Diminished 25‐OH vitamin D3 levels and vitamin D receptor variants are associated with susceptibility to type 2 diabetes with coronary artery diseases

Abstract Background Role of plasma vitamin D and genetic variants of its receptor (VDR) in susceptibility to different diseases has been documented. Various studies in different populations have been highlighted strong associations with diabetes and cardiovascular diseases. Vitamin D deficiency has been linked with the development of type 2 diabetes (T2D) and the onset of coronary artery diseases (CAD). However, the role of vitamin D in predisposition to CAD in patients with T2D is ill‐defined. Materials and Methods We enrolled 674 Chinese T2D patients, and based on clinical phenotype, patients were further categorized into patients with (n = 138) or without coronary artery disease (n = 536). Five hundred twenty‐one healthy subjects from similar geographical areas, free from diabetic or coronary disorders, were enrolled as controls. Serum levels of 25‐OH vitamin D were quantified by ELISA. Common VDR (FokI, TaqI, BsmI, and ApaI) polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). Results Patients with T2D displayed lower levels of 25‐OH vitamin D compared with healthy controls. Furthermore, T2D patients with CAD clinical phenotype had the lowest levels of vitamin D. Prevalence of FokI and TaqI mutants was significantly higher in diabetic patients when compared to controls. Interestingly, Tt genotype was more frequent in the artery disease group in comparison with T2D patients without heart involvement. Combined analysis of VDR polymorphisms and serum levels of vitamin D revealed a significant role in predisposition to T2D with or without CAD. Conclusions Lower vitamin D levels and variants of VDR polymorphisms (FokI and TaqI) are associated with susceptibility to T2D and clinical manifestation.


| INTRODUC TI ON
Diabetes is considered an important health-related complication in humans that results when the pancreas cannot produce a sufficient amount of insulin (Type 1 diabetes) or when the body cannot effectively utilize the insulin which it makes (Type 2 diabetes) (T2D). 1 It is estimated that by 2030, around 552 million people around the world will be diagnosed with T2D indicating the growing gravity of this health problem. 2 Despite such disease burden associated with socioeconomic issues, the pathogenesis of T2D is not yet completely understood. The most vital physiological feature of T2D is insulin resistance, which is characterized by the impaired response to insulin in insulin-sensitive tissues, and β-cell failure resulting in β-cell dysfunction and reduced β-cell mass. 3 Diabetes needs dedicated management by patients as well as health care professionals. An important clinical practice for diabetes is the aggressive management of risk factors to prevent complications that may lead to enhanced mortality. Among several such risk factors, a major one causing mortality in T2D subjects is cardiovascular disease (CVD). CVD in patients with T2D is often found to be more severe and complex and that leads to a higher rate of complications compared to those with non-diabetic phenotypes. 4 About one-fourth of total mortality in patients with T2D has been contributed to CVD clinical phenotype. 5 A multinational study including patients from twenty-eight countries revealed a prevalence of CVD in patients with T2D ranged from 21.6% to 34.2%. 6 Among different CVDs, the incidence of coronary artery diseases (CAD) increases along with age and occurs in younger age population with diabetes. 7,8 In patients with T2D, CAD possesses complexities characterized by small, diffuse, calcified multi-vessel disease. 9 An estimate showed about 75% of subjects with T2D die as a result of related heart diseases including CAD. 10 The human body synthesizes vitamin D with exposure to ultraviolet (UV) B rays, and the importance of vitamin D to maintain good health has been demonstrated elegantly. Vitamin D deficiency is widely prevalent across the globe, and the problem is more severe in elderly patients. 11 Deficient levels of vitamin D are associated with susceptibility to a wide range of diseases, including T2D and CVD. About 70%-90% of patients with T2D displayed deficient or insufficient plasma levels of vitamin D and believed to be associated with different clinical phenotypes of T2D. [12][13][14] An observational study including a more substantial number of women demonstrated an increased risk of T2D in those with lower levels of vitamin D. 15 Diminished vitamin D levels have been linked with impaired glycemic controls. 16,17 Supplementation of vitamin D in patients with T2D has shown promising findings by lowering blood cholesterols 18 and HDL levels. 19 There is growing evidence suggesting an association of lower vitamin D levels with susceptibility to CAD and mortality. 20,21 A recent report highlighted connotation between deficient vitamin D levels and severity of CAD. 22 In addition to vitamin D levels, vitamin D receptors (VDR) polymorphisms are also associated with genetic susceptibility to T2D and CAD patients. Vitamin D is believed to exert its effect through VDR, and genetic variations in the VDR gene lead to dysfunctional vitamin D signaling. Several studies have been executed in different populations to decipher the role of VDR polymorphisms in T2D [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] and CAD. [40][41][42][43][44][45] To the best of our knowledge, studies on the importance of vitamin D or VDR polymorphisms in T2D patients with CAD are lacking. In the present study, we enrolled patients with T2D from the Chinese cohort and investigated the importance of vitamin D and VDR in patients with T2D concerning CAD.

