Polycystic Ovarian Syndrome (PCOS) and its association with VDR gene variants Cdx2 (rs11568820) and ApaI (rs7975232): Systematic Review, Meta-analysis and In silico analysis

doi:10.21203/rs.3.rs-1744730/v1

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Systematic Review

Polycystic Ovarian Syndrome (PCOS) and its association with VDR gene variants Cdx2 (rs11568820) and ApaI (rs7975232): Systematic Review, Meta-analysis and In silico analysis

https://doi.org/10.21203/rs.3.rs-1744730/v1

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Objective: Polycystic ovary syndrome (PCOS) is a multifactorial and polygenic disorder of women in the reproductive-age group. Among various reasons, Vitamin D level and Vitamin D receptor (VDR) polymorphism have been investigated as one of the predisposing causes. The study aims to investigate the association between VDR polymorphism and PCOS susceptibility.

Methodology: Online databases (PubMed, Central Cochrane Library, Science Direct and Google Scholar) were searched up to 30th March, 2022. With suitable syntax. Odds Ratios (OR) with 95% Confidence Interval (CI) and Chi-square (χ2) tests were applied under different genetic models to find the association between Cdx2 G>A (rs11568820) and VDR ApaI C>A(rs7975232) polymorphisms with PCOS risk. All the statistical calculations were performed using Review Manager 5.3 software (Cochrane Collaboration, Oxford, UK). To understand molecular effect of variants in the untranslated region, in silico analysis was performed using Regulome DB, FATHMM-MKL and Haploreg v4.1 web tools.

Results: Total fifteen case-control studies were identified with 2114 PCOS patients and 1552 control subjects.

VDR variant Cdx2 (rs11568820) was found to be significantantly associated (OR= 0.40, 95% CI= 0.22-0.72, p= 0.002) after the odd one out analysis of one major study. VDR variant ApaI is significantly associated with PCOS under all four genetic models: Dominant Model (AA + AC vs CC): OR= 0.75, 95%CI= 0.62-0.91,p= 0.003; Recessive model (AA vs. AA+AC): OR= 0.78, 95%CI= 0.63-0.98, p= 0.03; Additive model(AAvsAA+CC):OR=0.70,95%CI=0.57-0.87,p=0.001 and Allele model (A vs C): OR= 0.83, 95%CI= 0.74-0.91, p=0.0002 indicating ‘A’ allele as risk factor. Subgroup analysis revealed significant association among Asian population. However, In silico analysis of the intronic variants have yielded contradictory results.

Conclusion: The study revealed VDR Cdx2 and ApaI polymorphism as a significant risk factor for PCOS.

Vitamin D receptor

Polymorphism

PCOS

Meta-analysis

Polycystic Ovarian Disease (PCOS) or Stein Leventhal Syndrome is the most frequent gynaecological, endocrine, and metabolic disorder found in premenopausal women which contributes to multiple health complications (Carmina & Lobo, 1999). Worldwide the prevalence of PCOS varies from 4% to 21%, depending on the population being investigated and the diagnostic criteria utilized (Lizneva et al., 2016). Ovulatory dysfunction, hyperandrogenic symptoms such as acne, alopecia, hirsutism, and polycystic ovarian morphology are the major characteristics of the disease (Fauser et al., 2012). PCOS patients have a higher prevalence of Type 2 Diabetes, hyperinsulinemia, insulin resistance, and obesity in addition to menstrual irregularity and hyperandrogenism (Legro et al., 2005; Maitra et al., 2001). Various susceptibility genes and environmental factors interact and influence the progression and appearance of the syndrome. There is evidence that Vitamin D deficiency may have a role in the development of insulin resistance and metabolic syndrome in PCOS patients and its levels have been linked to the severity of the disorder’s phenotype (Wehr et al., 2009). Vitamin D deficiency increases PTH production which is largely responsible for increased testosterone and menstrual abnormalities (Panidis et al., 2005; Thys-Jacobs et al., 1999).

