http://orcid.org/0000-0003-0444-2167
Figures
Abstract
Background
Polycystic ovary syndrome (PCOS), the commonest endocrine disorder affecting young women, appears to be a multigenic trait with contributing genes being unclear. Hence, analysis of polymorphisms in multiple candidate genes is required. Currently available genotyping methods are expensive, time-consuming with limited analytical sensitivity.
Aim
(i) Develop and validate high resolution melting (HRM) assay and allele-specific real-time quantitative PCR (AS-qPCR) for genotyping selected SNPs associated with PCOS.
(ii) Identify selected SNPs and their association with a Sri Lankan cohort of well-characterized PCOS.
Methods
DNA was extracted from women with well-characterized PCOS from adolescence (n = 55) and ethnically matched controls (n = 110). FTO (Fat mass and obesity associated gene; rs9939609), FSHB (Follicle stimulating hormone beta subunit; rs6169), FSHR (Follicle stimulating hormone receptor; rs6165/rs6166), and INSR (Insulin receptor; rs1799817) genes were genotyped using HRM assay. GnRH1 (Gonadotropin releasing hormone; rs6185), LHB (Luteinizing hormone beta subunit; rs1800447/rs34349826) and LHCGR (Luteinizing hormone/choriogonadotropin receptor; rs2293275) genes were genotyped using AS-qPCR method. Genotyping results were validated using Sanger sequencing.
Results
A significant association was observed within FTO gene polymorphism (rs9939609) and PCOS. Genotype frequency of FTO gene (rs9939609)—cases versus controls were TT-36.4% vs.65.4% (p<0.05), AT-23.6% vs.20.9%, AA-40% vs.13.6% (p<0.05). Genotype frequencies of the SNPs GnRH1 (rs6185), FSHB (rs6169), FSHR (rs6165 & rs6166), LHB (rs1800447 & rs34349826), LHCGR (rs2293275) and INSR (rs1799817) were not significantly different between cases and controls (p>0.05). Only the mutant alleles were observed for LHB rs1800447 and rs34349826 SNPs in both groups. The HRM and AS-qPCR assay results had 100% concordance with sequencing results.
Conclusions
FTO gene rs9939609 polymorphism is significantly more prevalent among Sri Lankan PCOS subjects while the other selected SNPs of HPG axis genes and INSR gene showed no association. HRM and AS-qPCR assays provide a reliable, fast and user-friendly genotyping method facilitating wider implication in clinical practice.
Citation: Branavan U, Muneeswaran K, Wijesundera S, Jayakody S, Chandrasekharan V, Wijeyaratne C (2018) Identification of selected genetic polymorphisms in polycystic ovary syndrome in Sri Lankan women using low cost genotyping techniques. PLoS ONE 13(12): e0209830. https://doi.org/10.1371/journal.pone.0209830
Editor: Alok Deoraj, Florida International University, UNITED STATES
Received: August 10, 2018; Accepted: December 12, 2018; Published: December 31, 2018
Copyright: © 2018 Branavan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was supported by grant no - 15-149, funder - National Research Council, Sri Lanka, http://www.nrc.gov.lk/, to Prof. CN Wijeyaratne. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder affecting women of reproductive age with a prevalence varying between 5–13%. PCOS typically presents during adolescence with a wide spectrum of phenotypes that are characterized by any combination of anovulation (amenorrhoea, irregular cycles), androgen excess (hirsutism, acne, alopecia) and polycystic ovaries on ultrasound [1].
Based on current research there is strong evidence that genetic factors play an important role in the etiology of PCOS. Despite several studies dissecting the variants of genes from multiple biological pathways, the impact of the mode of inheritance of PCOS on its pathophysiology remains unclear [2]. PCOS appears to be a multigenic trait, although the contributing genes remain undefined [3,4,5,6]. Candidate genes of steroid hormone biosynthesis and metabolism, gonadotropin and gonadal hormones action, obesity and energy regulation, insulin secretion and action have been studied and implicated in the pathogenesis of PCOS. Some recent studies have identified varying SNP sites to be associated with PCOS in differing populations. Tian et al. reported an association of FSHB gene variant (rs11031010) with PCOS in Han Chinese women [7]. Wang et al. proposed RAD54B gene (involved in homologous recombination and repair of DNA) may contribute to the hyperandrogenism of PCOS in the Han Chinese population [8]. Moreover, Cui et al. showed a direct correlation between the genotypes and PCOS phenotype and concluded that every feature of PCOS has a specific genetic association linked to the aetiological pathway [9]. Additionally, the recent Genome-wide association study (GWAS) by Chen et al. showed 3 SNP sites on THADA, DENND1A and TOX3 to be associated with PCOS [10]. Brower et al. evaluated whether the variants associated with PCOS in Han Chinese are also associated with PCOS in white Europeans and concluded that DENND1A, THADA, FSHR, INSR and YAP1 loci are likely to play important roles in the etiology of PCOS across populations [11]. Nevertheless, Shim et al. carried out a pathway-based analysis of a GWAS dataset to elucidate which biological pathways were associated with genes in PCOS. The conclusion was that oocyte meiosis is the top-ranking biological pathway associated with PCOS [12].
It is noteworthy that the underlying pathway of the pathophysiology in each of the phenotypes of PCOS may differ, based on the genetic and environmental contributions. There may be a number of interlinking factors that affects the expression of PCOS. A single cause for PCOS is very unlikely [13]. Hence, multiple candidate genes associated with PCOS should be screened to determine its exact genetic basis.
