Study of Methylation Status and Expression of Vitamin D Receptor Gene in Polycystic Ovarian Syndrome Patients of Kashmiri Population

Objective:
Downregulation of Vitamin D Receptor (VDR) gene expression resulting from epigenetics events, like methylation in promoter of VDR gene play an important role in pathogenesis, clinical and bio-chemical parameters of polycystic ovary syndrome, but the results from different studies are inconsistent.

We aim to determine the methylation status of promoter of VDR gene and VDR gene expression analysis in addition to the co-relation between promoter methylation and expression profile of VDR genes in PCOS patients in Kashmiri Population.

Methods:
This study was a case control, comprising of 100 women with PCOS and 100 normal healthy control women aged between 15-45 years. PCOS patients were selected after proper diagnosis using Rotterdam criteria (2004) and obtaining written consent.

DNA extraction was carried out by the non-enzymatic Salting out method and extracted DNA samples were subjected to bisulphite treatment using EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, California) to produces a chemical conversion of unmethylated cytosine to uracil, which is detected as a thymine after PCR. Methylation specific PCR (MS-PCR) was performed following the bisulfite conversion, using pre-designed sets of methylated and unmethylated primers to confirm the prominence of methylation in the promoter VDR region and to specially acknowledge methylated and unmethylated VDR fragments.

The VDR expression analysis was carried out following RNA extraction from the blood sample and processed ahead for cDNA synthesis using reverse transcriptase reaction and then relative expression analysis by RT-PCR using SYBR Green fluorescent dye.

Results:
We found that vitamin D level among test group was significantly lower compared to controls (13.95±9.60 VS. 16.63±7.46; p=0.026). Methylation was significantly higher in test group compared to controls (84% vs 62%; p<0.0001) with OR of 3.21. Furthermore, there exists a significant difference between the average cyclic threshold (CT) value of VDR gene among test and control groups (24.64 vs. 26.14;p < 0.0001). The average CT value corresponding to GAPDH (glyceraldehyde phosphate dehydrogenase) is 29.76 in test group and 32.11 in control group. The expression of VDR gene was 0.55 times down regulated in test group in comparison to controls.

Discussion/Conclusion:
In the present study, we have comprehensively shown that VDR hypermethylation is significantly associated with reduced vitamin D level and decreased expression level of VDR gene among PCOS women. Increased VDR methylation in response to decreased vitamin D levels could be involved in the impairment of VDR mediated effects resulting in the peculiar clinical and bio-chemical derangements in patients of polycystic ovary syndrome. However, the enhanced ligand availability results in decreased receptor expression to maintain homeostasis of vitamin D signaling. Hence, this, inverse association could have a significant role in the altered gene expression of VDR and thereby effecting the cell response in PCOS patients.