Biological actions of Vitamin D in different tissues are mediated by Vitamin D receptors (VDR). Variation in the binding capacity of the receptor due to genetic variants is one of the cause of Vitamin D deficiency (Christakos et al., 1996). Vitamin D receptor, a nuclear protein, is a DNA binding transcription factor that forms heterodimer with the Retinoid X Receptor (RXR) (Haussler et al., 1998). In humans, active vitamin D, and Vitamin D receptor complex control over 300 genes which are involved in glucose and lipid metabolism as well as blood pressure regulation (Bouillon et al., 2008).

The VDR gene is located on chromosome 12q13.11 and covers about 75kb with 11 exons (Uitterlinden et al., 2004). Two promoter regions, eight exons (2-9), and six untranslated exons (1a-1f) make up the genome (Holick et al., 2007). Polymorphisms in the Vitamin D receptor (VDR) have been linked to some of the clinical representations associated with PCOS (Reis et al., 2017). The gene is highly polymorphic, and its polymorphic markers namely FokI, BsmI, ApaI, TaqI, Tru9I, and Cdx2 have resulted in aslew of allelic variants that vary among ethnic groups (Vulcan et al., 2021). A previous meta-analysis with nine studies reported in 2019 has found the association of VDR ApaI (rs7975232) (C > A) gene polymorphism and PCOS outcome (Shi et al., 2019). Since then, many more relevant case-control studies have been reported.So far,no meta-analysis has been reported to find the association between Cdx2 (rs11568820) (G > A) gene polymorphism and PCOS. Further, Therefore, the present study has been carried out with an objective i) to investigate the association of VDR Cdx2 (rs11568820) gene polymorphism and PCOS susceptibility ii) to reanalyze ApaI (rs7975232) polymorphism with PCOS susceptibility with the help of updated data iii) Prediction of biological consequences of these variants through in silico tools.

Databases Search

Existing literature on the role of Vitamin D receptor polymorphism in Polycystic Ovarian Syndrome (PCOS) susceptibility was evaluated followed by Meta-analysis of original research. Eligible studies were systematically searched in PubMed, Google Scholar, Central Cochrane Library, and Science Direct databases, up to 30^th March, 2022, using the following Mesh of terms: “PCOS” OR “Polycystic Ovary Syndrome” OR “Polycystic Ovary Disease” AND “VDR polymorphism” OR “Vitamin D Receptor polymorphism”. To ensure that all relevant papers had been identified,the reference list of the selected papers were also checked (Fig.1).

Selection Criteria

The following were the specific inclusion criteria for search selection :i) Case-Control study, ii) Studies evaluated the association between Vitamin D receptor gene polymorphism and PCOS, iii) Report of at least one polymorphism in the VDR gene, iv) Availability of sufficient genotypic and allelic distribution for both cases and controls, v)Studies which considered the Rotterdam and/or NIH criteria for PCOS diagnosis.

Studies excluded if: i) Studies based on animal models, ii) Studies associated with diseases other than PCOS, iii) Meta-analysis, abstract, reviews, cohort studies, duplicated papers, iv) Insufficient data available for cases and controls.

Quality assessment

Newcastle-Ottawa Scale (NOS) was utilized for the qualitative assessment of the included studies.The scale is composed of three parameters:Selection,Comparability ,and Exposure,with a maximum value of 9. Three investigators (Komal Gupta, Richa Thakur, Priya Sharma) independently analyzed the quality using the NOS scale (Supplementary Table 1). A total score of 3, 4-6, 7, or 8 for each study is deemed low, medium, or high-quality study, respectively. Any disagreement about the assessment was settled by the fourth and fifth author (Pooja Kamra and Preeti Khetarpal).

Data extraction

The following information from each qualified study was extracted by three researchers independently and meticulously. i) Name of the first author with publication year ii) Ethnicity of the research population, iii) PCOS diagnosing criteria, iv) Age (in years) and BMI (kg/m²) of both cases and controls, v) Sample size of both patients and controls, vi) Genotypic frequency data with the Genotyping method used.

