Association of VDR Gene Variant (rs1544410) with Type 2 Diabetes in a Pakistani Cohort.

The present study was designed to measure the mean values of body mass index (BMI), random blood sugar/ fast ing blood sugar (RBS/FBS) tests, and Hb A1c and to investigate the role of a genetic variant rs1544410 in the VDR gene in a Pakistani cohort. For this purpose, a total of 917 samples including 469 diabetes mellitus type 2 (T2DM), 145 DM type 1 (T1DM), and 303 healthy control were collected. Out of the total sample set, 500 individuals (250 T2DM cases and 250 controls) were genotyped for rs1544410. It was found that 65 (26.0%) cases and 32 (12.8%) controls had homozygous AA, while 69 (27.6%) cases and 139 (55.6%) controls had heterozygous AG, and 116 (46.4%) cases and 79 (31.6%) controls had homozygous GG (χ2 = 41.81, p = 0.0001). In addition, a similar distribution of allele frequency was determined in cases and controls [p value = 0.866; odds ratio (OR) = 0.967; relative risk (RR) = 1.034]. A significant difference was observed in homozygous dominant [OR = 2.394 (1.501-3.816); RR = 1.46 (1.225-1.740); p = 0.003] and homozygous recessive models [OR = 2.970 (2.086-4.227); RR = 1.798 (1.501-2.154); p = <0.0001] analysis of rs1544410 in the VDR gene. These findings suggest that the VDR gene is associated with T2DM and genotype GG of genetic variant rs1544410 is the susceptible genotype in our Pakistani cohort.

Introduction

Diabetes mellitus type 2 (T2DM) is a multifactorial metabolic disorder, regulated by both genetic and environmental factors [1]. It is a chronic disease that is associated with the incapability of tissues such as liver and skeletal muscles to respond to insulin. Several genetic, as well as environmental factors, contribute to the etiology of T2DM [1]. One of them is vitamin D. Vitamin D deficiency appears to be related to the development of T2DM and metabolic syndrome [2]. Recent studies have shown that high vitamin D status offers protection against T2DM. Vitamin D has important biological functions including modifying insulin secretion and refining insulin resistance [3]. It is a hormone precursor and it has been found to be associated with various metabolic disorders [4]. Its deficiency results in reduced insulin secretion in humans. The frequency of T2DM is exponentially increasing at high rate globally [5]. The survey conducted by the National Diabetes Survey of Pakistan (NDSP 2016-2017) showed that the incidence of diabetes is about 26.3% in Pakistan [6]. It is already known that vitamin D deficiency is linked with glucose intolerance, insulin resistance, metabolic syndrome, and increases the risk for diabetes [7]. Vitamin D receptor (VDR) is a steroid family member that acts as a transcriptional activator of several genes [8]. The VDR primarily acts as a transcription factor. The VDR gene on situated on chromosome 12q12-14. It consists of eight protein-coding exons (exons 2 to 9) and six untranslated exons that spliced alternatively [9]. It has been investigated that polymorphisms in the VDR gene are associated with diabetes susceptibility. Previous studies have shown the connection of VDR polymorphism with T2DM, however, their frequency remained different across a diverse population [10].

Several techniques are being developed for the identification of single nucleotide polymorphisms (SNPs) but most of them are either expensive or have low sensitivity. Allele-specific polymerase chain reaction (PCR) is one of the less expensive and highly sensitive techniques widely used for the detection for the SNP in several species [11]. The aim of the current study was to detect the association between the rs1544410 polymorphism of the VDR gene and T2DM in Pakistani patients using allele-specific PCR.

Materials and Methods

Study Subjects. A total of 917 samples, which included 614 diabetes mellitus (DM) patients [469 T2DM and 145 DM type 1 (T1DM)] and 303 control samples’ data were collected from different hospitals of the District Swat, Khyber Pakhtunkhwa Province, Pakistan. A questionnaire was designed, and the patients were visited at hospitals to record various information such as random blood sugar/fasting blood sugar (RBS/FBS) tests, body mass index (BMI), age, family history and associated disease of these patients. Study participants were asked to sign a consent form provided in the questionnaire and approval was obtained from the ethics committee of the Department of Biotechnology, Abdul Wali Khan University Mardan, Marden, Khyber Pakhtunkhwa Province, Pakistan. All procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethics standards. Clinical profiling of the patients was carried out by performing different clinical assessments and diagnostic tests as given below.

