1801282 was not associated with PCOS in the studied population. Similar negative association
was observed in the Caucasian and Greek population and positive association shown from Italy and Korea. Earlier
studies have also confirmed the PPARG Pro12Ala variant allele association with higher insulin sensitivity,
high-density lipoprotein (HDL) levels [Deeb et al. 1998], decreased risk of T2DM [Hara et al. 2000] and decreased
incidence of CVD [Doney et al. 2004]. A meta-analysis reported lower insulin levels in Ala carriers considering
PCOS patients and controls as an entire group [San-Millan and Escobar-Morreale 2010]. Two additional
meta-analyses determined that the PPARG Pro12Ala variant may result in lower insulin levels, but exerts no effect
on HOMA-IR in PCOS patients [He et al 2010; Zhang et al. 2010]. One published in India found an association
towards PCOS at the allelic level [Dasgupta and Reddy 2013] but another study from Mumbai, India showed
significant difference in both allelic and genotypic frequency between PCOS and controls with reduced
susceptibility to PCOS [Shaikh et al. 2013]. Therefore the Ala variant of PPAR-G is thought to be associated with
reduced transcriptional activity which improves insulin action towards suppression of lipolysis in turn reducing the
production of free fatty acids and enables their storage in adipocytes [Taylor et al. 1990].
The variant rs3856806 located in exon 6 of PPAR-G is a functional synonymous SNP. For rs3856806 of PPAR-G
(His447His), neither the genotypes nor the alleles show any association towards PCOS but the mutant homozygous
TT genotype showed an increase in the right antral follicular count (p=0.020) in control women. Also to see the
effect of genotypes of SNPs together MDR analysis was carried out. The best MDR models for the studied SNP
rs1801282 and rs3856806 markers had a testing accuracy (TA) of 0.541 and cross‐validation consistency (CVC) of
10/10. However, this model was not significantly associated with PCOS (p=0.755). Each cell is labelled as high risk
if the ratio of the affected individual to the unaffected individual exceeds a threshold 1 and low risk if the threshold
is not exceeding. The higher risk group contains combinations of (corresponding to SNP rs3856806 and rs1801282)
CC‐CC, CC-CG, CT-CG, CT-GG, TT-CG. The homozygous GG of rs180282 signifies no risk and confers
protection towards PCOS over the other two genotypes. The pairwise linkage disequilibrium (LD) values (D’=0.289
and r2=0.22) between rs1801282 and rs3856806 revealed that these two SNPs are not in strong LD.
The minor allele frequency of rs7607759 of CAPN10 gene was 17.75% while Whites, Blacks, Hispanics,
Asian/Pacific Islanders reported 15.5%, 6.73%, 11.5% and 9.06% respectively [Wu et al. 2000]. Even though the
frequency is high in Indian population we found a negative association of rs7607759 genotype and allele frequency
of CAPN10 towards PCOS. We say that the SNP rs7607759 is not a susceptible locus linked to PCOS in our study
population. When genotypes of PCOS and control were compared with the phenotypes we found that the hirsutism
grades in control women with presence of AA/AG combination did show some significance but no phenotypic
relation was observed in PCOS women.
The genotypes of SNP rs2975766 did not show any association towards PCOS. The mutant homozygous AA
genotype was observed in 2 women with PCOS and 1 woman in the control group. This homozygous variant and the
heterozygous variant renders risk 2.12(0.19-23.65); p- 0.531 but was not significant enough to show an association.
When the genotypes of rs2975766, compared to the biochemical and other hormonal features of PCOS it was
revealed that increased levels of LH and LH/FSH ratio was observed in women with AA genotype compared to the
other genotypes. The mutant AA genotype may possibly indicate changes at the phenotypic level in PCOS women.
Studies from meta-analysis reported no association with the above studied variants of CAPN10 gene towards PCOS
[Shen et al. 2013]. The SNP’s rs760759 and rs2975766 of CAPN10 gene are the first of its kind to be studied in the
Indian population. The pairwise linkage disequilibrium (LD) values D’=0.211 and r2=0.87, between rs7607759 and
rs2975766 of CAPN10 gene revealed no significant LD between SNPs.
Conclusion
Out of all the SNPs studied, Gly972Arg of IRS1 gene showed an association with PCOS. Polymorphism of
Gly972Arg could play a contributory role in the pathophysiology and risk of PCOS. Further analysis of this SNP
variant should be evaluated in larger population and the same variant should be checked in different population
group to confirm its significance. Also, additional SNPs of IRS1 gene has to be studied to confirm the role of insulin
receptor substrate 1 gene towards PCOS. Apart from rs1801278, the other SNPs failed to show any association with
PCOS in the population we studied. The genotypes of analyzed SNPs strongly influenced the change in phenotype
by increasing weight gain, LH levels, LH/FSH ratio, ovarian volume and antral follicular count in PCOS women
Acknowledgement
We acknowledge funding from the Department of Biotechnology (DBT), Government of India
(Project Ref. No.BT/PR14090/GBD/27/275/2010)
Conflict of Interest
The authors declare no conflict of interest.