Statistical analysis

All the statistical analysis was performed using Review Manager 5.3 software (Cochrane Collaboration, Oxford, UK) and p<0.05 was considered statistically significant. The association strength of Cdx2 (rs11568820), ApaI (rs7975232) VDR polymorphism, and risk of PCOS was assessed by the Chi-square (χ2) test, pooled Odds Ratio (OR), and 95% Confidence Interval (CI). The strength of association was calculated under four genetic models: Dominant model, Recessive model, Additive and Allelic model. Heterogeneity was interpreted by Q test and I2 test. The results of OR were interpreted as: OR = 1, exposure to the factor is not a risk, OR < 1, exposure to the factor is associated with lower risk, and OR > 1, exposure to the risk is associated with higher risk. Heterogeneity among the studies was assessed by I2 value and Q-test. I2<50% and Q-test (p>0.05) indicate the absence of heterogeneity and a fixed-effect model was applie whereas If I2value > 50% and Q-test (p<0.05) random effect model was applied.

In silico analysis of Variants to predict Biological effects

In silico evaluation of the intronic variants using various web tools was performed using Regulome DB 2.0.3, FATHMM-MKL, and HaploReg v4.1 as these tools provide insight into the possible biological significance (Rojano et al., 2019). The Regulome DB links SNPs to known and expected regulatory elements in DNAase hypersensitive intergenic regions, transcription factor binding sites and promoter regions that have been biochemically characterized (Boyle et al., 2012). FATHHM-MKL using ENCODE annotations, is helpful in predicting the potential effects of regulatory variations. P-values more than 0.5 are projected to be harmful, whereas values less than 0.5 are predicted to be benign or neutral (Shihab et al., 2015).

The chromosomal location of the variants Cdx2 and ApaI and the reference and altered allele of these intronic variants were retrieved using database NCBI and ENSEMBL.

Non-coding variations was functionally annotated with the Haploreg database to provide conservation scores, promoter/enhancer histone marks, open chromatin position, and eQTL markers (Ward & Kellis, 2012).

Characteristics of studies selected

The present study describes the role of Cdx2 and ApaI VDR gene polymorphism and PCOS susceptibility. For the metananlysis, a total of 15 case-control studies were identified and included, which comprises 2114 PCOS patients and 1552 control subjects.

Association between VDR gene Cdx2 (rs11568820) and PCOS susceptibility

A total of five studies relevant to VDR rs11568820 polymorphism and risk of PCOS development have been included. The data extracted from these studies have been summarized in (Table 1) (Wehr et al., 2011); (Dasgupta et al., 2015); (Ali et al., 2018); (Malik et al., 2018); (Szafarowska et al., 2019), which include first author, publication year, methodology, ethnicity, genotypic data. Most of them had a Newcastle-Ottawa scale (NOS) score of more than 5. Statistical data synthesized from four different genetic models: Dominant model (AA + AG vs GG): OR=0.51, 95%CI=0.24-1.09, p=0.08, Recessive model (AA vs. AG+GG): OR=1.59, 95% CI=0.91-2.78, p=0.11, Additive model (AA vs. AA + GG): OR=1.30, 95%CI=0.73-2.31, p=0.37 and Allelic model (A vs. G): OR=0.67, 95%CI=0.41-1.10, p= 0.12 has not shown any significant results (Supplementary Fig 1). However, the correlation becomes significant (OR= 0.40, 95% CI= 0.22-0.72, p= 0.002) after a sensitivity analysis test with the exclusion of one major study by Wehr et al. 2011 (Fig 2).

