Association of Vitamin D deficiency and Vitamin D Receptor Gene Polymorphisms with Type 2 diabetes mellitus Saudi patients.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a global problem. Association of multiple genes in T2DM becomes a hot point recently. This study was aimed to evaluate association of vitamin D receptor gene polymorphisms with susceptibility to T2DM. SUBJECTS AND METHODS: One hundred T2DM Saudi male patients were included in this study and one hundred healthy Saudi men were used as control. For each individual, fasting blood glucose, cholesterol, HDL-C, LDL-C, HbA1c, insulin and 25-(OH) vitamin D were measured. In addition, Apal, BsmI and TaqI genotypes were performed for each subject. Data was analyzed by SPSS version 16, using Spearman's rho and ANOVA tests.
RESULTS: There was significant inverse correlation between 25-(OH) vitamin D level and T2DM (p<0.01). HbA1c was inversely correlated with 25-(OH) vitamin D level (P<0.05). Genotype study showed that tt of TaqI genotype was higher in T2DM group compared with control group (p<0.05). Moreover, tt genotype has higher HbA1c than both TT and Tt genotypes (p<0.05).
CONCLUSION: An association was confirmed between TaqI genotypes and T2DM but there is no correlation between BsmI, ApaI and T2DM. In addition, HbA1c is positively correlated with tt genotype of TaqI.

Background

Type 2 diabetes mellitus (T2DM) is still a global problem that affects individuals all over the world. Its prevalence in Saudi Arabia has increased in the last decade and affects the young at 30 years age1. T2DM prevalence and complications make it the first problem that affects Saudi's life. It is characterized by insulin resistance and alteration in its secretion leading to disturbance in carbohydrate, protein and lipid metabolism2. Both genetic and environmental factors influence T2DM development3. Vitamin D is responsible primarily for calcium hemostasis but also involved in diverse types of cells growth and differentiation4. Vitamin D deficiency enhances T2DM development and its supplementation induces glucose tolerance5. In non-obese diabetic mouse model, vitamin D plays important role in insulin release and normal glucose tolerance6. Vitamin D exerts its effect through binding to specific intracellulareceptor called vitamin D receptor (VDR)7. The VDR is a member of steroid/thyroid hormone receptor family which is expressed in different cells lines include pancreatic β-cell8. Vitamin D and VDR complex induce insulin secretion from pancreatic β-cells and this is may a procedure by which vitamin D induces glucose tolerance9. The VDR gene is located at 12q13 chromosome and many polymorphisms were indicated at this region. The most important polymorphisms are ApaI, TaqI and BsmI which covered by different studies in different area and ethics. The VDR gene polymorphisms become one of the candidate genes that influence T2DM development10. Our previous study which was done on Saudi obese males showed an association between these polymorphisms and insulin resistance in these patients11. Accordingly, we aimed in this study to investigate the association of VDR gene polymorphisms and T2DM in Saudi population.

Subjects and methods

Subjects

This project consisted of 100 T2DM male patients enrolled from diabetic center of King Abdul-Aziz specialist hospital in Taif and 100 healthy males used as control. Both groups matched in sex and ethics. All patients diagnosed according to the World Health Organization criteria (fasting blood glucose >126 mg/dL or 2-hour postprandial blood glucose >200 mg/dL)12. The diagnosis was confirmed by answering the questioner constructed by authors of this project that any patients in this study didn`t take any medications or vitamin supplementation and didn't suffering from any chronic that may alter vitamin D metabolism. All subjects in control group did not suffering from any chronic illness such as DM, hypertension, cardiac, hepatic, or urinary illness and did not use any medication affect vitamin D level. After an overnight fast, venous blood samples were collected in EDTA and plain tubes then separated immediately and stored at 4°C until the analysis.

Biochemical parameters

The plasma was used for biochemical marker measurement include glucose, triglyceride, total cholesterol, HDL-C, LDL-C, insulin and 25-(OH) vitamin D. Fasting blood glucose, triglyceride, cholesterol, HDL-C, LDL-C and HbA1c were measured in a Dimension autoanalyzer (Dade Behring Inc). Insulin and 25-(OH) vitamin D were measured by using ELISA technique13.