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Received 4 April 2016; in revised form 17 May 2016; accepted 4 July 2016;
Unedited version published online: 8 July 2016
Table 1. Descriptive analysis on variables between PCOS cases and control subjects
CONTROL
CASE
95% Confidence Interval
PARAMETERS
Mean
for Mean
95% Confidence
Mean
Interval for Mean
Lower
Upper
Lower
Upper
bound
bound
bound
bound
p-value
Age
27.52
26.91
28.13
26.92
26.26
27.58
0.190
BMI
24.18
23.67
24.68
25.14
24.45
25.84
0.028*
Waist-Hip
0.73
0.72
0.74
0.86
0.85
0.87
<0.001 **
LH
4.68
4.32
5.04
9.12
8.37
9.86
<0.001 **
FSH
7.11
6.75
7.46
6.18
5.86
6.49
<0.001 **
LH-FSH
0.71
0.65
0.77
1.62
1.47
1.77
<0.001 **
Estradiol
60.23
56.93
63.53
64.72
60.62
68.84
0.093
T. Testosterone
28.23
26.23
30.22
71.65
64.73
78.56
<0.001 **
Hirsutism
1.83
1.58
2.06
4.65
3.98
5.31
<0.001 **
Insulin
10.76
10.12
11.42
13.77
12.61
14.95
<0.001 **
Pg(mg/dl)
85.74
84.47
87.00
89.54
86.94
92.14
0.010*
P-pg(mg/dl)
110.19
108.56
111.84
116.00
111.02
102.98
0.030*
HOMA-IR
2.28
2.13
2.43
3.08
2.77
3.37
OV (Right)
4.79
4.18
5.39
10.33
9.32
11.34
<0.001 **
<0.001 **
OV (Left)
4.33
3.71
4.94
9.26
8.52
9.99
<0.001 **
AFC (Right)
6.07
5.71
6.42
10.43
9.89
10.97
<0.001 **
AFC (Left)
5.78
5.43
6.13
10.27
9.70
10.85
<0.001 **
BMI-body mass index, LH- luteinizing hormone, FSH- follicle stimulating hormone, T. Testosterone- total
testosterone, Pg- pre-prandial glucose, P-pg-post prandial glucose, HOMA-IR- homeostatic model assessment of
insulin resistance, OV-Ovarian volume, AFC-antral follicular count; Statistical analysis was performed using the
SPSS statistical software version 9.0. The continuous variables are expressed as mean ± standard deviation; the
significant difference obtained in PCOS subjects and controls were calculated and compared using ANOVA. Pvalue < 0.05 was considered to be statistically significant; *<0.05; **<0.01.
Table 2. Allele and genotype frequencies of INS rs689 in PCOS and control groups
Gene
Genotype
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
INS
p -Value
rs689
TT
124(73.37)
131(77.51)
Reference
0.667
TA
AA
TA+AA
41(24.26)
4(2.37)
45(26.63)
35(20.71)
3(1.78)
38(22.49)
0.81(0.48-1.35)
0.71(0.16-3.23)
0.81(0.49-1.31)
0.415
0.656
0.376
Reference
0.81(0.52-1.31)
0.365
HW:p
Allele frequency
T
A
MAF
0.780
289(85.50)
49(14.50)
0.14
0.711
297(87.87)
41(12.13)
0.12
Odds ratio -OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency, A, adenine; T,
thymine. A chi squared test was performed to evaluate the association between single nucleotide polymorphisms and
PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds ratio and 95%
confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05 was considered
to be statistically significant.
Table 3. Allele and genotype frequencies of INSR rs1799817 in PCOS and control groups
Gene
INSR
Genotype
rs1799817
CC
CT
TT
CT+TT
HW:p
Allele frequency
C
T
MAF
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
p -Value
22(13.02)
19(11.24)
Reference
0.600
67(37.64)
80(47.34)
147(86.98)
0.188
76(44.97)
74(43.79)
150(88.76)
0.938
1.31(0.65-2.63)
1.07(0.53-2.14)
1.18(0.61-2.27)
0.442
0.845
0.617
111(32.84)
227(67.16)
0.67
114(33.73)
224(66.27)
0.66
Reference
0.96(0.69-1.32)
0.806
Odds ratio - OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency; C, cytosine; T,
thymine. A chi squared test was performed to evaluate the association between single nucleotide polymorphisms and
PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds ratio and 95%
confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05 was considered
to be statistically significant.
Table 4. Allele and genotype frequencies of IRS1 rs1801278 in PCOS and control groups
Gene
IRS1
Genotype
rs1801278
GG
GA
AA
GA+AA
HW:p
Allele frequency
G
A
MAF
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
p -Value
50(29.59)
25(14.79)
Reference
0.003**
94(55.62)
25(14.79)
119(17.41)
0.0742
108(63.91)
36(21.30)
144(85.21)
<0.001
2.30(1.32-4.00)
2.88(1.43-5.80)
2.42(1.41-4.14)
0.002**
0.002**
0.001**
194(57.40)
114(42.60)
0.43
158(46.75)
180(53.25)
0.53
Reference
1.94(1.41-2.66)
<0.001**
Odds ratio - OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency; A, adenine; G,
guanine. A chi squared test was performed to evaluate the association between single nucleotide polymorphisms and
PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds ratio and 95%
confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05 was considered
to be statistically significant.