Assoociation between VDR gene ApaI (rs7975232) and PCOS susceptibility

Data has been summarized from thirteen relevant studies describing VDR ApaI (rs7975232) polymorphism and PCOS susceptibility (Table 2) ; (Al-baidani & Abdulhassan, 2017; Dasgupta et al., 2015; El-Shal et al., 2013; Jedrzejuk et al., 2015; Mahmoudi, 2009; Mahmoudi et al., 2015; Santos et al., 2018; Siddamalla et al., 2018; Sur & Chakravorty, 2015; Szafarowska et al., 2019; Wehr et al., 2011; Xavier et al., 2019); Meta-analysis results synthesized from four different genetic models: Dominant Model (AA + AC vs CC): OR= 0.75, 95% CI= 0.62-0.91,p=0.003, Recessive model (AA vs. AC+CC): OR= 0.78, 95% CI= 0.63-0.98, p= 0.03; Additive model (AAvs.AA+CC): OR=0.70, 95% CI= 0.57-0.87, p=0.001 and Allele model (A vs. C): OR= 0.83, 95%CI= 0.74-0.91, p=0.0002 and there is significant association between ApaI polymorphism and PCOS susceptibility under all genetic models (Fig. 3). Subgroup meta-analys when stratified by Asian and Non-Asian population revealed that VDR ApaI polymorphism significantly associated under Dominant model (AA + AC vs CC): OR= 0.69, 95% CI=0.53-0.90, p=0.006 among Asian population while no significant results were observed for Non-Asian population (Fig. 4).

Sensitivity analysis and publication bias

After performing the odd one-out sensitivity analysis for the VDR Cdx2 variant by the removal of one major study by Wehr et al. 2011, the gene variant is found to be significantly associated (OR= 0.40, 95% CI= 0.22-0.72, p= 0.002) with the PCOS risk. Funnel plot verified no publication bias and forest plot show the significant results as mentioned previously. For ApaI variant analysis, after removing one study by Jedrzejuk et al. 2015, heterogeneity changed from moderate (χ2= 21.91, df=12 (p=0.04), I2 =45%) to low (χ2 = 14.67, df=11 (p=0.20), I2 =25%) indicating this study contribute significantly to heterogeneity. Funnel plot and Forest plot (Supplementary Fig. 2) represented a symmetrical funnel plot indicating no publication bias.

The polymorphisms linked to PCOS in the meta-analysis were further investigated in-silico to determine their likely role and biological mechanism.

In silico analysis of the intronic variants

The variations discovered to be related with PCOS in the preceding meta-analysis were then tested in silico to determine their likely function and biological mechanism underlying such statistical relationships. Regulome DB analysis revealed and Cdx2 and ApaI as a transcription factor binding altering polymorphic variants with a score 5 and 3a respectively. The score 3a implies that there is transcription factor binding at ApaI and this overlap with some motifs. The SNP (rs7975232) is also within DNase peak region which implies it is within open chromatin and is less likely to affect binding of transcription factor, while Cdx2 (rs11568820) with score 5 has minimal binding evidence with the transcription factor. The FATHMM-MKL non-coding score for Cdx2 is found to be 0.92453 which predicts the SNP to be deleterious, while for ApaI this tool has not yielded any significant results.

By analysis of HaploReg v4.1 results it is found that the SNP Cdx2 overlaps with enhancer histone marks in two tissues namely, Gastrointestinal tract and Liver, and is found to change the motif of ERalpha-a and Hoxd10 gene which may be further analysed to know the effects of the given intronic variants. ApaI overlaps with an HMM predicted enhancers in 9 major tissue types. There is a cluster of enhancer activity is shown in mesenchymal, epithelial, digestive and other tissues and that is classified as a genetic enhancer by 25 state model of chromatin state and core 15 state model. Modification at H3K4me1, H2K4me3, H3K27ac all contribute to the chromatin state assignment in epithelial tissue while H3K4me1and H3K9ac are found to affect chromatin state in other tissues. The presence of black cells in the table indicates that DNase was not detected by Roadmap in many tissues but DNase 1 hypersensitivity is found in certain tissues like iPSC (induced Pluripotent Stem Cells). ChipSeq and motif data suggests SNP (rs7975232) is found to change the motif of zinc finger protein CTCF and HEN1 gene. (Supplementary Table 2).

To the best of our knowledge, the present study is the first attempt to access the correlation between Cdx2 polymorphism and PCOS development risk and the meta-analysis indicates that it is significantly linked with the PCOS risk. While for ApaI, the metanalysis includes more studies than the reported previously which the significanct association remining same. After subgroup analysis stratified by ethnicity, significant associations were observed between ApaI polymorphism and PCOS among Asian population while no such association found among the Non- Asian population.