Genotyping is now an important diagnostic means for elucidating most diseases. Among the advanced techniques available to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease screening. Their applicability mainly depends on three factors: cost, time and accuracy.
The aims of the study were to (i) develop and validate high resolution melting (HRM) assay and allele-specific real-time quantitative PCR for genotyping selected SNPs associated with PCOS and (ii) identify selected SNPs and their association with PCOS in Sri Lankan women.
Materials and methods
Recruitment of subjects
This study was approved by the Ethics Review Committee, Faculty of Medicine, University of Colombo, Sri Lanka. Written informed consent was obtained from all participants. Consecutive well characterized cases were recruited from the Endocrine Clinic of the University Obstetrics and Gynaecology Department, Colombo. Diagnosis of PCOS was based on the Rotterdam criteria [14], with diagnostic certification made by a single clinical lead. The sample size required for consecutive subjects with well characterized PCOS manifesting from adolescence was 55; and by selecting double this number of controls, the sample size of control subjects was 110 (Schlesselman case control study formula was used for sample size calculation [15]).
Inclusion criteria.
Inclusion criteria were women whose symptoms manifested from adolescent years (11–19 years WHO), with all 3 diagnostic criteria present from 16–19 years of age [16]. The lower limit of age selection was based on the mean age of menarche in Sri Lanka being 13 years and leaving an allowance of two additional years for regularization of menstruation [17].
Anovular PCOS or amenorrhoea/ oligomenorrhea: Anovular cycles are defined when the cycle length is more than 35 days, and the lack of demonstrable ovulation by mid cycle and luteal phase ultrasound scans, and mid-luteal serum progesterone [16]. Amenorrhoea—absence of menstrual periods for six months or more in a woman who has previously been menstruating. Oligomenorrhea—menstrual periods occurring at intervals of greater than 35 days, with only four to nine periods in a year.
Polycystic ovaries on ultrasound: defined by transvaginal or transabdominal ultrasound scan of ovaries, performed within the first 5 days from the onset of menstruation, and finding 24 or more follicles, measuring between 2 and 9 mm and/or an ovarian volume >10 cm3 [14].
Hyperandrogenism: Clinical evidence of hirsutism by modified Ferriman-Gallwey score (mFG) ≥8, serum testosterone (T) > 3.5nmol/L and/or free androgen index (FAI) >5 [16].
Exclusion criteria.
Exclusion criteria included inherited disorders of IR such as Rabson–Mendenhall syndrome, Cushing syndrome, hyperprolactinaemia, untreated primary hypothyroidism, congenital adrenal hyperplasia or an androgen secreting ovarian/adrenal tumor; those taking corticosteroid, antiepileptic or antipsychotic drugs, history of hormonal contraception within the previous 6 months, pregnancy and the first postpartum year.
Control sample: Concurrently asymptomatic, normo-androgenic, normal cycling since adolescence, non-medicated, consenting women of reproductive age in whom PCOS was objectively excluded by clinical, biochemical and ultrasound assessment, were recruited as controls. The control subjects were recruited from a single work setting where health promotion programs were conducted from 2012 (3 years before the study). Working women of similar ethnic and social background as the affected subjects were invited to participate in the study.
Clinical evaluation
Clinical evaluation was by a pre-tested questionnaire-based interview regarding: socio demographic factors, detailed menstrual and obstetric histories, infertility if relevant, the onset and degree of clinical symptoms of PCOS, drug history, family history of diabetes and other cardiovascular risk factors. Detailed physical examination included measurement of standing height to the closest centimeter [18] and weight in kilograms to calculate the BMI, waist and hip circumference and waist-to-hip ratio (WHR), resting blood pressure [19], hirsutism (FG score), frontal balding, distribution of acne and acanthosis nigricans [20].
Evaluation of the modified FG score was done by a single medically qualified clinical lead of the Department of Obstetrics and Gynecology, Faculty of Medicine, University of Colombo.
Ultrasound examinations were performed by a single trained medically qualified ultra sonographer under the supervision of the Radiology lead of the De Soysa Hospital for Women, Colombo.
Biochemical and endocrine evaluation
Two milliliters venous blood was collected into plain sterile tubes from each subject and serum was extracted. Serum kisspeptin and testosterone levels were measured with ELISA kits (Phoenix Pharmaceuticals Inc., Belmond, CA and Teco Diagnostics, USA respectively) as per manufacture recommendation. Serum kisspeptin and testosterone concentrations were determined using the standard curve of known concentrations. Fasting blood glucose was measured in all subjects. Routine laboratory tests performed to diagnose/monitor PCOS (follicular phase FSH, LH, thyroid stimulating hormone, and fasting blood glucose/75 g oral glucose tolerance test) which were carried out at the quality controlled laboratory of the National Hospital Colombo were recorded for PCOS subjects.
DNA extraction
DNA was extracted from blood samples (2ml) using genomic DNA extraction kit (Promega, USA), following the manufacturer’s protocol. The DNA samples were subsequently stored at −20°C.
Selection for study of candidate genes and SNPs of PCOS
The selection of SNPs for this study was based on screening OMIM, SNPedia and ClinVar and the highly susceptible SNPs for PCOS reported from Asia were selected for the study.
