One immunomodulatory agent that shows promise is vitamin D, known for its immune-stimulating properties, as it enhance the performance of the innate immune system and may contribute to immune tolerance. vitamin D deficiency has been linked to autoimmune diseases [19, 20].
In the present study, the quality and concentration of the extracted DNA were evaluated using a Nanodrop spectrophotometer and by measuring the 260/280 and 260/230 ratios. The typical 260/280 ratios for DNA and RNA generated by pure nucleic acids are 1.8 and 2.0, respectively. This ratio is affected by the buffer's pH and ionic potency used for measuring the blank and the sample [21]. Our results as per NanoDrop for DNA quality assessment indicated that the range of extracted DNA in both RA and HC group samples was (16.13 ng/ul to 151.89 ng/ul) and the purity fell within the range of good quality. According to Surzycki, DNA samples with an OD260/OD280 ratio between 1.6 and 2 were considered pure, while samples above or below this range were deemed to havie protein or RNA contamination, respectively [22].
The vitamin D receptor (VDR) is present in high concentrations in immune system cells such as dendritic cells, macrophages, and activated T and B lymphocytes, suggesting that vitamin D's regulatory role in the immune system operates through various mechanisms. Additionally,, vitamin D inhibits proinflammatory processes by binding to VDR and preventing immune cells from becoming overly active during an adaptive autoimmune response. It seems to regulate immune homeostasis by enhancing innate immunity while suppressing adaptive immunity. Moreever, vitamin D can alter the ratio of Th1 to Th2 and T regulatory cells [23, 24].
The last few decades have witnessed increased speculation about the potential role of hypovitaminosis D as a risk factor for multiple sclerosis, based on research findings linking MS and vitamin D status. There exist connection between MS susceptibility and certain gene variants that encode proteins involved in vitamin D metabolism, transport, and function [25]. Vitamin D3 inhibits the proliferation of cancer cells by binding to its nuclear receptors leading to apoptosis. As the VDR gene lacks consensus TATA and CAAT boxes, the VDR-Cdx2 polymorphism, found in the promoter region of exon 1e, affects the functional activity of the receptor [26]. The same author and his colleagues also studied the link between VDR-Cdx2 polymorphism and breast cancer in premenopausal Pakistani women. They hypothesized that young female patients with the GG genotype of the Cdx2-VDR gene polymorphism might have a raised risk for breast cancer. Although breast cancer and the VDR Cdx2 polymorphism were not significantly associated, women with the GG genotype were at an increased risk of breast cancer [26].
Due to its anti-inflammatory properties, vitamin D helps to prevent the onset of arthritis. Only after binding to its specific vitamin D receptor, which is encoded by the VDR gene, can the biologically active form of vitamin D's 1, 25(OH)2D3, exert its effect. Several human autoimmune diseases, mostly TH1- or TH17-intermediated, such as, rheumatoid arthritis, have been linked to the pathogenic impact of TH17 lymphocytes [27, 28].
Our result showed that the most common Cdx2 genotype in the Kurdish population of Iraq was GA, followed by the GG genotype. Additionally, no AA genotype was detected. Moreover, no significant associations were found among the genotypes; thus the Cdx2 hasn't influenced the risk of RA susceptibility among the Kurdish population. A study among the Turkish people investigated, VDR BsmI, TaqI and FokI gene polymorphisms, concluding that in the Turkish population, VDR polymorphisms do not affect rheumatoid factor positivity, susceptibility to RA, or erosive disease of RA [29]. It is well-known that G > A sequence variation in the 1a promoter region, known as the Cdx2 polymorphism, is linked to the transcriptional activity of the VDR gene [30].
The association between VDR gene polymorphisms and the risk of RA has been assessed, reporting conflicting results [31].In the same aspect, Pete et al. concluded that vitamin D and its receptor (VDR) significantly impact the onset of RA. When gender, age, and tobacco use were taken into account, false positives were avoided. Additionally, a trend for the risk of RA was found in the VDR FokI gene polymorphism. In contrast, there is no association between the VDR BsmI, TaqI, ApaI, and Cdx2 genes and the risk of developing RA. However, a haplotype analysis found a link between the haplotypes ACGAG and GTGCA and a higher risk of RA [32].
The GG genotype of Cdx2-VDR gene polymorphism may increase the risk of developing breast cancer in young female patients in South Pakistan [33].