| Study subjects
In the present study, a total of 674 subjects with T2D, visiting outpatient department (OPD) or admitted at the department of peripheral vascular disease, the first affiliated hospital of Xi'an Jiaotong University during the period of March 2015 to May 2019, were enrolled. Patients were considered to have confirmed CAD if they had experienced prior episodes of ST-elevation myocardial infarction or proven to have CAD angiographically. Age-and sex-matched healthy subjects hailing from a similar geographical area without a history of any heart-related anomalies or history of diabetes were enrolled as controls. Participants were divided into three groups, such as healthy controls (Group 1, n = 521), subjects with T2D without CAD (Group 2, n = 536), and subjects with T2D with CAD (Group 3, n = 138).
The staging for diabetes was performed on the basis of fasting blood sugar (FBS), postprandial blood sugar (PPBS), and glycosylated hemoglobin (HbA1c) levels according to American Diabetic Association 46 (FBS > 150 mg/dL, PPBS > 200 mg/dL, HbA1c > 7 mmol/mol). For all patients, standard care was followed for anti-diabetic treatment such as appropriate diet, oral hypoglycemic drugs with or without insulin, and only insulin treatment. Therapeutic intervention for CAD included multifactorial treatment such as the use of statins (95%), aspirin (86%), and blockade of the renin-angiotensin-aldosterone system (98%).
Different biochemical parameters such as triglycerides, total cholesterol, LDL, and HDL of patients and healthy controls were measured in the laboratory facility available in the hospital. Patients were categorized into three different groups based on vitamin D levels as deficient (0-20 ng/mL), insufficient (20-30 ng/mL), and sufficient (>30 ng/mL) according to guidelines of the Institute of Medicine and endocrine society. 47 Informed consent was collected from each participant, and the study protocol was approved by the Institutional Human Ethical Committee of the first affiliated hospital of Xi'an Jiaotong University.

| Collection of serum
About 2 mL of blood (without anticoagulant) was collected intravenously from each patient immediately after the enrollment of patients. Blood was also collected from healthy controls. Serum was separated after centrifuging blood at 2000rpm for 5 minutes and was stored at −20°C till further use.

| Determination of serum vitamin 25-OH vitamin D levels
Total vitamin D [25(OH)D] in sera was estimated using enzymelinked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer's instructions.

| Isolation of whole genomic DNA
About 200 microliters of whole blood were used for isolation of genomic DNA from patients with T2D and healthy controls. The genomic DNA was isolated by QIAamp DNA mini kit (Qiagen) according to manufactures directions.

| Genotyping of VDR polymorphisms
Common polymorphisms in vitamin D receptor (VDR) were genotyped by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP), as described earlier. 48 Briefly, four sets of primers were employed for amplification of DNA fragment containing respective polymorphic site as follows

| Baseline characteristics
The baseline characteristics for patients and healthy controls are shown in Table 1. Out of 674 subjects diagnosed with T2D, 138 patients (20.4%) had CAD. The average age of participants in the three study groups was comparable. No gender disparity among three study groups was noticed. As shown in Table 1, levels of FBS, PPBS, HbA1C (%), systolic blood pressure, diastolic blood pressure, and triglycerides were found to be significantly higher in diabetic subjects when compared to healthy controls. Although no difference was observed in total cholesterol and LDL levels among three study groups, diabetic patients had significantly lower levels of HDL as compared to healthy controls. Additionally, the prevalence of hypertension in patients with T2D was more frequent when compared to healthy controls.
Furthermore, we compared all biochemical parameters among T2D patients with or without CAD and observed significantly higher triglyceride levels in T2D patients with CAD compared to T2D patients without CAD phenotype. Furthermore, hypertension is more frequent in T2D patients with CAD than those without CAD. However, other biochemical parameters remained comparable among these two clinical groups (T2D patients with or without CAD).

| Serum levels of vitamin D in different clinical categories of enrolled subjects
25-OH vitamin D 3 levels were measured in the serum of subjects belonged to the three study groups, and results are shown in Figure 1.

| Variants of VDR polymorphisms are associated with susceptibility to T2D and/or clinical severity
We investigated the possible association of VDR polymorphisms (FokI, TaqI, BsmI, and ApaI) with susceptibility/ resistance against the development of type 2 diabetes. As shown in Table 3

| Association of Combined VDR polymorphisms and serum 25-OH vitamin D with T2D
Vitamin D shows its effect through vitamin D receptor (VDR).
Variants in the VDR gene lead to lower levels of VDR proteins or deformed receptors, which would lead to lowered signaling process. As we observed the significant association of VDR FokI and