Genotyping

Blood samples collected in EDTA tube and the DNA was extracted from peripheral blood leukocytes using the Thermo SCIENTIFIC DNA isolation kit (Thermo SCIENTIFIC). Genomic DNA was amplified and analyzed for VDR genotype by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) for, Apa1 Taq1 and BsmI genotypes using forward and reverse primers showed in Table 1.1. The PCR mix was contained 5 µL of each primer (10 pmol), 5 µL buffer, 1.5 µL MgCl2 (50 mM), 5 µL template DNA (50–100 ng), 5 µL dNTPs (2 mmol/L), Taq polymerase (MBI) 2 µL, H2O 26.5 µL. The DNA template was denatured at 95°C for 2 min. A total of 40 cycles of PCR were performed, consisting of denaturation step for 45 sec at 94°C, an annealing step for 45 sec at optimum temperature (67°C for ApaI/TaqI and 60°C for BsmI), and an extension reaction for 1 min at 72°C. A final extension step at 72°C for 2 min was added after the last PCR cycle. After amplification, the PCR products were digested by incubation with restriction enzymes. For ApaI polymorphism, the amplicon incubated with ApaI enzyme in 37°C for 5 minutes to get its three genotypes on 1.5% agarose gel designated AA, Aa and aa. Incubation of amplicon with TaqI at 65°C for 4 hours produced TT, Tt and tt on 2.5% agarose gel. BsmI genotypes were produced after incubation of amplicon with BsmI enzyme at 65°C for 15 min, then applied on 2% agarose gel14. The size of each genotypes ApaI, TaqI and/span>BsmI are shown in Amplified Table 1 and Fig 1, 2 and 3 respectively.

Table 1

Forward and reverse primers

SNP	Sequences	Restriction products (bp)
Apa1	Forward 5′-CAGAGCATGGACAGGGAGCAAG-3′ Reverse 5′-GCAACTCCTCATGGCTGAGGTCTCA-3′	Allele AA: 740 Allele Aa: 740+515+225 Allele aa: 515+225
Taq1	Forward 5′-CAGAGCATGGACAGGGAGCAAG-3′ Reverse 5′- GCAACTCCTCATGGCTGAGGTCTCA-3′	Allele TT: 495+245 Allele Tt: 495+290+ 245+205 Allele tt: 290+245+205
BsmI	Forward 5′-AGTGTGCAGGCGATTCGTAG-3′ Reverse 5′- ATAGGCAGAACCATCTCTCAG-3′	Allele BB: 360 Allele Bb: 360+191+169 Allele bb: 191+169

Figure 1

Detection of Apa1 polymorphism by PCR-RFLP method. Lane 1 and 5: homozygote (AA), Lane 3: heterozygote (Aa), Lane 2 and 4: homozygote (bb).M is 100-bp DNA marker.

Figure 2

Detection of Taq1 polymorphism by PCR-RFLP method. Lane 5 and 6: homozygote (TT), Lane 1, 2 and 4: heterozygote (Tt), Lane 3 homozygote (tt), M is 100-bp DNA marker.

Figure 3

Detection of BsmI polymorphism by PCR-RFLP method. Lane 3: homozygote (BB), Lane 2 and 4: heterozygote (Bb), Lane 1: homozygote (bb). M is 25-bp DNA marker.

Statistical analysis

Hardy-Weinberg equation was used to test whether the examined SNPs were in equilibrium. The SPSS software version 16 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The correlations were tested using Spearman's rho. ANOVA was used in comparisons performance. Both comparisons and correlations were considered statistically significant when p<0.05.