In view of deregulation of the hypothalamic pituitary gonadal (HPG) axis being linked to the pathophysiology of PCOS [21], we explored the potential for studying selected polymorphisms of genes associated with the HPG axis (Fig 1). The following HPG genes and their respective SNPs were selected: GnRH1 (rs6185), FSHB (rs6169), FSHR (rs616/rs6166), LHB (rs1800447/rs34349826) and LHCGR (rs2293275). Insulin resistance playing a central role in its pathophysiology with obesity and type 2 diabetes mellitus being common problems of PCOS [22], we also selected polymorphisms in the obesity associated gene (FTO—rs9939609) and Insulin receptor gene (INSR—rs1799817) that are reported to be more prevalent in Asians [23–25].
Primer designing for high resolution melt (HRM) assay and allele-specific real-time quantitative PCR (AS-qPCR)
Primers were designed using a similar methodology used by the SNPgen online tool (http://snp.biotech.edu.lk/) [26] and custom synthesized by Macrogen Inc, Korea. To increase the specificity and feasibility of an efficient allelic discrimination, the following technique was used in primer designing: designing of primers for HRM and allele specific real time quantitative PCR (AS-qPCR) were based on a modified version of CADMA (competitive amplification of differentially melting amplicons) [27]. This method uses 3 primers–two allele specific primers and one common primer. Of the 2 allele specific primers, one primer is designed to amplify only the mutated allele (mutant primer) and the other primer amplifies only the wild-type allele (wild type primer). The common primer was designed to amplify both wild type and mutant alleles (Table 1). Furthermore, secondary mismatches were introduced in the allele specific primer sequences to increase the melting temperature (temperature increasing mutations—G or C mismatches) of one amplicon (either mutant or wild type amplicon) and temperature decreasing mutations (A or T mismatches) were introduced to decrease the melting temperature of the corresponding amplicon. Furthermore, to increase the specificity, a single mismatch was introduced at the second base from the 3´end of each allele specific primer.
Since the HRM analysis works well for a melting temperature of around 60°C, SNPs with primers of melting temperature between 58–60°C were analyzed using the HRM method and primers with high melting temperature (above 60°C) analyzed using AS-qPCR. For the HRM analysis, all 3 primers (2 allele specific and 1 common primer) were added into one tube, whereas for allele specific PCR, each allele specific primer was added separately along with the common primer.
PCR conditions
Real-time quantitative PCR was performed using a thermocycler (BioRad CFX 96). The final reaction mix for each assay consisted of 2 μl template DNA (10 ng/μl), 2 μl Solis BioDyne master mix (5x HOT FIREPol EvaGreen HRM Mix), 0.5 μl wild-type primer (10 μM), 0.5 μl mutation primer (10 μM), 0.5 μl common primer (10 μM), and finally adding 4.5 μl ddH2O to a final volume of 10 μl.
PCR cycling conditions and HRM conditions were: Initial denaturation 95°C for 15 minutes, followed by 40 cycles of denaturation (95°C for 15s,) annealing (60°C for 20s; annealing temperature varies with each SNP) and extension (72°C for 20s). HRM was performed from 50°C to 95°C with a temperature increase of 0.2°C/s with 50 acquisitions/°C.
Each sample was run in duplicate and each genotyping was carried out with a set of sequentially confirmed control samples of known genotypes (wild type, heterozygous and homozygous alleles).
Data was analyzed using Precision Melt Analysis software version 1.3. HRM assay melt curve results were normalized to identify the genotype (Fig 2). For allele-specific real time PCR, the amplification plot and respective Cq values were used for genotype identification.
The primer sequences and annealing temperature for each SNP is listed in Table 1.
Sanger sequencing
Samples were randomly selected and the sequencing PCR was carried out on genomic DNA flanking the polymorphic site by using the sequence primer and the common primer from HRM or AS-qPCR and the amplified products were custom sequenced (Macrogen Inc, Korea) to validate the HRM and allele specific real time PCR methods. Primers used for sequencing are listed in Table 1.
Tetra ARMS and Taq man assay for FTO genotyping
Additionally, tetra ARMS PCR and TaqMan assay (predesigned SNP genotyping assay, Thermo Fisher Scientific) were carried out for FTO (rs9939609) genotyping to confirm the HRM assay results. Tetra ARMS PCR primer details and PCR conditions are given in the supplementary material (S1 Table).
Statistical analysis
Normal distribution of all clinical parameters was analyzed using the Kolmogorov-Smirnov test. Comparisons of normal distribution continuous variables between groups were analyzed using independent student’s t test. General clinical characteristics of cases and controls were expressed as mean ± standard deviation. The relative association between patients and controls for genotype and allele frequencies was assessed by Pearson’s χ2 test. The corresponding odds ratios (ORs) and confidence intervals (95% CIs) were calculated using SPSS version v.18.0 SPSS, Inc., Chicago, IL. The level of significance was set at 5%.
Results
Demographic, clinical and hormonal characteristics of women with PCOS and controls are summarized in Table 2. Cases and controls showed no significant differences in relation to demographic characteristics, indicating the comparability of the two groups. The exception was the age difference between the two groups, which was close to being statistically significant (p = 0.06). This may have been due to chance, however it is unlikely to have affected the risk, as age is usually not associated as a confounder with genes tested. Women with PCOS had significantly higher BMI and mFG score. Serum kisspeptin and testosterone concentrations were significantly higher in women with PCOS (kisspeptin– 4.873 nmol/L; testosterone—4.713 nmol/L) than controls (kisspeptin– 4.127 nmol/L; testosterone—3.415 nmol/L p<0.05).