Additionally, Pinho, Dias [34] investigated the distribution of VDR gene polymorphism in people with type 2 diabetes mellitus (DM2) and chronic periodontitis (CP), including FOKI, CDX2, and GATA. According to the findings, having these variants may result in less susceptibility to DM2 and CP. For the other SNP under investigation, no other significant group differences were discovered.
The relation of Cdx2 VDR gene polymorphisms with sensitivity to autoimmune thyroiditis was evaluated in a research study among the Caucasian Polish population, and it was discovered that the Cdx2 VDR gene is not a major susceptibility factor for the development of autoimmune thyroiditis [35]. A recent study indicated that RA patients frequently have vitamin D deficiency, and their 25(OH)D levels are significantly lower than those of healthy controls. Higher disease activity and disability scores were associated with lower vitamin D levels. Four VDR polymorphisms identified through genetic analysis did not increase the susceptibility to RA in the Lithuanian population [14].
Various vitamin D-related gene polymorphisms, including those in the VDR, may increase the risk of developing autoimmune diseases. For identifying the association between the VDR Cdx2 (rs11568820) polymorphism and autoimmune thyroid disease (AITD) in the Caucasian-Polish population. The CDX2 polymorphism's genotyping was conducted. There is no statistically significant difference between the genotype or allele distribution of the VDR Cdx2 polymorphism in the Caucasian-Polish population between AITD, AIT, or TAO, and the control group [30]. However, Maciejewsk et al. [36] discovered that TAO patients' VDR Cdx2 genotype distribution was significantly different from that of the AIT group.
Except for Cdx2, the VDR genetic variants were linked to the severity of PCOS symptoms but not the disease itself. These variants are not directly associated with PCOS risk factors or causes.; however, they could indirectly affect the development of PCOS at the biochemical level by regulating calcium or vitamin D levels [30].
Additionally, Ling et al. [37] showed that the serum 25(OH)D levels of Chinese women and the risk of osteoporosis and fracture in middle-aged and elderly individuals might be impacted by the Cdx-2 polymorphism in the VDR gene. Compared to GG genotype carriers, carriers of the AA and AG genotypes had higher odds of fracture. This study also found that women who were GG carriers had the highest 25(OH)D levels, AA carriers had the lowest, and AG carriers fell in the middle.
A subsequent study by Rehman et al. [38] in cancer patients found a significant association between the SNP of the VDR-Cdx2 gene polymorphism and the development of several diseases. According to the study's results, there is a significant link between the genotypes AG (32%), GG (66.8%), and several cancer types, including multiple myeloma (MM), rectal cancer, colon cancer, and gastric cancer (GC) (P 0.05). While in this study, the genotype AA (1.2%) was found to be less common.
In a research study involving Pakistani RA and osteoarthritis (OA) patients, it was found that serum vitamin D levels were not significantly low in the study groups. Still, that polymorphism on the VDR gene limits vitamin D's ability to act as an anti-inflammatory by changing the 1, 25(OH)2 D3 binding sites. As a result, 25(OH)2D3 dysfunction ultimately causes disease onset. They showed a significant correlation between the beginning of RA and OA in the population under study and the VDR gene polymorphisms (rs10735810, rs7975232, rs731236, and rs1544410). They proved that tryptophan is replaced with arginine when there is a polymorphism on rs10735810. Vitamin D could not convert into its active form and prevent disease onset because of polymorphism in the VDR gene. Consequently, in the Pakistani population, VDR gene polymorphism is a significant risk factor for the development of RA and OA [27].
Polymorphisms in the vitamin D receptor (VDR) are linked to many diseases, potentially impacting mortality. A prospective cohort study established a connection between the VDR gene polymorphisms (FokI, three haplotypes of the Cdx2 and GATA polymorphisms, and three haplotypes of the BsmI, ApaI, and TaqI polymorphisms) and mortality in older people. They found that osteoporotic fractures may contribute to the increased mortality risk associated with the Cdx2-GATA haplotype 1 allele [39]. In contrast, a separate genomic study identified that the HLA-DRB1*04 genotype was associated with RA susceptibility in the majority of Iraqi patients, conducting that the HLA-DRB1*04 allele significantly contributed to the onset of RA [40]. Various vitamin D-related gene polymorphisms, including those in the vitamin D receptor gene, may elevate the risk of emerging autoimmune diseases. Among RA patients, 41.7% suffered from co-existing autoimmune and non-autoimmune diseases, such as type 1 and 2 diabetes mellitus, hypothyroidism, asthma, psoriasis, and allergy. This result suggests RA patients' vulnerability and may indicate a connection between RA and other autoimmune diseases [41], and 78.3% of women have a family history of RA disorder [42]