VDR Gene Polymorphism and Rheumatoid Arthritis in Egyptian patients

 

Samar El-Sayed Mahmoud1*, Abeer M. El-Shafey2, A.T. Keshta1

1Biochemistry Division, Chemistry Department, Faculty of Science, Zagazig University.

2Rheumatology and Rehabilitation Department, Faculty of Medicine, Zagazig University.

*Corresponding Author E-mail: samaranas28@yahoo.com

 

ABSTRACT:

Background and objective: Rheumatoid arthritis (RA) is a systemic autoimmune multifactorial complicated disease that is related to widespread morbidity and mortality. The current study aimed to study the relation between vitamin D receptor (VDR) gene polymorphism rs731236 (TaqI) and rs7975232 (ApaI) and RA susceptibility. Subjects, Materials and Methods: This study included 50 individuals were divided into two groups: Healthy volunteers group (10) and RA group (40) from Rheumatology and Rehabilitation Department, Faculty of Medicine, Zagazig university hospitals. DNA was extracted from whole blood for all samples, and PCR was amplified using specific primers rs 731236 (TaqI) and rs 7975232 (ApaI) VDR restriction digestion was carried out for TaqI and ApaI genotyping, respectively. Results: There were non-significant difference in VDR gene polymorphism between RA patients & control subjects. Conclusion: TaqI is more specific than ApaI but Taq1 and Apa1 gene polymorphism not used as a diagnostic marker for RA disease, so there is no relation between VDR and RA.

 

KEYWORDS: Rheumatoid arthritis, Gene polymorphism, VDR, TaqI, ApaI.

 

 


INTRODUCTION:

Rheumatoid arthritis (RA) causes symptoms that range from numbness and tingling to paralysis and sudden death. It result from joint damage that RA causes the disease process itself, or medications that treat it. Blood vessels: RA can cause inflammation of your blood vessels. It shows up as spots on the skin that look like ulcers. It characterized via way of means of joint swelling, with synovitis taking place symmetrically in small joints1. Joint swelling is a reflection of synovial membrane inflammation following immune activation.

 

The normal synovial compartment is infiltrated with the aid of using leukocytes and the synovial fluid is inundated with pro-inflammatory mediators that have interaction to provide an inflammatory cascade in cells with the cells of the innate immunity, inclusive of monocytes, macrophages, and adaptive immune cells as T-lymphocytes and B-lymphocytes. Monocytes/ macrophages have been found to massively infiltrate synovial membranes and are central to the pathophysiology of inflammation2-3-4.

 

RA is consider a major Cause of disability and premature mortality5-6, as common in women than men 7. Various genetic, hormonal, immunological and environmental factors implicated as factors in the evolution of RA8-9 . Vitamin D is the fat soluble vitamin. It is obtained by endogenously through in skin induced by ultraviolet radiation from sun light and exogenously through dietary sources which includes of milk products, oily fishes, cod liver oil and egg yolk all are rich sources for vitamin D10-11. The starring function of vitamin D carries out the activities through vitamin D receptor (VDR) Taq1 rs731236 and Apa1 rs 7975232 which known to be associated with autoimmune disease including RA, systemic lupus erythematous, Addison's disease and type 1diabetes12. Vitamin D has a crucial function in modulating immune function that is supported of VDRs in peripheral mononuclear blood cells. VDR gene encoded for VDR is positioned on chromosome 12 and its polymorphism is detected by the presence or absence of a restriction site13-14. Numerous variations on VDR were identified in patients of multiple disorders as RA, where in VDR is not able to bind to 1, 25-OH-D, as VDR gene polymorphism results in disorder of 1, 25(OH) 2D3 ultimately disease onset15-16.

 

 

The purpose of the current study is assessment of two type of VDR gene polymorphism (TaqI rs731236 and ApaI rs7975232) in RA patients, as well as determination possible association between VDR gene polymorphism and RA susceptibility in Egyptian population.

 

MATERIAL AND METHODS:

Subjects:

This study included 50 adult individuals, 43 femals and 7 males, age ranged from (40-70 years). These samples were divided into two groups: Group (I): Healthy control group with no evidence of any rheumatologic disease or chronic medical illness. Group (II): Rheumatoid arthritis (RA) patients were selected from Rheumatology and Rehabilitation Department, Faculty of Medicine, Zagazig University Hospitals. The study was conducted according to the guidelines of the Faculty of Medicine, Zagazig University.

 

Sampling: 2ml whole blood collected in EDTA-lined tubes For DNA extraction.

 

Chemical and reagents:

DNA purification kit purched from QIAGEN (Cat No 51104).

 

Primers from Willofort Manf.ID 223090245, 223090246, 223090247 and 223090248.Purchase order 3060734.