Table 5. Allele and genotype frequencies of IRS2 rs1805097 in PCOS and control groups
Gene
IRS2
Genotype
rs1805097
GG
GA
AA
GA+AA
HW:p
Allele frequency
G
A
MAF
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
p -Value
84(49.70)
83(49.11)
Reference
0.611
65(38.46)
20(11.83)
85(50.30)
0.184
71(42.01)
15(8.88)
86(50.89)
0.973
1.11(0.70-1.74)
0.76(0.36-1.58)
1.02(0.67-1.57)
0.664
0.461
0.913
233(68.93)
105(31.07)
0.31
237(17.12)
101(29.88)
0.30
Reference
0.95(0.68-1.31)
0.738
Odds ratio - OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency; A, adenine; G,
guanine. A chi squared test was performed to evaluate the association between single nucleotide polymorphisms and
PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds ratio and 95%
confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05 was considered
to be statistically significant.
Table 6. Results of association test with PPAR-G gene polymorphisms rs1801282 and rs3856806 in PCOS and
control group
Gene
PPAR-G
Genotype
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
p -Value
3(1.78)
4(2.37)
Reference
0.767
CG
GG
CG+GG
HW:p
Allele frequency
C
G
MAF
rs3856806
CC
41(24.26)
125(73.96)
166(98.22)
0.86
36(21.30)
129(76.33)
165(97.63)
0.44
0.65(0.13-3.14)
0.77(0.16-3.52)
0.75(0.16-3.38)
0.598
0.740
0.702
47(13.91)
291(86.01)
0.86
44(13.02)
294(86.98)
0.87
Reference
1.07(0.69-1.68)
0.735
117(69.23)
123(72.78)
Reference
0.357
CT
TT
CT+TT
HW:p
Allele frequency
C
T
MAF
49(28.99)
03(1.78)
52(30.77)
0.405
40(23.67)
06(3.55)
46(27.22)
0.237
0.78(0.48-1.27)
1.90(0.47-7.78)
0.84(0.53-1.35)
0.094
0.363
0.471
283(83.73)
55(16.27)
0.16
286(84.62)
52(15.43)
0.15
Reference
0.94(0.62-1.41)
0.751
rs1801282
CC
Odds ratio - OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency; G, guanine; C,
cytosine; T, thymine. A chi squared test was performed to evaluate the association between single nucleotide
polymorphisms and PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds
ratio and 95% confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05
was considered to be statistically significant.
Table 7. Results of association test with CAPN10 gene polymorphisms rs7607759 and rs2975766 in PCOS and
control group
Gene
CAPN10
Genotype
Controls
(n=169)
Cases
(n=169)
OR
(95%CI)
p -Value
108(63.91)
118(69.82)
Reference
0.248
AG
GG
AG+GG
HW:p
Allele frequency
A
G
MAF
rs2975766
GG
55(32.54)
6(3.55)
61(36.09)
0.756
42(24.85)
09(5.33)
51(30.18)
0.052
0.69(0.43-1.13)
1.37(0.47-3.98)
0.76(0.49-1.21)
0.141
0.558
0.247
271(80.18)
67(19.82)
0.20
278(82.25)
60(17.75)
0.18
Reference
0.87(0.59-1.28)
0.490
157(92.90)
148(87.57)
Reference
0.255
GA
AA
GA+AA
HW:p
Allele frequency
G
A
MAF
11(6.51)
1(0.59)
12(7.10)
0.118
19(11.24)
2(1.81)
21(12.43)
0.139
1.83(0.84-3.98)
2.12(0.19-23.65)
1.85(0.88-3.91)
0.121
0.531
0.099
325(96.15)
13(3.85)
0.04
315(93.20)
23(6.80)
0.07
Reference
1.82(0.91-3.66)
0.086
rs7607759
AA
Odds ratio - OR, CI- confidence interval, HW- hardy-Weinberg, MAF- minor allele frequency; A- adenine; G,
guanine. A chi squared test was performed to evaluate the association between single nucleotide polymorphisms and
PCOS; the genotypes were verified to comply with the Hardy – Weinberg equilibrium; odds ratio and 95%
confidence interval were calculated to assess the relative risk; *.p<0.05, **.p< 0.01; p-value < 0.05 was considered
to be statistically significant.