Fasting Blood Sugar (FBS) and Random Blood Sugar (RBS) Tests. Blood samples were collected from patients in EDTA-containing vacutainer tubes and processed for RBS and FBS tests. The blood samples were centrifuged for 1 min. at 4000 rpm to extract serum. Then 10 μL serum was mixed with1000 mL glucose reagents. These test tubes were then left in a water bath for 5 min. After incubation, the mixture was put in the Microlab 330 machine (TLITech Group, Puteaux, France) to record the reading. Individuals with RBS values ranging from 70-170 and FBS value ranging from 7-115, were considered normal, while readings higher than 170 for RBS and higher than 115 for FBS, were considered as abnormal and diabetic. The remaining blood samples were stored at –20 °C until needed for further analysis.

Hb A. In this test, 100 μL Hb A1c-buffer was mixed with 5 μL blood sample in a test tube. The tubes were placed in a water bath for 12 min. These tubes were placed in the Ichroma™ machine (BodiTech Med, Inc., Chuncheon-si, Gangwond-do, Korea) to record the reading.

Body Mass Index. The BMI of these patients was calculated from their body weight and height using a universal formula (BMI = weight/height). The normal BMI value ranges from 19-25. The BMI value higher than this range was considered to be obese.

Glucose Tolerance Test (GTT). The GTT was done by measuring the FBS for each patient after 60, 90 and 120 min. Similarly, the sugar level in the urine of each patient was measured (Table 1).

Table 1

Upper and lower ranges and mean values of different variables in cases and controls.

Category	Age in Years (mean/range)	BMI (kg/m2) (mean/range)	RBS/FBS (mg/dL) (mean/range)	Hb A1c (%) (age mean)
T1DM (males)	40.0 (5-67)	22.3 (12.7-32.2)	196.7 (122.0-384.0)	9.4 (6.8-15.5)
T1DM (females)	43.1 (15-62)	24.6 (16.0-37.2)	219.1 (8161.0-402.0)	9.6 (7.3-12.1)
T2DM (males)	57.4 (32.0-100.0)	23.4 (13.5-39.8)	245.0 (135-510.0)	9.6 (6.7-13.7)
T2DM (females)	5.5 (29.0-91.0)	22.1 (12.3-37.3)	?269.7 (144.0-510.0)	9.8 (6.9-12.7)
Controls	44.0 (10.0-100.0)	21.2 (14.5-33.7)	112.3 (70.0-168.0)	–

BMI: body mass index; RBS: random blood sugar; FBS: fasting blood sugar.

Genomic DNA Extraction and Primer Designing. Genomic DNA of 250 T2DM patients and 250 control individuals were extraverted using the organic phenol-chloroform method. After extraction, DNA was quantified and then stored at –20 °C for future experiments. Genomic DNA sequence was retrieved from the National Center for Biotechnology Information (NCBI), and primers for allele-specific PCR were designed using online bioinformatics tools.

Genotyping of the . For genotyping of the VDR gene variant (rs1544410), an allele-specific PCR technique was used for both case and control samples. Two forward primers, each specific to a particular allele (VDR-F1:5’-GCC ACA GAC AGG CCT GCA-3’) VDR-F2: 5’-GCC ACA GAC AGG CCT GCG-3’) and one common reverse primer (VDR-R: 5’-GTC ACT GCA CAT TGC CTC CAA-3’) was used for genotyping of VDR in the selected samples. The amplified PCR products were run on a 2.0% agarose gel and the data was noted for each allele.