Validation of HRM assay and allele-specific real-time quantitative PCR
The FTO, FSHR, FSHB and INSR genes were genotyped using HRM method (Fig 2) and GnRH1, LHB and LHCGR genes were genotyped using AS-qPCR method. Samples were randomly selected and validated by Sanger sequencing.
Fig 3 illustrates the amplification plot of SNP–rs2293275 (INSR). Cq values <30 indicates positive amplification of the target. If the Cq value of one primer set is > 30, it indicates the absence of the respective allele in the sample; thereby homozygous towards the other allele (S2 Fig). When both primer sets show Cq value < 30, the sample is heterozygous. The allele specificity of Cq values <30 was decided based on several trials with different human DNA samples for all SNPs and validated by Sanger sequencing.
Comparing the results of the two genotyping methods (HRM and AS-qPCR), there was 100% accordance between the results of these two assays and the sequencing results. In samples evaluation all duplicates had similar results, indicating consistency and reproducibility of the assay.
Genotype frequencies
Genotype distribution of all the studied polymorphisms of 55 women with PCOS and 110 ethnically matched controls are shown in Table 3.
The genotype frequencies of the SNPs GnRH1 (rs6185), FSHB (rs6169), FSHR (rs6165 & rs6166), LHB (rs1800447 & rs34349826), LHCGR (rs2293275) and INSR (rs1799817) were not significantly different between cases and controls (p>0.05). However, in the FTO gene rs9939609 polymorphism, a higher frequency of A allele (mutant allele) was observed in the PCOS group, while the frequency of T allele (normal allele) was significantly higher in the control group (Cases—AA = 40%, AT = 23.6%, TT = 36.4%; Controls—AA = 13.6%, AT = 20.9%, TT = 65.4%; p<0.05). AA genotype was positively associated with PCOS in our sample (OR = 5.28; 95% CI = 2.320–12.016; p<0.05). A significant correlation was also found between FTO gene and BMI (chi square value = 17.05, p<0.05).
Meanwhile, GnRH1 (rs6185) homozygous mutant CC genotype was not detected among cases or controls. In terms of FSHR (rs6165 and rs6166) and LHCGR (rs2293275) polymorphisms, heterozygous genotype (GA) was more frequently observed in the population studied. Although no statistically significant difference was observed in genotype distribution between cases and controls in rs2293275 (LHCGR) polymorphism (p>0.05), mutant homozygotes (GG) were observed at a higher frequency than the normal wild type (AA) genotypes in both groups. Moreover, in rs1799817 (INSR) polymorphism, TT and CT genotype frequencies were found to be considerably less in cases and controls than those of their wild type genotype (CC). Furthermore, only homozygous mutant genotypes, CC and GG were present in both cases and controls in LHB gene polymorphism rs1800447 and rs34349826 respectively (Table 3). The latter two polymorphisms are in linkage disequilibrium (rs1800447/rs34349826), while the wild type and heterozygous genotypes were not detected.
Discussion
There is a growing demand for rapid and reliable genotyping in chronic conditions of unclear aetiology, such as PCOS, and in particular among differing ethnic groups. In recent years, conventional polymerase chain reaction (PCR) techniques have been replaced by quantitative real-time PCR (qPCR). The benefits of qPCR in relation to conventional PCR include speed, reproducibility and quantitative ability. Additional operational advantages of qPCR include greater sensitivity and reproducibility, with the potential to replace conventional PCR in routine diagnostic practice. It is noteworthy that HRM and AS-qPCR have been used by some lead centers for genotyping [27–30]. To the best of our knowledge this is the first report on SNPs of selected genes associated with PCOS among Sri Lankan women and also the first study to use HRM and AS-qPCR for genotyping.
We acknowledge that cases and control were not strictly age matched, where the mean age of controls is higher than that of PCOS subjects (p = 0.06). Our explanation for this marginally older cohort of controls is that we sought volunteers from a large work setting; when we had to ensure they were truly normal with regular cycles over a period of time, had achieved fertility and clearly did not express even the milder phenotypes of PCOS. We propose that this age difference is unlikely to impact on their genetic makeup and thereby the findings of this study.
The HRM method uses mutation introduced primers that produce amplicons which differ in nucleotide content based on the genotype. The nucleotide content is reflected in the melting properties of the amplicon, detectable through HRM analysis. Furthermore, a mismatch introduced in the two allele specific primers at the second base from the 3´terminus, increases the specificity of PCR amplification and weakens non-specific amplifications and thereby limits false amplification. In addition, by increasing the melting temperature of one allele specific primer and decreasing the melting temperature of another allele specific primer, the HRM method allows sufficient difference in the melt curve to distinguish between homozygous and heterozygous alleles.
Recent genome-wide association studies reported that FTO gene variants are associated with PCOS, mostly in Asians [31]. We found the FTO gene rs9939609 polymorphism to be associated with PCOS in Sri Lankan women (OR = 3.316; 95% CI = 1.687–6.515; p<0.05). Furthermore, we repeated the FTO genotyping using Tetra ARMS PCR and TaqMan assay to confirm our findings, which mirrored the results by HRM method (S1 Fig). Table 4 compares results of this study with other reports. Barber et al. [32], Yan et al. [33], Sokkary et al. [34] and Farhan et al. [35] reported an association between the FTO rs9939609 variant and PCOS, which mirror our findings. Meanwhile, Tan et al. [36], Wehr et al. [37] and Ramos et al. [38] showed no association between SNPs in FTO and the PCOS phenotype.