 

Methods:

VDR gene polymorphism was determined according to method of Kilic17. Extracted DNA from each sample was stored at - 20C for further genetic analysis. Two polymorphic sites rs731236 and rs7975232 were selected. The targeted DNA fragment was amplified for PCR in a 10l reaction mixture by using following primers: for rs7975232 polymorphic site "Forward primer (F.P): (5'-CAG AGC ATG GAC AGG GAG CAA G-3') and Reverse primer (R.P): (5'-GCA ACT CCT GGC TGA GGT CTC A-3') and for rs731236 Polymorphic site (F.P): (5'-CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA-3') and (R.P): (5'CAC TTC GAG CAC AAG GGG CGT TAG C-3'). The PCR reaction included for rs7975232 ApaI an initial denaturation at 95C for 5 minute, followed by 35 cycles at 95C for 20 seconds, primer annealing at 57C for 40 seconds and extension at 72C for 45 seconds, a final extension at 72C for 5 minutes. And for rs731236 TaqI an initial denaturation at 95C for 2 minutes, followed by 30 cycles of denaturation at 96C for 30 seconds, primer annealing at 56C for 59 seconds and extension at 72C for 135 seconds, a final extension at 72C for 5 minutes.

 

VDR genotyping for rs731236 in exon 9 and rs7975232 in intron 8 was performed by direct sequencing method 18and restriction fragment length polymorphism (PCR-RFLP) 19. The direct sequencing confirmed the primer sequence.

 

The PCR products were digested using restriction enzymes: TaqI (rs731236) and ApaI (rs7975232). Briefly, a reaction mixture of 10l containing 1 l buffer, 1 l of restriction enzyme, 5l PCR product and 3 l water. The tubes were incubated at 37C overnight for ApaI and 65C for 15 minutes for TaqI. Digested samples were run on 1.5% agarose gel and visualized on the gel documentation system (Bio Doc.Imagining system).

 

Statistical Analysis:

Data was statistically analyzed with SPSS software (SPSS Inc.) version 20.0. Variables were expressed as mean standard deviation (SD). Categorical variables were analyzed using the Chi-square test or Fisher's exact test when appropriate and expressed as numbers (%). Differences between variables were analyzed using analysis of variance for two groups. A two- sided P value. The diagnostic performances of markers were determined using area under receiver operating characteristic (AUC). The optimal cut-off points have been determined, and then the corresponding sensitivity and specificity had been stated20.

 

RESULTS:

The distribution of genotypes for genes TaqI and ApaI in patients and healthy controls were tested for deviation. Differences in genotypic and allelic distribution between patients and healthy controls and polymorphism distribution in patients were determined by Chi-square or Fisher exact test.

 

Figure 1 Genetics patterns of TaqI polymorphism Lane 1, 4, 5,7,12 and 13 is heterozygous mutant (Tt): 2000, 1800 and 200bp. Lane 2, 6, 14, 17 and 18 is homozygous mutant (tt): 1800 and 200bp. Lane 3, 8, 9, 10, 11, 15, 16 and 19 is homozygous mutant (TT): 2000bp.As, the PCR product size before digestion was 2000 and after digestion was 1800 and 200 bp.

 

Figure 1: Genetics patterns of TaqI polymorphism on the electrode gel with staining of ethidium bromide and UV light.

Figure 2 Genetics patterns of ApaI polymorphism Lane 2, 4, 5 and 10 is heterozygous mutant (Aa): 740, 530 and 210 bp. Lane 9, 11 and 12 is homozygous mutant (aa): 530 and 210 bp. Lane 1, 3, 6, 7 and 8 is homozygous mutant (AA): 740 bp. Where the PCR product size before digestion is 740 and after digestion is 530 and 120 bp.

 

 

Figure 2: Genetics patterns of ApaI polymorphism on the electrode gel with staining of ethidium bromide and UV light.

 

Genotyping frequencies for TaqI and ApaI polymorphism showed in Table 1 and Table 2.

 

Table 1 illustrated Genotyping frequencies for TaqI. The genotyping frequency t/t increased in patients as compared to controls, while genotyping frequencies T/t and T/T were decreased in patients compared to control, but these differences were not significant, (p = 0.297).

 

Table 1: Genotype frequencies of TaqI among control and patients:

 

TaqI

P value

 

tt

Tt

TT

 

Control

No

1

3

5

0.297

%

11.1%

33.3%

55.6%

Patients

No

10

5

11

%

38.5%

19.2%

42.3%

 

Table 2 illustrated Genotyping frequencies for ApaI. The genotyping frequency a/a, A/a, and A/A were decreased in patients as compared to controls, but these differences were not significant, (p = 0.74).

 

Table 2: Genotype frequencies of ApaI among control and patient.