Table 8. Genotype – phenotype correlation of rs689 of INS gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
INS
T/T
T/A
A/A
(rs689)
(n=131)
(n=35)
(n=3)
Age
27±4.4
26.97±3.96
22.67±1.53
BMI
25.3±4.6
24.32±4.15
Waist(cms)
82.31±15.70
Hip(cms)
CONTROLS (n=169)
p-Value
T/T
T/A
A/A
p-Value
(n=124)
(n=41)
(n=4)
0.230
27.46±4.05
27.75±4.17
26.75±1.71
0.859
27.55±3.89
0.343
24.17±3.25
24.38±.61
22.58±3.02
0.589
79.71±11.35
87.0±12.3
0.548
64.08±13.95
63.95±13.69
61.25±12.82
0.922
96.36±18.38
90.94±12.32
99.33±11.93
0.238
87.78±13.65
85.36±14.37
83.75±19.36
0.664
Waist/hip ratio
0.85±0.04
0.87±0.04
0.87±0.02
0.020*
0.73±0.07
0.74±0.06
0.74±0.04
0.410
LH
8.55±4.44
11.06±5.96
11.25±6.53
0.020*
4.73±2.42
4.58±2.25
4.30±1.65
0.891
FSH
6.22±2.14
6.02±1.92
6.02±1.16
0.874
7.21±2.37
6.72±2.22
8.06±1.81
0.366
LH/FSH
1.50±0.83
2.04±1.28
1.80±0.77
0.011*
0.71±0.42
0.73±0.39
0.52±0.13
0.597
Estradiol
63.74±27.96
68.86±24.49
59.39±5.23
0.577
58.92±20.86
62.99±24.21
72.73±19.4
0.297
Testosterone
72.15±48.61
70.27±34.51
65.81±14.79
0.953
28.91±14.43
26.97±4.79
19.96±4.79
0.321
Insulin
13.83±8.30
13.98±5.16
8.86±1.19
0.537
10.71±4.34
11.01±4.27
9.95±3.11
0.862
HOMA-IR
3.12±2.14
3.02±1.31
1.94±0.32
0.583
2.27±0.98
2.32±0.95
2.14±0.74
0.930
Hirsutism
4.63±4.37
4.68±4.43
5.0±5.29
0.989
1.84±1.61
1.87±1.52
1.00±0.81
0.566
PG
90.30±18.23
86.74±12.51
89.0±14.0
0.552
85.86±8.42
85.22±8.34
87.25±4.85
0.854
PPG
117.49±35.36
110.28±21.74
117.66±17.78
0.514
109.50±9.36
111.34±13.14
120±21.60
0.118
ET(mm)
5.53±3.09
5.53±2.96
3.66±3.21
0.579
4.00±2.16
3.88±1.61
5.55±1.80
0.300
OV(right)
10.89±77.03
7.93±4.40
13.70±5.70
0.042*
4.53±3.75
5.50±4.52
5.69±5.09
0.362
OV(left)
9.40±4.99
8.59±3.95
10.75±9.35
0.591
4.47±4.43
3.95±2.87
3.53±2.40
0.719
AFC(right)
7.21±6.46
9.51±5.86
13.0±0.00
0.058
0.95±2.73
1.75±3.94
2.00±4.00
0.306
AFC(left)
7.03±6.53
9.40±6.08
13.0±0.00
0.054
0.94±2.76
1.46±3.39
1.00±2.00
0.613
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 9. Genotype – phenotype correlation of rs1799817of INSR gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
INSR
C/C
C/T
T/T
(rs1799817)
(n=19)
(n=76)
(n=74)
Age
26.05±3.86
27.04±4.47
27.02±4.33
BMI
21.31±4.26
25.35±4.66
Waist(cms)
78.63±12.96
Hip(cms)
CONTROLS (n=169)
p-Value
C/C
C/T
T/T
p-Value
(n=22)
(n=67)
(n=80)
0.652
27.04±2.90
27.68±4.46
27.51±3.94
0.813
25.13±4.56
0.677
23.23±3.52
24.13±3.30
24.48±3.27
0.289
82.45±13.38
82.08±16.68
0.599
59.41±11.25
63.77±14.51
65.42±13.67
0.192
92.89±14.97
95.90±15.73
95.28±19.42
0.796
82.81±13.40
87.19±15.24
87.53±12.79
0.355
Waist/hip ratio
0.85±0.05
0.86±0.04
0.86±0.04
0.402
0.71±0.05
0.72±0.07
0.74±0.07
0.215
LH
11.70±5.60
8.90±4.84
8.68±4.64
0.049*
4.97±1.99
4.96±2.41
4.36±2.37
0.247
FSH
6.11±1.90
6.35±2.21
6.02±1.99
0.618
7.05±2.12
7.31±2.24
6.96±2.46
0.663
LH/FSH
1.96±0.67
1.53±0.91
1.63±1.06
0.221
0.74±0.28
0.73±0.39
0.68±0.46
0.754
Estradiol
65.75±27.85
63.80±25.96
65.42±28.25
0.922
55.31±22.35
60.84±19.78
61.07±23.15
0.524
Testosterone
72.60±24.73
71.65±60.06
71.39±30.25
0.995
26.35±14.57
28.06±12.68
28.88±13.24
0.723
Insulin
11.36±4.51
14.05±7.67
14.11±8.31
0.350
10.14±3.53
10.98±4.15
10.76±4.60
0.728
HOMA-IR
2.59±1.10
3.20±2.13
3.07±1.99
0.495
2.24±0.92
2.29±0.94
2.28±1.01
0.966
Hirsutism
5.73±4.33
4.56±4.05
4.46±4.71
0.514
1.64±1.65
1.91±1.67
1.81±1.49
0.775
PG
93.63±24.46
90.64±15.09
87.36±16.81
0.275
88.77±10.01
84.67±8.96
85.80±7.04
0.132
PPG
127.78±60.05
114.56±21.74
114.46±32.54
0.252
110.23±10.50
110.16±12.19
110.22±9.71
0.999
ET(mm)
4.98±2.84
5.13±3.29
6.02±2.80