In the current study, GnRH1 (rs6185) gene polymorphism had an approximately equal distribution of wild type (GG) and heterozygous (GC) alleles between PCOS and controls subjects; while the homozygous mutant allele (CC) was not present in cases and controls. Meanwhile, FSHB gene polymorphism (rs6169) showed a similar distribution of wild type (CC) and heterozygous (CT) alleles in PCOS and controls subjects. Nonetheless, the frequency of FSHB gene homozygous mutant (TT) allele was 37.3% among the control subjects compared to 29.1% in patients. We also found no significant association between GnRH1 and FSHB gene polymorphisms with PCOS (Table 3) (p>0.05). The few studies reported (Table 4) to identify any association of rs6185 (GnRH1) and rs6169 (FSHB) polymorphisms with PCOS, also failed to find any association [39, 40]. Therefore, we conclude that GnRH1 (rs6185) and FSHB (rs6169) gene mutations are uncommon in Sri Lankan subjects with well characterized PCOS manifesting from adolescence.
We also observed that the polymorphisms of FSHR rs6165 and rs6166 had no significant association with PCOS. A few researchers [41, 42] have reported an association between FSHR gene polymorphisms (rs6165 and rs6166) and PCOS, while the majority failed to find any association [43–47] (Table 4). Meanwhile, Valkenburg et al. [39] concluded that FSHR gene variants were strongly associated with the severity of PCOS and its clinical features, but not with the disease risk.
Studies have identified two common mutations in the LHB gene associated with PCOS; one in codon 8 (rs1800447) and other in codon 15 (rs34349826) and that these two polymorphisms (rs1800447 & rs34349826) exist in complete linkage disequilibrium [48]. Meanwhile, some have shown this LHB gene variant to represent a universal polymorphism [48, 49]. However, the LHB gene variant has been reported to be so far less common in Asians [49, 50]. Conversely, Batista et al. [51] showed that LHB gene polymorphisms (rs1800447/rs34349826) were associated with elevated testosterone levels in women with PCOS. In our analysis, LHB gene polymorphisms (rs1800447 and rs34349826) were detected only as homozygous mutant genotypes (CC and GG respectively) in both PCOS and control subjects. It is noteworthy that in the rs1800447 polymorphism, the point mutation in codon 8 causes amino acid replacement from Trp to Arg; while in rs34349826 polymorphism the point mutation in codon 15 causes amino acid replacement from Ile to Thr [48]. The resulting alterations of the amino acids, Trp (hydrophobic) to Arg (hydrophilic) and Ile (hydrophobic) to Thr (hydrophilic) may themselves introduce significant conformational changes to the LH protein. Since our study population have only mutated genotypes of LHB gene polymorphisms (rs1800447 and rs34349826), it can be argued that these variant LH proteins have little or no impact on the phenotype of PCOS in our study population. This study also suggests that the LHCGR polymorphism (rs2293275) is most unlikely to be associated with the pathogenesis of PCOS. Conversely, the majority of studies [52–54] indicate that rs2293275 polymorphism in exon 10 of the LHCGR gene variant is strongly linked with PCOS (Table 4), while there are only a few reports that rs2293275 polymorphism is not associated with PCOS [39].
We did not find significant differences in the genotype distribution between cases and controls of rs1799817 polymorphism of the INSR gene (p>0.05). This is consistent with findings of Lee et al. [55], where no association was found between rs1799817 polymorphism and PCOS susceptibility in a Korean population [55]. On the other hand, an Indian study showed polymorphism of INSR gene as a susceptibility factor in patients with PCOS, especially in non-obese PCOS patients [24]. Yet, another Indian study reported the INSR gene SNP (rs1799817) was associated with increased insulin resistance in Indian women with PCOS [25]. However, we did not find any relationship between clinical and biochemical characteristics and the INSR gene polymorphism (rs1799817) among women with PCOS (p>0.05). Such discrepancies in results may be due to variations in study design, sampling technique and sample size, along with demographic and genetic differences among study populations.
Most importantly, we have found that serum kisspeptin levels were significantly higher in adult subjects with PCOS having the well characterized phenotype from adolescence than ethnically matched controls (Table 2). In addition, our study of the Kiss 1 and the GPR54 receptor genes revealed 2 and 5 SNPs respectively (manuscript in preparation), although these SNPs were not associated with PCOS.
Therefore, in summary, it can be assumed that selected SNPs of HPG axis genes are not likely to be associated with PCOS in Sri Lankan women. These findings, no doubt require validation by further large scaled studies.
Conclusions
This is the first study of Sri Lankan women with well characterized PCOS carried out to explore any association with multiple SNPs of different pathophysiological pathways implicated in the aetiology of PCOS. The rs9939609 variant of FTO gene is significantly associated with PCOS among Sri Lankan women, reflecting its effect on central adiposity. Meanwhile, the gene polymorphisms representing the HPG axis (GnRH1, FSHB, FSHR, LHB and LHCGR) and INSR gene polymorphism do not show any significant association with PCOS. Interestingly, only homozygous mutant genotypes were present in LHB gene polymorphism (rs1800447 and rs34349826) in both PCOS and control subjects.