Results

The results were represented by Table 1, 2 and 3. Average of population age, BMI, FBG, HbA1c, triglyceride, cholesterol, HDL-C, LDL-C, insulin and 2-(OH) vitamin D were represent in Table 2. Age, weight, BMI, FBG, HbA1c, triglyceride and insulin were significantly higher in T2DM patients compared with control group (P<0.01). Our focusing parameter 2-(OH) vitamin D showed a significant higher level in T2DM group compared with control group (P= 0.003). Based on VDR genotypes, With respect to examined VDR SNPs, all examined genotypes were in Hardy-Weinberg equilibrium (Table 3). The result showed significant differences in TaqI in both groups.. The tt genotype was significantly more frequent in T2DM group than control group. The TT genotype of Taq1 was more frequent in control group than T2DM group. Moreover, the tt allele was more frequent in T2DM group while TT allele was more frequent in control group. The results were not confirmed any significant differences between T2DM group and control group in both BsmI and ApaI genotypes of VDR (Table 3).

Table 2

Comparison of biochemical parameters between type 2 diabetes mellitus patients and control using t-test (Mean±SD)

	Control N=100	T2DM patients N=100	P value
Age	25.25±6.30	38.25±10.36	0.004**
Weight (kg)	62.9±5.4	89.8±7.2	0.001**
BMI	26.49±2.04	30.7±1.81	0.003**
FBS (mg/dl)	90.33±7.31	152.68±30.66	0.002**
TG (mg/dl)	158.44±21.22	183.59±33.49	0.008**
Cholesterol (mg/dL)	198.21±16.49	223.75±17.32	0.241
HDL (mg/dL)	51.96±7.73	47.65±5.01	0.566
LDL (mg/dL)	102.32±9.44	123.91±22.56	0.311
Insulin (pmol/L)	17.83±2.32	8.22±6.03	0.004**
25-H-Vit-D (ng/mL)	37.41±8.12	18.09±4.17	0.003**

Table 3

Comparison of ApaI, TaqI and BsmI genotypes and allelic frequencies between type 2 diabetes mellitus patients and control

Genotypes ApaI	Control (100) %	T2DM (100) %	P value	Hardy- Weinberg P value
AA Aa aa	30 (30%) 48 (48%) 22 (22%)	32 (32%) 47 (47%) 21 (21%)	0689 0.815 0.809	0.735$
Allele A Allele a	108 (54%) 92 (46%)	111 (55.5%) 89 (44.5%)	0.665 0.571
Genotype TaqI
TT Tt tt	53 (53%) 36 (36%) 11 (11%)	24 (24%) 34 (34%) 42 (42%)	0.041* 0.602 0.013*	0.208$
Allele T Allele t	142 (71%) 58 (29%)	82 (41%) 118 (59%)	0.036* 0.011*
Genotypes BsmI
BB Bb bb	50 (50%) 38 (38%) 12 (12%)	45 (45%) 41 (41%) 14 (14%)	0.417 0.632 0.801	0.263$
Allele B Allele b	138 (69%) 62 (31%)	131 (65.5%) 69 (34.5%)	0.406 0.558

In Hardy-Weinberg equilibrium

Table 4 represents a comparison of all biochemical parameters between each genotype of ApaI, TaqI and BsmI in all individual included in this study. The results showed a significant higher HbA1c value in tt genotype of TaqI (P=0.029). According to BsmI genotypes, BB genotype showed a significant higher 2-(OH) vitamin D level (P=0.036)

Table 4

Comparison between fasting blood glucose, HbA1c, insulin and 25-(OH) vitamin D in each genotypes of ApaI, TaqI and BsmI in both groups

ApaI genotypes	AA	Aa	aa	P value
FBG	136.21±20.64	129.88±30.46	142.11±36.83	0.309
HbA1c	7.99±2.08	8.12±3.22	8.33±2.06	0.752
Insulin	18.03±4.36	20.45.03±6.36	19.79±5.36	0.601
25-(OH) vit D	31.14±7.22	37.11±4.22	35.66±5.63	0.447
TaqI genotype	TT	Tt	tt	P value
FBG	140.24±28.01	126.44±35.19	125.34±21.79	0.335
HbA1c	5.21±2.11	7.89±1.57	10.55±1.85	0.029*
Insulin	17.26±4.04	21.69±7.28	24.81±2.36	0.466
25-(OH) vit D	29.49±8.22	32.65±4.28	27.56±7.23	0.486
BsmI genotype	BB	Bb	bb	P value
FBG	111.20±20.21	120.66±18.93	133.67±30.51	0.298
HbA1c	6.34±2.22	6.45±3.06	6.93±2.45	0.814
Insulin	19.84±5.55	20.59±4.77	22.35±6.71	0.619
25-(OH) vit D	40.21±3.22	38.58±5.92	37.73±4.44	0.671

