Relationship between vitamin D deficiency, vitamin D receptor gene variants, & the risk of coronary artery disease among South Indians: A case-control study

Materials & Methods

This case-control study conducted between March 2020 and December 2021 was conducted by the department of Pathology, Sri Jayadeva Institute of Cardiovascular Sciences and Research (SJICR), Bengaluru, Karnataka, India, after obtaining clearance from the Institutional Medical Ethics Committee. Written informed consent for using samples for research was obtained from all the individuals before sampling. A detailed proforma with demographic variables and clinical details was used to document data for all study participants. Total 250 CAD patients who presented at SJICR, Bengaluru, were recruited in this study. Patients above 18 yr of age, with a clinical diagnosis of CAD with echocardiographic and ECG evidence, undergoing coronary angiography (CAG) were included in the study. The study population comprised both diabetic and non-diabetic patients. Terminally ill patients, those with hepatic/renal failures/malignancy, patients reactive to HIV, HBsAg, HCV & VDRL, and those on vitamin D or calcium supplements were excluded. The control group (n=260) was recruited from healthy volunteers who were attending to the hospital patients and blood donors during the study period. Individuals with any history of CVDs and those taking calcium or vitamin D supplements were excluded.

Results

Correlation of vitamin D levels and CAD risk factors

Vitamin D plays a crucial role in regulating blood pressure and blood glucose levels, which are significant risk factors for CAD¹³. Hence, we did a correlation analysis of vitamin D levels and CAD risk factors (blood pressure and blood glucose) for cases and controls. However, our analysis did not reveal any significant correlation between vitamin D levels and either blood pressure or blood glucose levels in cases and controls (Fig. 1A-C for cases; Fig. 2A-C for controls).

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Fig. 1.

The image presents three scatter plots (A, B, and C) showing the relationships between vitamin D levels (ng/ml) and CAD risk factors: Systolic blood pressure (Plot A), Diastolic blood pressure (Plot B), and Blood glucose levels (Plot C). The correlation coefficients (r) and P values for each relationship are provided. A P value <0.05 is considered significant.

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Fig. 2.

The image presents six scatter plots (A to F) showing the relationship between vitamin D levels (ng/ml) and various blood lipid parameters: Total Cholesterol (Plot A), Triglycerides (Plot B), VLDL (Plot C), HDL (Plot D), LDL (Plot E), and the TC/HDL ratio (Plot F). The correlation coefficients (r) and P values for each relationship are provided. A P value <0.05 is considered significant.

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Linkage disequilibrium analysis of VDR variants

The comparison of linkage disequilibrium (LD)patterns between cases and controls revealed a distinct difference in the genetic correlations among the four SNPs analysed (TaqI, BsmI, ApaI, and FokI) within a 34 kb genomic block. The case group demonstrated a higher LD between ApaI (rs7975232) and FokI (rs2228570), r² = 92 and between BsmI (rs1544410) and ApaI, r² = 87. Moderate LD is also observed between TaqI (rs731236) and ApaI (r² = 47) and between TaqI and BsmI (r² = 41). The deep red colour in the LD plot for cases underlines the strong linkage among certain SNP pairs, indicating the existence of a tightly linked haplotype in affected individuals (Fig. 3A). In contrast, the control group exhibited markedly lower LD values across most SNP pairs, with the strongest LD observed between ApaI and FokI (r² = 54), which is reduced compared to the case group. The values of LD between other pairs, such as TaqI and BsmI or TaqI and ApaI, are minimal and between r²=1 to 4. The overall light intensity of the control LD plot reflects that the genetic correlations are relatively weak, which suggests a lack of closely linked haplotypes in non-affected individuals (Fig. 3B).

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Fig. 3.

Linkage disequilibrium (LD) plot: ‘A’ represents the LD pattern observed in cases; ‘B’ represents LD pattern observed in controls. The deep red colour in the LD plot underlines the strong linkage, whereas the light intensity of the LD plot reflects the weak linkage.

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Discussion

This is a case-control study carried out on CAD patients in a tertiary cardiac centre in Southern India. We studied the prevalence and association between VDD and VDR gene variants with various risk factors for CAD and its severity.