Vitamin D Receptor gene or calcitriol gene has an important role in the regulation of protein functioning and signaling related to uptake of Vitamin D and other metabolic pathways. Recent studies have linked VDR gene polymorphism with Vitamin D deficiency, obesity, insulin resistance, (Rahmadhani et al., 2017) metabolic syndromes in PCOS (Santos et al., 2018) and osteoporosis risk in postmenopausal women (Zhang et al., 2021). VDR plays an important role in the pathogenesis of PCOS by mediating the action of Vitamin D and also affecting the insulin signaling pathways (Pittas et al., 2007). Another study investigatedthat lower vitamin D was independently associated with risk of PCOS but the genetic variationof VDR had no effect (Lone et al., 2020), whereas a recent study in a model organism has investigated the role of Vitamin D3 in the ovary in PCOS indicating reduced levels of Vitamin D3 concentration and implication in the PCOS pathogenesis (Grzesiak et al., 2021). The findings of the VDR gene as a candidate gene that plays a role or affects PCOS pathogenesis have been contradictory.

There were many limitations in this study like the language constrain as only articles in English have been included. Sample size for Cdx2 analysis was lower than ApaI. Moreover, the genes alone do not play the only role, there are other factors that affect the functioning and characteristics of a disease such as environmental factors like geographical location, duration of exposure to sunlight, diet,etc.

Bioinformatic evaluation for the functional characterisation of the intronic variants using available web tools, has generated inconsistent results. Regulome DB analysis for the non coding variants revealed that the SNP ApaI is likely to affect the binding of transcription factor and may play a functional role in PCOS pathogenesis, while in silico analysis using FATHMM-MKL have shown a high non-coding score for Cdx2 with deleterious effect. Thus, both the SNPs are significantly associated with PCOS risk with in silico analysis is helpful but not conclusive.

Acknowledgment

The authors would like to thank financial support for this work by ICMR New Delhi [54/08/2019/HUM-BMS, Project id 2019-1384]. DST-FIST support (SR/FST/LS-I/2017/49-C) to the Department of Human Genetics & Molecular Medicine is acknowledged.

Disclosure Statement

The authors declare no conflict of interest.

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Table 1. Characteristics of included studies on vitamin D receptor gene Cdx2 (rs11568820) polymorphism and PCOS

Reference	Ethnicity	Genotyping Method	PCOS diagnostic Criteria	Sample Size		Age, Years		BMI, kg/m2		Genotype
				Sample Size		Age, Years		BMI, kg/m2		Case			Control
				Case	Control	Case	Control	Case	Control	GG	GA	AA	GG	GA	AA
Wehr et al., 2011	Europe	SNP genotyping	Rotterdam	545	145	23–31	26–36	24.2 (21.2-29.0)	24.4 (20.9–29.2)	346	176	23	96	46	3
Dasgupta et al., 2015	India	ASM-PCR	Rotterdam	250	250	NA	NA	NA	NA	190	52	8	107	143	0
Hamdi et al., 2018	Iraq	ASM-PCR	Rotterdam	45	43	18-34	18-34	NA	NA	24	14	7	16	20	7
Malik et al., 2018	Pakistan	TARMS-PCR	Rotterdam	80	78	19-31	18-28	NA	NA	63	10	7	57	12	9
Szafarowska et al., 2019	Poland	Real-time PCR	Rotterdam	75	23	25-43	27–42	21.50 (19.95- 23.50)	NA	52	16	7	10	12	1

NA: not available; SNP: Single nucleotide polymorphism; ASM-PCR: allele specific multiple-polymerase chain reaction; TARMS-PCR: tetra-primer amplification refractory mutation system-polymerase chain reaction.