Moreover, HRM and allele specific real time PCR are simple, fast, cost-effective and efficient genotyping techniques, feasible in many diagnostic units, as real time PCR instruments are standard equipment in most molecular diagnostic laboratories. The limitation of these two assays is that initially samples need to be validated by sequencing method to confirm the results. However, by applying samples of known genotypes as a reference, the two assays can be used for reliable genotyping of samples of unknown status.
Supporting information
S1 File. Genotype results of the study population.
Genotype data of the cases and controls.
https://doi.org/10.1371/journal.pone.0209830.s001
(XLSX)
S2 File. Demographic and clinical characteristic of study population.
Demographic, clinical and hormonal data of the cases and controls.
https://doi.org/10.1371/journal.pone.0209830.s002
(XLSX)
S1 Fig. Genotyping of FTO (rs9939609) polymorphism by TaqMan assay and Tetra ARMS-PCR.
Allelic discrimination plot of FTO (rs9939609) polymorphism by TaqMan assay and detection of rs9939609 gene polymorphism by Tetra ARMS-PCR (PCR products on 2% agarose gel).
https://doi.org/10.1371/journal.pone.0209830.s003
(DOCX)
S2 Fig. Genotyping of GnRH1 and LHB polymorphism by AS-qPCR.
Amplification plot of GnRH1 and LHB genes single nucleotide polymorphisms.
https://doi.org/10.1371/journal.pone.0209830.s004
(DOCX)
S1 Table. Primer details and PCR conditions for Tetra ARMS PCR of FTO rs9939609 polymorphism.
https://doi.org/10.1371/journal.pone.0209830.s005
(DOCX)
Acknowledgments
We thank all the staff of the Professorial Obstetrics and Gynaecology Department and Professorial Unit of De Soysa Hospital for Women, Colombo for their ready assistance and support with ultrasound scanning, all staff of the Reproductive Biology and Endocrinology Laboratory, at the Department of Obstetrics and Gynecology, Faculty of Medicine and the patients and their families for their wonderful cooperation. We also thank the staff of Bank of Ceylon, Head office, Colombo for their voluntary participation for the study. We specially thank Dr. Anoma Senanayake for her excellent support during the ultrasound examinations. We also thank Dr. Sumudu Jayasinghe and Dr. Dakshila Galappathi for their excellent support in coordinating the patient evaluation and sample collections.
References
- 1.
Zawadski JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. Polycystic Ovary Syndrome, Boston: Blackwell Scientific Publications. 1992;377–84.
- 2. Morreale EHF, Ramırez LM, Millan JLS. The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome. Endocr Rev 2005;26:251–82. pmid:15561799
- 3. Kandarakis ED, Piperi C, Spina J, Argyrakopoulou G, Papanastasiou L, Bergiele A, et al. Polycystic ovary syndrome: the influence of environmental and genetic factors. Hormones Rev 2006;5(1):17–34.
- 4. Hussein ZAM, Harun S. Construction of a polycystic ovarian syndrome (PCOS) pathway based on the interactions of PCOS-related proteins retrieved from bibliomic data. Theoretical biology & medical modeling 2009;6:18.
- 5. Xu Y, Li Z, Ai F, Chen J, Xing Q, Zhou P, et al. Systematic Evaluation of Genetic Variants for Polycystic Ovary Syndrome in a Chinese Population. PLoS ONE 2015; 460 10(10): e0140695. https://doi.org/10.1371/journal.pone.0140695. pmid:26474478
- 6. Dunaif A. Perspectives in Polycystic Ovary Syndrome: From Hair to Eternity. The Journal of Clinical Endocrinology & Metabolis 2016;101(3):759–68.
- 7. Tian Y, Zhao H, Chen H, Peng Y, Cui L, Du Y, et al. Variants in FSHB Are Associated With Polycystic Ovary Syndrome and Luteinizing Hormone Level in Han Chinese Women. J Clin Endocrinol Metab 2016;101(5):2178–2184. pmid:26938199
- 8. Wang Z, Li T, Xing X, Gao X, Zhang X, You L, et al. Replication study of RAD54B and GREB1 polymorphisms and risk of PCOS in Han Chinese. Reprod Biomed Online 2013; 27(3):316–321. pmid:23876972
- 9. Cui L, Li G, Zhong W, Bian Y, Su S, Sheng Y, et al. Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature. Hum Reprod 2015;30(3):732–736. pmid:25586784
- 10. Chen L, Hu LM, Wang YF, Yang HY, Huang XY, Zhou W, et al. Genome-wide association study for SNPs associated with PCOS in human patients. Exp Ther Med 2017;14(5):4896–4900. pmid:29104669
- 11. Brower MA, Jones MR, Rotter JI, Krauss RM, Legro RS, Azziz R, et al. Further investigation in europeans of susceptibility variants for polycystic ovary syndrome discovered in genome-wide association studies of Chinese individuals. J Clin Endocrinol Metab 2015;100(1):E182–6. pmid:25303487
- 12. Shim U, Kim HN, Lee H, Oh JY, Sung YA, Kim HL. Pathway Analysis Based on a Genome-Wide Association Study of Polycystic Ovary Syndrome. PLoS One. 2015;10(8):e0136609. pmid:26308735
- 13. Jakubowski L. Genetic aspects of polycystic ovary syndrome. Endokrynologia Polska 2005;56(3):285–93. pmid:16350721
- 14. Rotterdam ESHRE/ASRM-465 Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19–25.