| D ISCUSS I ON
The present investigation aimed to examine the association of vitamin D level and its receptor variants with susceptibility of type 2 diabetic and clinical severity in the Chinese population. We observed diminished vitamin D levels in T2D patients with or without CAD when compared to healthy controls. Vitamin D-deficient frequent in patients with T2D and interestingly highly prevalent in T2D patients with CAD clinical phenotype compared with diabetic patients without CAD. Furthermore, with subjects to coronary artery diseases.
Furthermore, TaqI and FokI variants and minor alleles were significantly associated with predisposition to T2D. It is important that the combined analysis of vitamin D levels and VDR polymorphisms revealed their association with the development of T2D with or without CAD manifestations in the Chinese population.
Cholesterols are believed to play a significant role in heart-related disorders and T2D. In the present study, no significant difference was marked in total cholesterol level and LDL among three study groups, the HDL level was found to be markedly lower in diabetic subjects as compared to healthy controls. However, such a difference was not noticed between T2D patients with or without CAD. Earlier epidemiological studies have demonstrated the association of lower HDL cholesterol with increased risk of type 2 diabetes 53,54 and our data were also in line with these observations. Furthermore, we observed higher levels of triglycerides in patients with T2D when compared with healthy controls corroborating with earlier findings 55,56 and triglycerides has been served as a potent marker of type 2 diabetes. 57 Interestingly, the levels of triglyceride were significantly high in T2D patients with CAD compared with diabetic patients without CAD.
Enhanced level of triglycerides has been considered as an independent predictor for CVD, and a fasting triglyceride level of more than 150mg/dl is mostly considered as an accepted criterion for defining individuals at high risk of CVD. 58,59 However, the role of triglycerides as a risk factor for developing CAD in type 2 diabetics is still unclear. 60  presented in many studies. Vitamin D deficiency is related to endothelial dysfunction and enhanced the risk of CVD. 66,67 The deficiency of this micronutrient has also been linked with cardiovascular events such as myocardial infarction, congestive heart failure, and sudden cardiac death. 66,68 Experimental evidence showed the presence of vitamin D receptors in the heart of rats and believed to play major role in intracellular calcium homeostasis. 69  fibrosis in rats. 70 The role of vitamin D in T2D patients with CAD remains conflicting. A report in Italian patients with T2D showed an inverse correlation between lower vitamin D levels and CVD in patients with T2D. 71 In contrast, no significant association of vitamin D levels and severity of coronary atherosclerosis was observed in Polish patients. 72  correlation with susceptibility to T2D. However, the association of FokI was not proved in Tunisian, 28 Caucasian, 24 Brazilian, 31 and Egyptian 25 patients with T2D. The mechanism of how VDR FokI and TaqI variants associated with T2D susceptibility is not known.
Possibly subjects with VDR FokI and TaqI variants suppress vitamin D signaling and predisposed to the development of T2D.
Various studies have been conducted in different populations to understand the possible association of VDR polymorphisms with a predisposition to CAD. Although a recent meta-analysis showed the absence of association between VDR polymorphisms (FokI, TaqI, BsmI, and ApaI) with susceptibility to CAD, reports from Iranian, 43 Egyptian, 43 and Chinese 41 cohort showed a significant association of FokI variant with susceptibility to CAD. Our study was also corroborated with earlier observations: variants of FokI and TaqI were significantly higher in T2D patients with CAD phenotype. Interestingly, TaqI heterozygous mutant was significantly higher in T2D patients with CAD phenotype when compared to those without CAD. These observations altogether suggesting the importance of VDR polymorphisms on predisposition to CAD in patients with T2D.
Nowadays, combined analysis of vitamin D and VDR polymorphisms has been the most accepted form of association investigation on various diseases. In the present study, we observed heterozygotes or homozygous mutants of FokI and TaqI polymorphism and insufficient or deficient vitamin D levels with a predisposition to T2D development. It is important that combined genotype and serum vitamin D levels for FokI (Ff/deficient) and TaqI polymorphism (Tt/ insufficient, TT/deficient and Tt/deficient) were significantly more prevalent higher in T2D patients with CAD phenotype compared to those without CAD, indicating an important role of combined vitamin D and VDR variants with development of CAD in patients with T2D. Although the exact mechanism of how these genotypes phenotypes combination associated with CAD predisposition in patients with T2D is not known, possibly lower vitamin D and variants in VDR significantly altered vitamin D signaling process could lead to heart-related deformities.
The present study has several limitations, and those need to be disclosed. Firstly, we have considered only four SNPs in the VDR gene, and other polymorphisms in the VDR gene are not included.
Secondly, the number of samples included in the current investigation is limited. Thirdly, the mechanism of how VDR variants diminished vitamin D signaling is not investigated in this study.
In conclusion, lower levels of vitamin D and genetic variants of the VDR gene (FokI and TaqI) are associated with susceptibility to T2D and CAD. Furthermore, the combined analysis revealed a significant role of both serum vitamin D and VDR polymorphism on predisposition to patients with T2D or diabetic with CAD phenotype.
However, more studies in other population including more samples are required for validation of our findings.