Discussion

Recently, T2DM affects young people and its complications are increased in Saudi Arabia. Vitamin D is steroid derived vitamin and has a large variety of functions. It is involved in growth, bone strength, immune and nervous function. Low levels of vitamin D can involve in development of various chronic diseases such as cancers, osteoarthritis and cardiovascular disease15. Vitamin D deficiency is becoming a global problem in all countries including sunny areas. A study done by Alharbi et al., showed that 98% of all Saudi men included in their study have vitamin D deficiency and this may occur as a result of less exposure of people to sun light due to high temperatures of this area during spring, summer and autumn16. Several studies found that low vitamin D levels are associated with T2DM. Mercedes and his colleagues found that low vitamin D levels are associated with T2DM in Spanish people17. Also another study done by Romero and Lozano found that vitamin D deficiency is common in T2DM in patients in Southern Spain18. The results are in agreement with the previous studies, which showed that 97% of T2DM patients have vitamin D deficiency and the group has a lower level of this vitamin compared with normal people. In 2017, Al-Hazmi and his colleagues also found that vitamin D deficiency is dominant in obese people11. also found that vitamin D deficiency is dominant in obese people11. In the same year, Mahmodenia et al., also found that vitamin D deficiency is common in obese and T2DM Iranian patients19. Both obesity and vitamin D insufficiency induce insulin resistance20. Vitamin D supplementation induces insulin production by pancreatic β-cells and It also improves insulin resistance and impaired glucose tolerance in T2DM patients21. Vitamin D produces its signals either directly by binding to VDR in β-cells or indirectly by induction of release of calcium from these cells22.

Several studies found inverse correlation between insulin level and 25-(OH) vitamin D level in T2DM patients23. This observation was not confirm in this study, but an inverse correlation between HbA1c and 25-(OH) vitamin D was confirmed in this study as previous studies concluded24,25. Previously, several studies were done to evaluate association of VDR with different chronic diseases. Results of previous studies to indicate association of VDR genotypes and T2DM are conflict. In 2011, Reza et al found a significant difference in TaqI genotype between T2DM patients and control group in Iranian population. Their result showed that TT genotype is decreased in T2DM patients group, but did not confirm any difference in TaqI allele26. Obesity is a predisposing factor for T2DM and previously, our group found that tt genotype of TaqI is more frequent in obese people11. Simlarly, this study showed that tt genotype of TaqI is more frequent in T2DM patients group.

In addition, t allele is more frequent in T2DM patients group than control group. Other studied done on Turkish population found no association between TaqI genotype of VDR and T2DM27. About ApaI genotype, Oh et. al., found that ApaI genotype is associated with T2DM in American population28. On the other hand, Dilmec et al found no association between this APaI genotypes and T2DM27. The finding also did not confirm association of AqaI genotype and T2DM. Association between BsmI genotype and T2DM is reported by Speer et al., and Ortlepp et. al.,29,30. The study findings are inconsistent with those of Mackawy et al who found no association between BsmI genotype and T2DM31. The reason for differences in results of studies that focus on association of VDR gene polymorphisms with T2DM may be due to genetic, ethical and environmental differences in population. The strength of this study was studying of more than one genotype in vitamin D receptor and the subjects were matched in both race and sex. Moreover, The study demonstrated the correlation of vitamin D genotypes with some diabetic parameters such as FBS, HbA1c, and insulin. The limitation of this study was that it used different ages for T2DM patients.

Recommendation and conclusion

An association was confirmed between TaqI genotypes and T2DM but there is no correlation between BsmI, ApaI and T2DM among Saudi population. In addition, HbA1c is positively correlated with tt genotype of TaqI. This is the first study done on Saudi population to evaluate association of VDR gene polymorphisms with T2DM. Vitamin D supplementation is recommended to preventing insulin resistance and T2DM.