- 15.
Schlesselman J. Case-Control Studies: Design, Conduct, Analysis. New York: Oxford University Press. 1982;354.
- 16. Boteju WMM, Karunarathna GDKN, Udayangani SAD, Silva KGH, Wijeyaratne CN. Markers of hyperandrogenism in South Asians with polycystic ovary syndrome. Sri Lanka Journal of Diabetes, Endocrinology and Metabolism 2014;4:3–8.
- 17. Balasooriya S, Fernando MA. Seasonal variation of menarche in Sri Lanka. Ceylon Med J 1988;33:105–109. pmid:3266107
- 18.
Stewart A, Marfell-Jones M. International Society for the Advancement of Kinanthropometry. 2011;New Zealand: Lower Hutt.
- 19.
AAMI Standards Philosophy and Strategy—Key Elements—Standards—Association for the Advancement of Medical Instrumentation. Available from: http://www.aami.org/
- 20. Wijeyaratne CN, Seneviratne RdeA, Dahanayake S, Kumarapeli V, Palipane E, Kuruppu N, et al. Phenotype and metabolic profile of South Asian women with polycystic ovary syndrome (PCOS): results of a large database from a specialist Endocrine Clinic. Hum Reprod 2011;26(1):202–13. pmid:21098627
- 21. Allahbadia GN, Merchant R. Polycystic ovary syndrome and impact on health. Middle East Fertility Society Journal 2011;16:19–37.
- 22. Rojas J, Chávez M, Olivar L, Rojas M, Morillo J, Mejías J, Calvo M, Bermúdez V. Polycystic Ovary Syndrome, Insulin Resistance, and Obesity: Navigating the Pathophysiologic Labyrinth. Int J Reprod Med 2014; 2014: 719050. pmid:25763405
- 23. Li H, Kilpelainen TO, Liu C, Zhu J, Liu Y, Hu C, et al. Association of genetic variation in FTO with risk of obesity and type 2 diabetes with data from 96,551 East and South Asians. Diabetologia 2012;55:981–95. pmid:22109280
- 24. Mukherjee S, Shaikh N, Khavale S, Shinde G, Meherji P, Shah N, et al. Genetic variation in exon 17 of INSR is associated with insulin resistance and hyperandrogenemia among lean Indian women with polycystic ovary syndrome. European Journal of Endocrinology 2009;160:855–62. pmid:19211708
- 25. Gangopadhyay S, Agrawal N, Batra A, Kabi BC, Gupta A. Single-Nucleotide Polymorphism on Exon 17 of Insulin Receptor Gene Influences Insulin Resistance in PCOS: A Pilot Study on North Indian Women. Biochem Genet 2016;54(2):158–68. pmid:26721804
- 26.
Kajan M. High Resolution Melt (HRM) Based Quantitative Real-Time Polymerase Chain Reaction (qPCR) Primer Design Tool for Point Mutation Detection. University of Colombo School of Computing. https://doi.org/10.13140/RG.2.2.24229.40166, 2018.
- 27. Borgbo T, Sommer Kristensen L, Lindgren I, Yding Andersen C, Hansen LL. Genotyping common FSHR polymorphisms based on competitive amplification of differentially melting amplicons (CADMA). J Assist Reprod Genet 2014;31:1427–36. pmid:25241129
- 28. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, et al. Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons. Clinical Chemistry 2004;50(7):1156–64. pmid:15229148
- 29. Słomka M, Sobalska-Kwapis M, Wachulec M, Bartosz G, Strapagiel D. High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies. Int J Mol Sci 2017;18(11):2316.
- 30. Dhas DB, Ashmi AH, Bhat BV, Parija SC, Banupriya N. Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR. Indian J Med Res 2015;142(5):555–62. pmid:26658590
- 31. Li T, Wu K, You L, Xing X, Wang P, Cui L, et al. Common variant rs9939609 in gene FTO confers risk to polycystic ovary syndrome. PLoS One 2013;8(7):e66250. pmid:23840863
- 32. Barber TM, Bennett AJ, Groves CJ, 510 Sovio U, Ruokonen A, Martikainen H, et al. Association of variants in the fat mass and obesity associated (FTO) gene with polycystic ovary syndrome. Diabetologia 2008;51(7):1153–58. pmid:18478198
- 33. Yan Q, Hong J, Gu W, Zhang Y, Liu Q, Su Y, et al. Association of the common rs9939609 variant of fto gene with polycystic ovary syndrome in Chinese women. Endocrine 2009;36(3):377–82. pmid:19859840
- 34. Osman HG, El-Refaeey AAA, El-Sokkary AMA, El-Sokkary MMA, El-Saeed RA. A Study on Fat Mass and Obesity Associated (FTO) Gene rs9939609 Variant in Egyptian Women with Polycystic Ovarian Syndrome. Australian Journal of Basic and Applied Sciences 2014;8(13):98–104.
- 35. Al-Tu'ma FJ, HadiFarhan N, Al-Safi WG. Association between Fat Mass and Obesity Geners9939609 Polymorphism with PCOS Women in Iraqi Population. Ijppr.Human 2015;5(1):62–72.
- 36. Tan S, Scherag A, Janssen OE, Hahn S, Lahner H, Dietz T, et al. Large effects on body mass index and insulin resistance of fat mass and obesity associated gene (FTO) variants in patients with polycystic ovary syndrome (PCOS). BMC Medical Genetics 2010;11:526 12.