0.145
4.14±2.12
3.70±1.74
4.24±2.24
0.265
OV(right)
9.80±6.41
9.37±5.39
11.44±7.70
0.150
5.43±6.27
4.48±2.73
4.87±4.09
0.611
OV(left)
9.30±4.36
8.73±4.76
9.79±5.09
0.413
4.85±3.08
4.03±2.32
4.43±5.28
0.679
AFC(right)
7.76±6.27
7.18±6.04
8.42±6.73
0.497
1.82±3.61
0.85±2.56
1.26±3.34
0.419
AFC(left)
7.73±7.01
7.41±6.36
7.82±6.54
0.923
1.64±3.00
0.58±2.03
1.33±3.43
0.189
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 10. Genotype – phenotype correlation of rs1801278 of IRS1 gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
IRS1
G/G
G/A
A/A
(rs1801278)
(n=25)
(n=108)
(n=36)
Age
26.76±3.87
27.06±4.58
26.61±3.95
BMI
24.81±4.94
25.00±4.34
Waist(cms)
83.04±81.38
Hip(cms)
CONTROLS (n=169)
p-Value
G/G
G/A
A/A
p-Value
(n=50)
(n=94)
(n=25)
0.846
28.20±4.25
26.97±4.05
28.20±3.30
0.148
25.79±4.99
0.617
24.15±3.78
23.99±3.13
24.94±3.08
0.451
81.38±15.11
82.47±16.20
0.848
63.56±14.09
63.85±13.97
65.36±12.96
0.860
95.12±12.87
95.00±17.28
96.31±20.20
0.925
85.54±12.52
86.84±15.22
89.08±11.19
0.584
Waist/hip ratio
0.87±0.05
0.85±0.04
0.85±0.04
0.194
0.74±0.07
0.73±0.06
0.73±0.07
0.854
LH
9.64±5.91
8.75±4.59
9.86±5.08
0.426
4.68±2.25
4.51±2.23
5.32±2.93
0.314
FSH
5.63±1.77
6.36±2.21
6.00±1.81
0.233
7.06±2.33
7.16±2.24
6.97±2.74
0.925
LH/FSH
1.84±1.14
1.50±0.85
1.82±1.11
0.108
0.71±0.34
0.68±0.44
0.83±0.45
0.285
Estradiol
60.49±17.87
64.31±26.49
68.90±33.40
0.476
56.71±18.14
58.34±67.91
47.72±65.02
0.152
Testosterone
72.16±30.33
72.41±52.47
69.02±29.91
0.927
27.85±10.76
28.75±14.73
26.99±11.43
0.817
Insulin
14.15±12.78
13.27±4.94
15.04±9.74
0.475
11.18±4.56
10.23±3.92
11.96±4.82
0.141
HOMA-IR
3.27±2.90
2.96±1.41
3.27±2.61
0.622
2.41±1.07
2.15±0.86
2.51±1.07
0.149
Hirsutism
3.92±2.90
4.66±4.40
5.11±4.44
0.580
1.90±1.33
1.82±1.77
1.72±1.24
0.895
PG
95.16±26.62
88.94±16.12
87.43±9.95
0.186
86.86±9.14
85.42±8.04
84.72±7.59
0.492
PPG
120.56±55.77
116.86±29.97
110.26±15.18
0.439
111.17±12.38
109.87±10.69
109.48±7.52
0.741
ET(mm)
5.15±2.68
5.51±3.12
5.71±3.15
0.781
3.64±2.02
4.13±1.93
4.32±2.44
0.288
OV(right)
9.88±4.64
10.49±7.27
10.13±5.95
0.899
5.31±5.67
4.68±3.12
4.15±2.55
0.456
OV(left)
8.06±4.05
9.64±5.08
8.96±4.67
0.320
4.33±2.35
4.45±5.11
3.85±1.76
0.805
AFC(right)
8.48±6.21
7.51±6.41
8.14±6.47
0.739
1.06±3.03
0.90±2.65
2.40±4.35
0.094
AFC(left)
8.32±6.72
7.44±6.56
7.69±6.19
0.830
0.94±2.62
0.77±2.36
2.44±4.60
0.036*
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 11. Genotype – phenotype correlation of rs1805097 of IRS2 gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
IRS2
G/G
G/A
A/A
(rs1805097)
(n=83)
(n=71)
(n=15)
Age
27.06±3.91
26.56±4.33
27.86±6.34
BMI
24.54±4.46
25.24±4.43
Waist(cms)
79.86±14.82
Hip(cms)
CONTROLS (n=169)
p-Value
G/G
G/A
A/A
p-Value
(n=84)
(n=65)
(n=20)
0.529
27.36±3.82
27.40±4.17
28.55±4.46
0.480
28.01±4.89
0.024*
23.92±3.28
24.36±3.51
24.69±2.93
0.552
82.95±13.55
87.67±19.33
0.123
62.16±14.29
65.23±13.42
67.60±12.22
0.187
93.48±17.19
96.25±15.70
100.80±23.84
0.267
84.95±15.19
87.78±12.19
91.25±12.59
0.145
Waist/hip ratio
0.85±0.04
0.86±0.04
0.87±0.03
0.265
0.73±0.06
0.73±0.07
0.73±0.04
0.630
LH
9.21±4.98
8.73±4.92
10.45±4.38
0.455
4.67±2.31
4.71±2.40
4.64±2.50
0.993
FSH
6.34±2.07
5.91±2.07
6.53±2.14
0.346
7.01±2.34
7.08±2.29
7.62±2.43
0.574
LH/FSH
1.61±1.01
1.63±0.97
1.63±0.57
0.990
0.73±0.41
0.72±0.44
0.63±0.29
0.604
Estradiol
66.12±30.32
62.24±23.37
68.80±24.52
0.563
58.72±21.22
60.74±22.63
64.91±21.13
0.507
Testosterone
64.30±30.19
78.62±61.12
79.31±15.34
0.119
26.47±11.63
30.48±14.56
28.25±13.87
0.183
Insulin