Table 2. Characteristics of included studies on vitamin D receptor gene ApaI (rs7975232) polymorphism and polycystic ovary syndrome (PCOS)

Reference	Ethnicity	Genotyping Method	PCOS diagnostic Criteria	Sample Size		Age, Years		BMI, kg/m2		Case Genotype			Control Genotype
				Sample Size		Age, Years		BMI, kg/m2		Case			Control
				Case	Control	Case	Control	Case	Control	AA	AC	CC	AA	AC	CC
Mahmoudi et al. (2009)	Iran	PCR	NIH Criteria	162	162	19-42	18-54	28.04	26.43	58	68	36	49	90	23
Wehr et al (2011)	Europe	SNP Genotyping	Rotterdam	543	145	23–31	26-36	24.2 (21.2–29.0)	24.4 (20.9-29.2)	142	274	127	48	60	37
El Shal et al (2013)	Egypt	PCR-RFLP	Rotterdam	150	150	24-35	23-35	30.3 ± 5.4	29.4 ± 5.7	63	65	22	68	64	18
Dasgupta et. al. (2015)	India	PCR-RFLP	Rotterdam	250	250	NA	NA	NA	NA	118	120	12	121	117	12
Dipanshu et al (2015)	India	PCR-RFLP	Rotterdam	125	82	25.78	26.86	27.3± 7.5	23.7 ± 4.7	23	70	32	32	29	21
Jedrzejuk et al (2015)	Poland	PCR-mini sequencing	Rotterdam	92	85	20–35	20-35	28.7 ± 7.4	27.4 ± 6.3	19	52	19	32	49	17
Mahmoudi et al (2015)	Iran	PCR-RFLP	NIH Criteria	35	35	28.43	30.29	26.98	26.30	15	11	9	8	21	6
Huabin Cao et al (2016)	China	PCR-RFLP	Rotterdam	120	120	18-27	20-26	NA	NA	22	58	40	39	55	26
Ismail et al (2017)	Iraq	Real-time PCR	Rotterdam	50	50	15-45	15-45	NA	NA	21	28	1	21	23	6
Siddamalla et al (2017)	India	PCR-RFLP	Rotterdam	95	130	23-30	22-31	24.10±4.20	23.73±3	42	21	32	70	35	25
Santos et al (2018)	Brazil	Real-time PCR	Rotterdam	191	100	16-30	17-33	72.6%	53.4%	61	88	41	36	48	16
Xavier et al (2018)	Brazil	PCR-RFLP	Rotterdam	99	99	26-36	22-37	29.4 (8.4)	23.0 (5.4)	41	51	7	42	47	10
Szafarowska et al, 2019	Poland	Real-time PCR	Rotterdam	75	23	25–43	27-42	21.50	NA	37	35	3	13	9	1

NA: Not Available; PCR: Polymerase Chain Reaction; PCR-RFLP: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism; SNP: Single Nucleotide Polymorphism

No competing interests reported.

SupplementaryFig1.docx
Supplementary Fig. 1 Forest plots of the VDR polymorphism Cdx2 (rs11568820) G>A and PCOS in the overall populations: a) Dominant model (AA+AG vs. GG); b) Recessive model (AA vs. AG+GG); c) Additive model (AA vs. AA+GG); d) Allele model (A vs. G)
SupplementaryFig.225may.doc
Supplementary Fig. 2 Funnel plot of publication bias and forest plot for sensitivity analysis in the selection of studies on the ApaI (rs7975232) polymorphism. Publication bias and sensitivity analysis was assessed under the Dominant model (AA + AC vs. CC). SE represents Standard Error; OR represents Odds Ratio
SupplementaryTable1.docx
Supplementary Table 1. Quality assessment of the included studies by NOS scale
SupplementaryTable225may.docx
Supplementary Table 2. In-silico analysis results by Regulome DB, FATHMM-MKL, and HaploReg v4.1

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published 30 Apr, 2024

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Polycystic Ovarian Syndrome (PCOS) and its association with VDR gene variants Cdx2 (rs11568820) and ApaI (rs7975232): Systematic Review, Meta-analysis and In silico analysis

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Introduction

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Assoociation between VDR gene ApaI (rs7975232) and PCOS susceptibility

Sensitivity analysis and publication bias

Discussion

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