- 37. Wehr E, Schweighofer N, Möller R, Giuliani A, Pieber TR, Obermayer-Pietsch B. Association of FTO gene with hyperandrogenemia and metabolic parameters in women with polycystic ovary syndrome. Metabolism 2010;59:575–80. pmid:19913856
- 38. Ramos RB, Spritzer PM. FTO gene variants are not associated with polycystic ovary syndrome in women from Southern Brazil. Gene 2015;560:25–29. pmid:25592819
- 39. Valkenburg O, Uitterlinden AG, Piersma D, Hofman A, 532 Themmen AP, de Jong FH, et al. Genetic polymorphisms of GnRH and gonadotrophic hormone receptors affect the phenotype of polycystic ovary syndrome. Hum Reprod 2009;24(8):2014–22. pmid:19403562
- 40. Tong Y, Liao WX, Roy A, Ng SC. Association of AccI polymorphism in the follicle stimulating hormone b gene with polycystic ovary syndrome. Fertility and Sterility 2000;74(6):1233–36. pmid:11119757
- 41. Dolfin E, Guani B, Lussiana C, Mari C, Restagno G, Revelli A. FSH receptor Ala307Thr polymorphism is associated to polycystic ovary syndrome and to a higher responsiveness to exogenous FSH in Italian women. J Assist Reprod Genet 2011;28(10):925–30. pmid:21792664
- 42. Du J, Zhang W, Guo L, Zhang Z, Shi H, Wang J, et al. Two FSHR variants, haplotypes and metaanalysis in Chinese women with premature ovarian failure and polycystic ovary syndrome. Mol Genet Metab 2010;100(3):292–95. pmid:20399696
- 43. Gu BH, Park JM, Baek KH. Genetic variations of follicle stimulating hormone receptor are associated with polycystic ovary syndrome. Int J Mol Med 2010;26(1):107–12. pmid:20514429
- 44. Unsal T, Konac E, Yesilkaya E, Yilmaz A, Bideci A, Ilke Onen H, et al. Genetic polymorphisms of FSHR, CYP17, CYP1A1, CAPN10, INSR, SERPINE1 genes in adolescent girls with polycystic ovary syndrome. J Assist Reprod Genet 2009;26(4):205–549. pmid:19387820
- 45. Mohiyiddeen L, Salim S, Mulugeta B, McBurney H, Newman WG, Pemberton P, et al. PCOS and peripheral AMH levels in relation to FSH receptor gene single nucleotide polymorphisms. GynecolEndocrinol 2012;28(5):375–77.
- 46. Tong Y, Liao WX, Roy AC, Ng SC. Absence 553 of mutations in the coding-regions of follicle-stimulating hormone receptor gene in Singapore Chinese women with premature ovarian failure and polycystic ovary syndrome. HormMetab Res 2001;33:221–26.
- 47. Fu L, Zhang Z, Zhang A, Xu J, Huang X, Zheng Q, Cao Y, Wang L, Du J. Association study between FSHR Ala307Thr and Ser680Asn variants and polycystic ovary syndrome (PCOS) in Northern Chinese Han women. J Assist Reprod Genet 2013;30(5):717–721. pmid:23536150
- 48. Furui K, Suganuma N, Tsukahara S, Asada Y, Kikkawa F, Tanaka M, et al. Identification of two point mutations in the gene coding luteinizing hormone (LH) b558 subunit, associated with immunologically anomalous LH variants. J ClinEndocrinolMetab 1994;(78):107–13.
- 49. Haavisto AM, Pettersson K, Bergendahl M, Virkamäki A, Huhtaniemi I. Occurrence and biological properties of a common genetic variant of luteinizing hormone. J ClinEndocrinolMetab 1995;80:1257–63.
- 50. Nilsson C, Pettersson K, Millar RP, Coerver KA, Matzuk MM, Huhtaniemi IT. Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. International Collaborative Research Group. FertilSteril 1997;67:998–1004.
- 51. Batista MC, Duarte Ede F, Borba MD, Zingler E, Mangussi-Gomes J, dos Santos BT, et al. Trp28Arg/Ile35Thr LHB gene variants are associated with elevated testosterone levels in women with polycystic ovary syndrome. Gene 2014;550(1):68–73. pmid:25111116
- 52. Capalbo A, Sagnella F, Apa R, Fulghesu AM, Lanzone A, Morciano A, et al. The 312N variant of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) confers up to 2·7-fold increased risk of polycystic ovary syndrome in a Sardinian population. Clinical Endocrinology 2012;77(1):113–19. pmid:22356187
- 53. Ha L, Shi Y, Zhao J, Li T, Chen ZJ. Association 574 study between polycystic ovarian syndrome and the susceptibility genes polymorphisms in Hui Chinese women. Plos One 2015;10(5):e0126505. pmid:25978310
- 54. Bassiouny YA, Rabie WA, Hassan AA, Darwish RK. Association of the luteinizing hormone/choriogonadotropin receptor gene polymorphism with polycystic ovary syndrome. Gynecological Endocrinology 2014;30(6):428–30. pmid:24592983
- 55. Lee EJ, Yoo KJ, Kim SJ, Lee SH, Cha KY, Baek KH. Single nucleotide polymorphism in exon 17 of the insulin receptor gene is not associated with polycystic ovary syndrome in a Korean population. Fertility and Sterility 2006;86:380–84. pmid:16806206