14.51±9.81
13.27±4.87
12.11±4.40
0.418
10.81±4.08
10.92±4.69
10.10±3.79
0.754
HOMA-IR
3.27±2.55
2.91±1.18
2.80±1.22
0.447
2.32±0.97
2.27±0.98
2.17±0.89
0.838
Hirsutism
4.711±4.44
4.36±4.30
5.66±4.45
0.572
1.83±1.67
1.83±1.49
1.80±1.47
0.996
PG
89.59±16.96
88.41±14.34
94.60±27.77
0.447
86.33±9.07
84.51±7.07
87.25±8.54
0.285
PPG
115.31±32.25
113.84±14.34
130.06±68.29
0.213
111.02±12.22
108.89±8.84
111.0±10.29
0.464
ET(mm)
5.42±2.68
5.59±3.50
5.50±2.90
0.940
4.05±1.94
3.94±2.34
4.12±1.39
0.919
OV(right)
9.93±5.97
10.36±7.68
12.39±4.58
0.418
5.12±4.81
4.42±3.11
4.65±2.33
0.563
OV(left)
9.22±4.79
9.21±5.17
9.68±3.91
0.939
4.37±2.68
4.48±5.71
3.61±2.03
0.698
AFC(right)
7.08±6.37
8.61±6.31
7.80±6.59
0.338
0.95±2.47
1.46±3.76
1.15±3.13
0.611
AFC(left)
6.90±6.57
8.38±6.27
8.06±6.85
0.358
0.79±2.21
1.38±3.55
1.20±3.24
0.466
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 12. Genotype – phenotype correlation of rs1801282 of PPAR-G gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
PPAR-G
C/C
C/G
G/G
(rs1801282)
(n=4)
(n=36)
(n=129)
Age
22.5±1.50
26.77±4.07
27.11±4.39
BMI
26.47±3.84
24.23±4.13
Waist(cms)
84.75±10.99
Hip(cms)
CONTROLS (n=169)
p-Value
C/C
C/G
GG
p-Value
(n=3)
(n=41)
(n=125)
0.085
27.33±1.53
27.75±4.17
27.45±4.04
0.912
25.32±4.68
0.357*
23.89±1.83
24.37±3.61
24.12±3.27
0.904
79.53±11.25
82.42±15.79
0.545
67.33±4.93
63.95±13.69
63.92±14.02
0.915
97.50±10.41
90.58±12.33
96.54±18.45
0.183
93.00±7.00
85.36±14.36
87.10±13.87
0.582
Waist/hip ratio
0.86±0.02
0.87±0.04
0.85±0.04
0.009*
0.72±0.04
0.74±0.06
0.73±0.07
0.418
LH
9.82±6.05
10.94±5.91
8.58±4.46
0.036*
3.96±1.84
4.58±2.25
4.73±2.41
0.812
FSH
5.81±1.04
5.95±1.94
6.25±2.14
0.703
8.00±2.22
6.72±2.22
7.22±2.36
0.404
LH/FSH
1.62±0.73
2.04±1.26
1.50±0.83
0.011*
0.47±0.11
0.74±0.39
0.71±0.42
0.568
Estradiol
54.64±10.41
69.40±24.35
63.73±28.02
0.408
78.45±19.17
62.98±24.20
58.89±20.78
0.198
Testosterone
59.46±17.53
70.02±34.05
72.48±48.90
0.830
21.12±5.14
26.96±8.61
28.81±14.42
0.476
Insulin
9.82±2.14
14.15±5.19
13.79±8.35
0.566
10.36±3.67
11.01±4.27
10.69±4.33
0.909
HOMA-IR
2.07±0.37
3.04±1.29
3.12±2.15
0.580
2.23±0.88
2.32±0.95
2.27±0.97
0.960
Hirsutism
4.00±4.76
4.78±4.40
4.64±4.38
0.942
1.00±1.00
1.87±1.52
1.83±1.61
0.650
PG
86.75±12.28
86.55±12.38
90.46±18.32
0.458
86.00±6.67
85.22±8.34
85.88±8.39
0.889
PPG
113.25±16.99
110.55±21.48
117.61±35.59
0.517
110.0±10.00
111.34±13.14
109.83±10.00
0.741
ET(mm)
3.75±2.63
5.48±2.92
5.56±3.11
0.508
5.93±2.00
3.88±1.61
4.01±2.16
0.248
OV(right)
11.17±6.88
7.90±4.32
10.98±7.05
0.046*
3.25±1.69
5.50±4.52
4.59±3.81
0.362
OV(left)
9.49±8.04
8.49±3.94
9.46±5.01
0.571
3.22±2.84
3.95±2.87
4.47±4.42
0.695
AFC(right)
13.00±0.00
9.42±5.81
7.17±6.48
0.043*
0.00±0.00
1.76±3.94
1.01±2.79
0.328
AFC(left)
13.00±0.00
9.36±6.00
6.97±6.56
0.035*
0.00±0.00
1.46±3.39
0.96±2.76
0.522
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05, ** p <0.01
Table 13. Genotype – phenotype correlation of rs3856806 of PPAR-G gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
PPAR-G
C/C
C/T
T/T
(rs3856806)
(n=123)
(n=40)
(n=6)
Age
27.08±4.44
26.50±4.06
26.33±4.27
BMI
24.97±4.36
25.72±5.04
Waist(cms)
80.94±14.71
Hip(cms)
CONTROLS (n=169)
p-Value
C/C
C/T
TT
p-Value
(n=117)
(n=49)
(n=3)
0.717
27.68±4.17
26.86±3.59
32.0±2.00
0.073
24.81±5.74
0.655
23.92±3.28
24.67±3.16
26.51±6.85
0.194
84.40±15.43
83.67±13.71
0.424
63.06±14.04
66.16±12.62
64.66±23.71
0.418
94.32±16.61
97.92±19.51
97.67±16.25
0.493
85.71±14.14
89.46±12.79
85.0±21.28
0.277
Waist/hip ratio
0.86±0.04
0.86±0.04
