Association of Vitamin D receptor gene BsmI polymorphism with type 2 diabetes mellitus in Pakistani population

Background Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder with strong genetic components. The reported association of vitamin D receptor (VDR) gene polymorphisms varies among ethnic groups. Objectives The present study was conducted to determine association of vitamin D receptor gene BsmI (rs1544410 A>G) polymorphism with type 2 diabetes mellitus in Pakistani population. Methods Blood samples were collected from 150 T2DM patients and 100 non-diabetic engaged by convenient sampling method. After collection of demographic data, assessment of fasting glucose (FG), vitamin D, HbA1c, renal function tests, liver function tests and lipid profile was done. Candidate gene polymorphism was analyzed by DNA amplification with polymerase chain reaction and endonuclease digestion. Results Biochemical parameters were significantly different among case and control groups. Associations of BsmI genotype with T2DM, related complications and biochemical variables were not significant. Conclusion The current study did not provide evidence for the association of VDR gene BsmI polymorphism with T2DM in Pakistani population.


Introduction
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder with strong genetic components.Candidate genes and variants for T2DM risk present in specific genome parts are involved in disease onset, associated pathways and functions. 1,24][5] Genetic polymorphism of vitamin D receptor (VDR) gene can affect insulin secretion, causing insulin resistance, affect vitamin D syn-thesis, transportation and action. 6][9] Vitamin D binding protein (DBP) is the gene product that mediates vitamin D action.Cholcalciferol enters blood stream through binding to DBP.VDR gene genotype BsmI polymorphism found in intron 8is related with onset of type 2 diabetes mellitus. 10,11Al-Daghri et al. 12 stated that BsmI SNP is significantly more common in T2DM patients.4][15][16] Consequently, association between BsmI SNP and risk of T2DM in different ethnic groups is not conclusive.SNP relates to disease susceptibility and response to treatment. 179][20] However, researchers overlooked the role of VDR gene SNP in local population especially with reference to diabetes mellitus.Progress in identification of novel VDR gene variants predisposing to diabetes mellitus in Pakistan has been limited.Therefore, current research was conducted to assess the association of VDR gene BsmI polymorphism with T2DM in Pakistani population.

Research design
Study was conducted from March, 2015 to January, 2016 at Clinico-Medical Biochemistry Lab., Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan and Molecular Labs., Department of Medical and Dentistry, Southmead Hospital, University of Bristol, Bristol, United Kingdom.Sampling was conducted within Pakistan and included consented 150 T2DM patients and 100 non-diabetic engaged by convenient sampling method.Graduates Studies and Research Board (GSRB), University of Agriculture, Faisalabad, Pakistan granted ethical approval.

Sample collection and bioassays
After collection of demographic data,fasting blood samples were taken in EDTA-coated vacutainers.Assessment of fasting glucose (FG), vitamin D, HbA1c, renal function tests,liver function tests and lipid profile was done by kits (Merck, Germany) as per manufacturer's guidelines using Dade Behring clinical chemistry system(dimension auto-analyzer, Siemens, USA) and Diastat auto-analyzer (Randox, UK).

DNA extraction and quantification
After genomic DNA extraction, the quantity and quality of DNA was assessed by Nanodrop (Nanophotom-eter™, Implen, Germany).Purity of the DNA was assessed by measuring optical density (OD) as OD260/ OD280. 22

Biochemical assays
Results are presented in table l.Elevated FG, HbA1c, BMI, blood pressure and decreased vitamin D (P < 0.05) levels were evident in case group as compared to controls with negative correlation between vitamin D and HbA1c.Liver function tests inferences were similar among both groups.Regarding RFTs and lipid profile, T2DM sample showed appreciably (P < 0.05) higher concentrations than control participants.Within the each group, associations of biochemical variables with vitamin D were negligible.Based upon the physician-diagnosed complications, diabetic subjects were sub-divided into CP (cardiac patients), NP (nephropathy patients), RP (retinopathy patients) and HP (hypertensive patients).Among these groups, non-significant differences in clinical and biochemical profiles were acquired, an observation that can be justified by the fact that most of the patients were either in initial complication phases or were using oral hypoglycemic medications.Therefore, data was simplified and presented as case and control participants.

BsmI polymorphisms
After amplification of intron 8 by PCR (figure 1a), heterozygosity of intron 8 was confirmed by enzymatic digestion (Figure 1b).Distribution of genotype allele frequencies and carriage rate of BsmI among diabetic patients, T2DM sub-groups and control group was non-significant (table 2).Effect of VDR gene polymorphisms on metabolic parameters in terms of probability to clarify their association underlying the diabetic complications are mentioned in table 3. VDR gene BsmI polymorphism was related non-significantly (p>0.05) to the diabetic complications in the present study.

Discussion
Although numerous genes have role in T2DM, 24,25 VDR gene plays a dominant role in onset and progression of T2DM. 11,14,26BsmI polymorphism found in intron 8 of VDR gene may be involved in pathogenesis of T2DM. 27revalence of vitamin D deficiency in T2DM group was higher as compared to control subjects, showing significant association of BsmI with T2DM in the present study.However, Santos et al. 27 observed conflicting results in Brazilian population.Contrary to current findings, Dilmec et al. 14 found significant association of BsmI to T2DM onset.While Israni et al. 28 suggested potential role of BsmI polymorphisms.Wang et al. 30 studied significant association of BsmI polymorphism with T2DM onset.
Review of literature indicates conflicting results regarding the impact of VDR gene polymorphisms on T2DM pathogenesis in different populations 14,26,30 either supporting or contrasting current findings.Heterogeneity in different populations and limited knowledge of underlying mechanism may be responsible for these discrepancies. 31,32tatistically non-significant relationship between BsmI polymorphism and T2DM in current project is supported by earlier study. 33This study suggests that the BsmI may be related with susceptibility to T2DM subjects but genetic contribution of VDR gene polymorphism for the development or existing diabetic complications is not clear.In addition, vitamin D receptor gene consists of many single nucleotide polymorphisms (SNPs).To investigate whether functional changes of VDR gene may be potential risk for T2DM, future studies should focus on population or case-control studies along with family linkage with multiple SNP study .34 Figure 1a-b showed typical results in current study that correlate with previously studied results.It was performed to check the action of restriction enzyme.BsmI polymorphisms among T2DM complications groups were scored in figures 3-5.Three enzymatic digested fragments of BsmI (76, 115 and 191 bp) indicated heterozygosity (Bb) of BsmI genotype.Differences of BsmI genotypes of VDR gene were significant between T2DM and normal groups (p < 0.01).No substantial association was found between biochemical parameters and BsmI restriction site (p > 0.01).