Results

Prevalence of Study Subjects. Clinical and demographic data such as family history, gender, and ages of the patients, associated diseases and Hb A1c levels of 917 study subjects (459 T2DM, 145 T1DM, 303 controls) were analyzed. The upper and lower ranges of these variables along with the mean/average were calculated at gender level. In male T1DM patients, the mean age was 40 years (5-57), mean BMI was 22.3 (12.7-32.2), mean FBS/RBS was 196.7 (122-384) and Hb A1c was 9.4 (6.8-15.5). Whereas in female T1DM patients, the mean age was 43.1 years (15-62), mean BMI was 24.6 (16-37.2), RBS/FBS was 219.8 (161-402) and HbA1c was 9.6 (7.3-12.7). In male T2DM patients, the mean age was 57.4 years (32-100), mean BMI was 23.4 (13.5-39.8), mean FBS/RBS was 245 (135-510) and Hb A1c mean was 9.6 (6.7-13.7). Similarly, in female T2DM patients, the mean age was 55.4 (29-91), mean BMI was 22.1 (12.3-37.3), mean FBS/RBS was 269.7 (144-510) and Hb A1c was 9.8 (6.9-12.7). In the control samples, the mean age was 44 years (10-100), the mean BMI was 21.2 (14.5-33.7) and the mean FBS/RBS was 112.3 (70-168) (Table 1).

Association of . A genetic variant (rs1544410) in the VDR gene was mapped in 250 T2DM patients and 250 controls, as shown in Figures 1 (A) and (B). The frequencies of genotype and allelic distribution and the effect of homozygous dominant and recessive models were determined in cases vs. controls. It was found that 65 (26.0%) cases and 32 (12.8%) controls had homozygous AA, while 69 (27.6%) cases and 139 (55.6%) controls had heterozygous AG, and 116 (46.4%) cases and 79 (31.6%) controls had homozygous GG (χ2 = 41.81, p = 0.0001). However, through allele frequency distribution analyses, we determined that 199 (39.8%) cases and 203 (40.6%) control individuals had allele A and 301 (60.2%) cases and 297 (59.4%) control individuals had allele G [OR = 0.967 (0.751-1.246); RR= 0.984 (0.866-1.116); p value = 0.846] (Table 2). The effect of the major allele on the association of VDR with T2DM was checked through homozygous dominant model analysis. Homozygous AA was found in 65 (26.0%) cases and 32 (12.8%) in controls. Similarly, AG+GG was found 185 (74.0%) and 218 (87.20%) in cases and controls, respectively (OR = 2.394 (1.501-3.816); RR = 1.46 (1.225-1.740); p = 0.003). Moreover, the effect of the minor allele on the association of VDR with T2DM was assisted through recessive model analysis. Where homozygous GG was observed in 116 (46.4%) cases and 79 (31.28%) controls and AG+AA was seen in 134 (53.6%) cases and 271 (68.3%) controls (OR = 2.970 (2.086-4.227); RR = 1.798 (1.501-2.154); p = <0.0001) (Table 2).

Figure 1

Representative gel pictures depicting genotypes in T2DM cases (A) and controls (B), and controls (C).

Table 2

Genotype and allele frequencies VDR gene SNP (rs1544410) using codominant, additive, homozygous dominant, homozygous recessive models.

Statistical Model	Genotype	Cases (n=250) (%)	Controls (n=250) (%)	χ2 (df=2)	OR (95% CI)	RR (95% CI)	p Value
Genotype Frequency/ Codominant Model	AA	65 (26.0)	32 (12.8)	41.81	–	–	0.0001
	AG	69 (27.6)	139 (55.6)
	GG	116 (46.4)	79 (31.56)
Homozygous Dominant Model	GG	116 (46.4)	79 (31.6)	–	2.970	1.798	<0.0001
	AG+AA	134 (28.6)	271 (68.3)		(2.086-4.227)	(1.501-2.154)
Homozygous Recessive Model	AA	65 (26.0)	32 (12.8)	–	2.394	1.460	0.003
	AG+GG	185 (74.0%	218 (87.2)		(1501-3.816)	(1.225-1.740)

SNP: single nucleotide polymorphism; OR: odds ratio; 95% CI: 95% confidence interval; RR: relative risk.