0.85±0.04
0.654
0.73±0.07
0.74±0.06
0.75±0.11
0.785
LH
9.08±4.89
9.09±5.14
10.07±4.23
0.891
4.76±2.43
4.47±2.23
4.88±1.04
0.751
FSH
6.30±2.15
5.88±1.97
5.63±0.96
0.441
7.12±2.24
7.09±2.62
6.98±0.89
0.993
LH/FSH
1.59±0.96
1.67±0.98
1.83±0.75
0.778
0.73±0.45
0.67±0.31
0.69±0.11
0.760
Estradiol
64.14±27.46
66.62±25.59
64.25±32.22
0.881
60.01±21.55
59.69±21.78
77.71±28.89
0.373
Testosterone
68.73±30.41
82.02±75.64
62.35±36.13
0.241
28.13±14.08
28.02±10.47
35.18±17.98
0.654
Insulin
13.96±6.67
13.57±10.58
11.44±4.66
0.725
11.01±4.53
10.27±3.71
9.33±2.91
0.506
HOMA-IR
3.11±1.85
3.07±2.43
2.40±1.12
0.693
2.33±1.02
2.18±0.83
1.95±0.79
0.561
Hirsutism
4.65±4.42
5.15±4.36
1.33±1.36
0.137
1.80±1.53
1.93±1.72
1.00±0.00
0.581
PG
89.29±17.88
91.23±15.72
83.33±6.83
0.552
85.59±8.53
86.20±7.96
84.0±6.93
0.852
PPG
115.20±34.67
119.27±28.83
110.67±13.44
0.732
109.54±10.38
111.04±10.76
122.33±22.23
0.104
ET(mm)
5.49±3.15
5.52±2.96
5.50±1.52
0.999
4.09±1.99
3.90±2.15
2.70±2.34
0.459
OV(right)
10.69±7.37
9.36±3.97
9.28±4.58
0.509
5.07±4.39
4.04±2.57
6.00±5.43
0.272
OV(left)
9.25±4.89
9.14±4.63
10.18±6.48
0.888
4.08±2.50
4.94±6.46
3.59±3.09
0.449
AFC(right)
7.36±6.46
9.27±5.87
6.50±7.12
0.228
0.83±2.63
1.75±3.77
5.00±5.00
0.020*
AFC(left)
6.98±6.53
9.75±5.86
6.67±7.31
0.059
0.85±2.49
1.59±3.75
1.33±1.53
0.319
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 14. Genotype – phenotype correlation of rs7607759 of CAPN10 gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
CAPN10
A/A
A/G
G/G
(rs7607759)
(n=118)
(n=42)
(n=9)
Age
27.28.±4.57
26.38±3.76
24.67±2.69
BMI
25.24±4.50
25.18±4.94
Waist(cms)
82.24±14.12
Hip(cms)
CONTROLS (n=169)
p-Value
A/A
A/G
G/G
p-Value
(n=108)
(n=55)
(n=6)
0.140
27.85±4.07
27.14±3.96
25.00±3.09
0.170
23.68±3.56
0.616
24.25±3.11
23.84±3.67
26.05±3.55
0.286
81.64±17.03
77.89±14.29
0.697
64.75±13.76
61.96±14.29
68.83±7.03
0.326
95.85±16.49
94.69±19.84
90.89±16.08
0.688
88.07±13.87
84.16±14.27
87.67±7.76
0.235
Waist/hip ratio
0.86±0.04
0.86±0.04
0.86±0.05
0.838
0.73±0.06
0.73±0.07
0.78±0.05
0.170
LH
8.76±4.58
9.63±5.24
11.45±6.93
0.211
4.74±2.45
4.68±2.26
3.61±0.85
0.521
FSH
6.05±2.00
6.56±2.38
6.09±1.44
0.399
7.06±2.17
7.34±2.65
5.75±1.86
0.273
LH/FSH
1.61±0.94
1.60±0.99
1.88±1.13
0.695
0.72±0.43
0.69±0.39
0.68±0.24
0.922
Estradiol
64.28±27.12
63.77±28.47
75.02±18.00
0.502
61.26±21.94
58.51±21.49
57.54±22.42
0.715
Testosterone
72.12±52.48
68.06±20.11
82.26±31.48
0.686
27.52±11.21
29.64±16.65
27.95±9.81
0.625
Insulin
13.28±4.86
15.28±12.88
13.23±5.52
0.347
11.05±4.50
10.36±3.94
9.35±2.96
0.451
HOMA-IR
2.92±1.27
3.55±3.29
2.95±1.25
0.209
2.35±1.01
2.18±0.91
1.94±0.63
0.401
Hirsutism
4.63±4.48
4.07±3.62
7.56±5.38
0.095
2.06±1.61
1.36±1.39
2.00±2.00
0.028*
PG
88.52±17.09
92.21±18.21
90.44±11.38
0.483
86.20±8.71
86.20±8.71
85.0±7.81
0.613
PPG
113.59±32.72
123.24±35.25
113.88±13.63
0.258
110.51±10.79
110.51±10.79
109.74±10.88
0.869
ET(mm)
5.32±2.77
5.89±3.79
6.06±2.81
0.501
4.02±2.08
4.02±2.08
4.03±2.01
0.976
OV(right)
10.32±7.14
9.95±4.96
12.25±7.04
0.645
4.94±3.93
4.94±3.93
4.54±4.15
0.818
OV(left)
9.28±4.99
8.95±4.37
10.35±5.71
0.732
4.40±4.52
4.40±4.52
4.11±2.88
0.831
AFC(right)
7.74±6.33
7.76±6.55
8.56±6.73
0.934
1.38±3.27
1.38±3.27
0.89±2.86
0.409
AFC(left)
7.19±6.32
8.57±6.82
8.89±6.97
0.417
1.27±3.04
1.27±3.04
0.80±2.78
0.411
LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment of insulin resistance, PG- pre-prandial glucose, PPGpost-prandial glucose, ET- endometrial thickness, OV- ovarian volume, AFC- antral follicular count, * p <0.05.