Although both controls and cases had VDD in our study, the mean vitamin D level was significantly lower in patients. Also, 90 per cent of patients were deficient compared to 63 per cent of controls. We also found that VDD was associated with 5.7-fold increased risk of CAD. These findings suggest a strong association between VDD and CAD risk in our study group. In a meta-analysis of 44,717 participants across 65 studies from five South Asian countries, including India, Pakistan, Bangladesh, Nepal, and Sri Lanka, the prevalence of VDD was 67 per cent among Indians. The average level of vitamin D ranged from 4.7 to 32 ng/ml, with a weighted mean of 19.15 ng/ml¹⁵. The meta-analysis by Yan et al⁹, which included 13studies from China, Iran, Brazil, Egypt, Poland, Croatia, and Germany, concluded that low plasma vitamin D levels are associated with CAD⁹. A study from the Northern part of India on first incident acute myocardial infarction reported a higher prevalence of VDD and insufficiency and much lower mean values of vitamin D (98.3% of the cases had below normal values with a mean level of 6 ng/ml; 95.8% of controls had below normal values with a mean of 11.1 ng/ml). A 4.5-fold risk of myocardial infarction was also noted among individuals with severe (<10 ng/ml) VDD¹⁶. Collectively, these findings suggest that low vitamin D levels may be a significant risk factor for CAD, particularly in populations with a high prevalence of VDD. Our partial correlation analysis indicated that differences in vitamin D levels between cases and controls remain significant even after adjusting for key confounders, underscoring the potential role of vitamin D deficiency in CAD. Vitamin D plays a crucial role in regulating blood pressure and blood glucose levels, which are significant risk factors for CAD^17,18. Our correlation analysis suggests that there are weak negative correlations between vitamin D levels and CAD risk factors, but none of these relationships are statistically significant. In a previous study from India, higher levels of vitamin D have been associated with lower incidences of CAD⁸, but our findings do not support a strong correlation between vitamin D levels and CAD risk factors. VDD can affect lipid profiles by influencing low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, and total cholesterol levels in CAD patients^19,20. Additionally, VDD may impact blood sugar levels and the TC/HDL ratio²¹. Consistent with the previous findings our analysis revealed a trend for a negative correlation of vitamin D levels with cholesterol and triglyceride levels. However, other variables did not correlate. These findings suggest that maintaining adequate levels of vitamin D may have a positive impact on lipid profiles in CAD patients.

There are conflicting findings among various studies on VDR variants and their association with CAD^22,23. In the present study, the allelic frequency of the TaqI variant was significantly different between cases and controls. Also, a significant difference was found in the allelic distribution of the FokI variant in males with VDD compared to controls. Under the dominant inheritance model (Tt + tt vs TT), the TaqI variant suggested a potential protective association with an odds ratio of 0.6. This association remained significant even after adjusting the odds ratio for age, smoking, and alcohol. Upon gender stratification, the protective association persisted significantly in both males and females, even after adjusting for age, smoking, and alcohol. These findings suggest that the protective association of the ‘t’ allele against CAD is consistent across genders and remains significant after controlling for potential confounders.

The presence of the ‘ff’ genotype of Fok I conferred a 5.9-fold risk of CAD in our cohort of males. In contrast, a previous study on 40 South Indian CAD subjects did not show any significant association of CAD with the FokI variant²⁴. This discrepancy in the results may be due to differences in sample size, genetic heterogeneity within the population, or variations in environmental factors that were not accounted for in either study. Hence, it is important to consider other genetic variations that may contribute to the development of CAD. Also, a case-control study from China did not find any significant association of FokI (rs2228570) and BsmI (rs1544410) with CAD²⁵.A recent meta-analysis⁹ evaluated thirteen studies using trial sequential analysis (TSA) for the association of four common VDR polymorphisms for CAD susceptibility. In their analysis, on stratification by race, the White people were found to have an increased CAD risk in BsmI (‘b’ allele), FokI (‘f’allele), and TaqI (t allele) polymorphisms and a protective association of ‘a’ allele of ApaI; Asians exhibited only 60 per cent risk in ‘ff’ genotype of FokI and no association with TaqI and BsmI polymorphisms⁹.The present study correlates with these findings except for TaqI polymorphism, which has shown a protective association in our study. A meta-analysis of 158 studies has reported an increased risk of CAD in patients with TaqI polymorphism and also suggested a possible protective role of FokI polymorphism²⁶. Their observations are in total contrast to our study findings. Overall, our findings suggest that the VDR TaqI variant may play a role in reducing the risk of CAD, regardless of gender. However, the VDR FokI variant conferred a risk of CAD in our cohort of males. These findings indicate that genetic variations in the VDR gene may have differing effects on CAD risk based on gender.