 

 

ApaI

P value

aa

Aa

AA

Control

No

1

2

5

0.74

 

%

12.5%

25.0%

62.5%

Patients

No

8

10

17

 

%

22.9%

28.6%

48.6%

 

 

The genotyping frequencies for TaqI and ApaI polymorphism between the two groups as there were no significant difference in genotype frequency between control and patients groups (p=0.297, p=0.74) respectively.

ROC curve of TaqI gene and ApaI gene in two groups were summarized Table 3 and figure 3, 4.

 

Area under ROC curve (AUC) for TaqI was (0.620) and for ApaI was (0.580) the sensitivity was plotted as a function of 1-specificity. Each point on the ROC plot represents a sensitivity / specificity pair corresponding to a particular decision threshold. An AUC of 1.0 is characteristic of an ideal test, whereas an AUC of 0.5 or less indicates a test of no diagnostic value.

 

Table 3: ROC curve of TaqI gene and ApaI gene in two groups

Area Under the Curve

Test Result Variable(s)

Area

Std. Errora

Asymptotic Sig.b

Asymptotic 95% Confidence Interval

Lower Bound

Upper Bound

TaqI gene

0.620

0.101

0.291

0.421

0.818

ApaI gene

0.580

0.109

0.483

0.367

0.794

a. Under the nonparametric assumption

b. Null hypothesis: true area = 0.5

 

 

Figure 3: Diagram shows the sensitivity and specificity for TaqI to diagnose RA.

 

Figure 4: Diagram shows the sensitivity and specificity for ApaI to diagnose RA.

 

DISCUSSION:

RA is a chronic, inflammatory and auto immune disease that has affected an approximate of 1-2% of adult population worldwide21-24 in which environmental and genetic factors can play a major role. Several genes have been indicated in the pathogenesis of RA25, with 50% of RA risk attributable to genetic factors26. Vitamin D cause specific changes in cell and organ functions and thus lead to risk for chronic disease of different etiology.27 As vitamin D consider natural immunoenhancers28, there was a close relationship between VDR gene polymorphism and the immunological action. The active form of 1, 25 Dihydroxy vitamin D3 (1, 25(OH) 2D3, calcitriol) plays an important role in activating monocytes, stimulating cell mediated immunity and suppressing lymphocyte proliferation29-30. It restrains the suppression of T lymphocytes and the production of cytokines31. Also, numerous actions on dendritic, T/B cells leading to the reduction the production of numerous cytokines (TNF-, IL-17, IL-6, and IL-1, and the inhibition of IL-2 and interferon) that have crucial in etiopathogenesis of RA 32. Vitamin D mediates its function by binding to the vitamin D receptor (VDR), which is encoded by the VDR gene a member of nuclear hormone receptors superfamily detected in various immune cell types that is also present on chondrocytes and synovial cells in RA affected joints33.

 

Current study confirmed that VDR gene polymorphism ApaI (rs7975232) and TaqI (rs731236) aren't involved in RA susceptibility. This may be due to ethnic variation such as climate where North Africa differs from Europe in sunny climate. VDR gene polymorphism differs by geographical altitude. Various studies also reported that, there was no significant association for TaqI and ApaI gene polymorphism with RA risk34. The present study was a line to study by Maalej et al.,35 who conducted on French population did not show any significant result for TaqI gene polymorphism with RA. Also, ATEŞ et al.,36 carried out the study on Turkish population suggested that vitamin D receptor polymorphisms do not play a role in either RA susceptibility. Also Tizaoui et al.,34 was agreed with this present study as showed that no significant association was observed for VDR ApaI and TaqI polymorphism with RA risk (P> 0.05). Meanwhile, another studies demonstrated risk factor of TaqI and ApaI gene polymorphism with RA disease such as study by Mossad et al.,37 confirmed that ApaI, BsmI, TaqI polymorphism may be a susceptibility risk factor for RA and by Wang et al.,38 showed that polymorphism of TaqI, BsmI, FokI not ApaI in VDR gene might be involved in the development of RA and Atoum et al., 39 showed that rs (731236) TaqI VDR tt genotype may be possible risk factors for RA.

 

CONCLUSION:

TaqI is more specific than ApaI but Taq1 and Apa1 gene polymorphism not used as a diagnostic marker for RA disease, so there is no relation between VDR and RA. The study recommended further studies on RA disease with other gene polymorphism.

 

ACKNOWLEDGMENT:

Authors are thankful to all patients and control volunteers, Scientific and Research Center Molecular Biology Unit, Faculty of Medicine, Zagazig University.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 09.06.2021 Modified on 11.07.2021

Accepted on 02.08.2021 RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(3):1128-1132.

DOI: 10.52711/0974-360X.2022.00189