Table 15. Genotype – phenotype correlation of rs2975766 of CAPN10 gene in PCOS and control subjects
PARAMETERS
CASES-PCOS (n=169)
CAPN10
G/G
G/A
A/A
(rs2975766)
(n=148)
(n=19)
(n=2)
Age
27.01±4.37
26.53±4.15
24.00±2.83
BMI
25.34±4.59
23.84±4.17
Waist(cms)
82.39±14.81
Hip(cms)
CONTROLS (n=169)
p-Value
G/G
G/A
p-Value
(n=157)
(n=11)
0.570
27.50±4.02
27.91±4.50
0.885
22.96±6.29
0.320
24.04±3.21
26.14±4.55
0.119
78.47±15.21
74.50±14.85
0.436
63.92±13.75
66.91±13.92
0.246
95.65±17.36
93.56±17.21
85.0±18.38
0.622
87.06±13.97
84.54±12.49
0.339
Waist/hip ratio
0.86±0.04
0.84±0.04
0.87±0.02
0.050
0.73±0.07
0.78±0.06
0.011*
LH
8.71±4.46
11.96±7.04
12.74±4.58
0.013*
4.58±2.24
6.15±3.37
0.084
FSH
6.24±2.02
5.75±2.59
5.99±1.68
0.630
7.13±2.34
6.97±2.22
0.522
LH/FSH
1.52±0.86
2.34±1.39
2.10±0.17
0.002*
0.69±0.38
0.96±0.66
0.114
Estradiol
64.13±27.05
69.84±28.04
60.65±24.53
0.674
60.05±21.84
65.18±19.71
0.359
Testosterone
69.66±46.94
85.68±33.06
85.03±9.23
0.325
28.12±13.14
30.52±14.05
0.667
Insulin
13.99±8.02
11.68±4.44
18.05±1.20
0.346
10.69±4.29
11.23±3.94
0.267
HOMA-IR
3.15±2.07
2.42±0.89
4.09±0.38
0.242
2.26±0.96
2.36±0.94
0.236
Hirsutism
4.53±4.34
5.78±4.76
3.00±0.00
0.432
1.86±1.59
1.18±1.16
0.149
PG
90.11±17.98
84.95±8.31
91.50±2.12
0.462
85.74±8.21
85.27±10.29
0.863
PPG
116.74±34.72
110.37±12.28
115.00±7.07
0.729
109.76±10.18
116.36±17.20
0.147
ET(mm)
5.44±3.17
6.08±2.14
4.80±0.28
0.654
4.04±2.08
3.74±1.55
0.96
OV(right)
10.30±6.66
10.84±6.77
7.48±6.81
0.789
4.71±3.63
6.24±7.52
0.373
OV(left)
9.08±4.74
10.69±5.88
8.33±2.85
0.386
4.28±4.08
5.10±4.03
0.715
AFC(right)
7.60±6.37
8.42±6.26
15.50±3.53
0.198
1.17±3.16
1.27±2.19
0.926
AFC(left)
7.42±6.48
8.74±6.65
12.50±0.71
0.400
1.05±2.94
1.36±2.50
0.884
The A/A (A/A=1) genotype were not included in the control group because only one subject among controls had this
genotype. LH-luteinizing hormone, FSH- follicle stimulating hormone, HOMA-IR-homeostatic model assessment
of insulin resistance, PG- pre-prandial glucose, PPG-post-prandial glucose, ET- endometrial thickness, OV- ovarian
volume, AFC- antral follicular count, * p <0.05.
Table 16. Results from multifactor dimensionality reduction analysis
No. of loci
Gene Variants
CVC
TA
Odds Ratio
(95%CI)
1
rs3856806
9/10
0.514
1.16(0.25-5.39)
0.845
2
rs1801282
10/10
0.526
1.24(0.31-4.83)
0.755
CVC- cross validation consistency, TA- testing accuracy, CI- confidence interval.
p- value
Figure Title:
Figure 1 The distribution of high risk and low risk genotypes of PPAR-G gene between rs1801282 and rs3856806
SNPs in PCOS and control women by MDR analysis.
Figure Legend:
Figure 1 The best two ways SNP-SNP interaction. The high risk genotype combinations are in dark grey and lowrisk genotype combinations in light grey. Higher the intensity of grey shades higher the risk. Each box represent cell.
And the bars inside the cell represent the number of subjects. The left bar denote controls and the right bar represent
cases.