Discussion

It is predicted that there will be an alarming increase in the incidence of diabetes from 382 million (8.3%) in 2013 to 592 million (10.1%) in 2035. The previous study has shown a strong correlation between the VDR polymorphisms and T2DM-associated metabolic parameters [12]. To briefly explain, the human VDR gene is located on chromosome 12q13.1. The VDR gene consists of coding and non coding exons that is spliced alternatively [13, 14, 15]. Genetic polymorphism in the VDR gene may play an important role in increased β cells capacity of secretion, and thus have an association with T1DM and T2DM [16].

In this study, a total of 917 samples, which included 614 DM patients (469 T2DM and 145 T1DM) and 303 control samples’ data were collected from different hospitals of District Swat, Khyber Pakhtunkhwa Province, Pakistan. A questionnaire was designed, and the patients were visited at hospitals to record various information such as RBS/FBS, BMI, age, family history and associated disease of these patients. For each variable, the upper and lower ranges and the mean/ average was calculated in both males and females. Then a genetic variant rs1544410 in the VDR gene was genotyped in 250 T2DM patients and 250 control subjects using the (amplification refractory mutation system) ARMS-PCR method. In the current study, we determined the mean/average values for certain variables in our data stated at the gender level. Our findings are consistent with the observations who studied the association of BMI with T2D in the health records’ system in the United States. They recruited 12,179 T2DM and 25,177 healthy controls and concluded that BMI is strongly associated with the risk of being diagnosed with T2DM [17]. In another case-control study, the risks of T2DM focused on the physical activity of individuals. This study was conducted on 279 males and 119 females in Tokyo and was reported that family history of diabetes and smoking are the risk factor for the prevalence of T2DM [18]. Through genotype distribution, we found that 65 (26%) cases had homozygous AA, 69 (27.6%) cases had heterozygous AG, 116 (46.4%) cases had homozygous GG. Whereas, in control subjects, AA was found in 32 (12.8%), AG in 139 (55.6%), GG in 79 (31.6%). A significant difference was observed at the genotype level in cases and controls (χ2 = 41.81, p = 0.0001). However, through allele frequency distribution analysis, we determined an insignificant difference between T2DM cases and controls (p value = 0.866; OR = 0.967; RR = 1.034).

The effect of the major allele on the association of the VDR gene with T2DM was checked through homozygous dominant model analysis. Homozygous AA was found in 65 (26.0%) cases and 32 (12.8%) in controls. Similarly, AG+GG was found in 185 (74.0%) cases and in controls that was 218 (87.20%). Thus, a significant effect of <0.05 was observed (OR = 2.394 (1.501-3.816); RR = 1.46 (1.225-1.740); p = 0.003). Moreover, the effect of the minor allele on the association of the VDR gene with T2DM was assisted through recessive model analysis. Homozygous GG was present in 116 (46.4%) cases and 79 (31.28%) controls, whereas, AG+AA was 134 (53.6%) in cases and 271 (68.3%) in controls (OR = 2.970 (2.086-4.227); RR = 1.798 (1.501-2.154); p = <0.0001). Our data suggest that the VDR gene BsmI (rs1544410) genetic variant is associated with the risk of T2DM in a Pakistani cohort.

To date, more than 25 different polymorphisms have been mapped to the VDR locus. There are several reports that these VDR polymorphisms are associated with T2DM and insulin secretion [19, 20, 21]. In addition, VDR polymorphisms are related to metabolic syndrome, metabolic changes related to obesity [22]. The association of the VDR gene polymorphisms and T2DM in older people living in a community of Santiago de Chile, Chile, were previously established through a case-control study on 138 T2DM patients and 172 control subjects with ages ranging from 60-79 years. They further suggested that the C allele (TC+ CC) of the VDR-FokI gene is a possible risk factor for T2DM in older people living in a community in Santiago de Chile, Chile [10].

In conclusion, the findings of the current study suggest that genotype GG of genetic variant rs1544410 of the VDR gene is the most susceptible genotype to T2DM, and thus, obesity in patients of the Pakistani cohort. Although, the sample size of our study cohort was small, an extensively large case-control study with a huge sample set is needed to further confirm these findings and to be applied for the management and proper therapeutic intervention by the clinicians.