Overall, the analysis of VDR gene variants revealed distinct associations with CAD under specific conditions. The TaqI variant demonstrated a potential protective effect under the dominant inheritance model, with the association remaining significant even after adjusting for age, smoking, and alcohol. Gender stratification showed that the protective effect persisted in both males and females, even after accounting for these covariates. In contrast, the BsmI variant showed no significant association with CAD, irrespective of gender or covariate adjustments. Similarly, the ApaI variant did not exhibit any association with CAD across various models of analysis, including gender stratification. On the other hand, the FokI variant displayed a significant association in the recessive model, where the ‘f’ allele notably increased the risk of CAD, particularly in males, with a marked 5.9-fold higher risk even after adjusting for vascular risk factors. This comparison highlights the differing roles of VDR gene variants in CAD susceptibility and the influence of gender and other covariates on these associations.

Then, we were interested in knowing whether TaqI and FokI variants maintain their protective and risk effects under the context of VDD. A measure of the odds ratio was calculated to assess the combined effects of VDD and VDR gene variants FokI and TaqI. We found that neither FokI ‘ff’ nor the TaqI ‘tt’ genotype significantly alters the risk in the context of VDD entirely. The lack of significance suggests that multiple factors likely contribute to the outcome. Firstly, there may be a threshold effect for vitamin D levels below which the impact of VDR gene variants becomes negligible. Secondly, if VDD is severe enough, the relative contribution of the VDR variants might be overshadowed by the overall deficiency. Biological systems often have compensatory mechanisms that can mitigate the effects of genetic variations. For example, individuals with ‘ff’ or ‘tt’ genotypes might have other compensatory pathways that help maintain some level of VDR activity or vitamin D metabolism.

The comparison of LD patterns between cases and controls revealed a distinct difference in the genetic correlations among the four SNPs analysed (TaqI, BsmI, ApaI, and FokI). These differences in LD patterns suggest that certain SNPs, particularly ApaI and FokI, may be associated with the cases. In contrast, the weaker LD in controls implies a more random distribution of alleles. The observed patterns warrant further investigation to explore the functional roles of these SNPs and their contribution to disease susceptibility.

Although our study focused on VDR variants, we recognise the importance of other genes involved in vitamin D metabolism, such as CYP27B1, which encodes the enzyme responsible for converting 25-hydroxyvitamin D into its active form, 1,25-dihydroxyvitamin D (calcitriol)²⁷. Variants in CYP27B1 can lead to reduced activity of this enzyme, impairing the activation of vitamin D even when precursor levels are normal²⁸. Loss-of-function mutations in CYP27B1 have been associated with conditions such as vitamin D-dependent rickets type 1 (VDDR1), characterised by bone deformities and growth retardation due to insufficient calcitriol levels^29,30. Including the analysis of CYP27B1 variants in future studies could provide a more comprehensive understanding of vitamin D metabolism and its role in CAD, particularly when VDR function is normal but calcitriol synthesis may be disrupted.

Some limitations of this study were that it did not comprehensively account for environmental and dietary factors, such as diet, sun exposure, and lifestyle, which can significantly influence vitamin D levels and CAD risk. Furthermore, the role of other genetic factors in conjunction with VDR variants, which might provide a more comprehensive understanding of CAD development and progression were not explored.

In conclusion, while our study provides valuable insights into the association between VDR gene variants and CAD risk, more research is necessary to fully comprehend the underlying mechanisms and potential treatment options.