1. Introduction

Type 1 diabetes mellitus (T1DM) is a multi-factorial autoimmune disorder, characterized by the destruction of pancreatic beta cells resulting in the deficiency of insulin secretion ^[1], caused by complex interaction between genetic and environmental factors, accounting for 5-10% of diabetes cases worldwide ^[2], and it is common among children and young adults and usually developed early under age 30 years.

It is known that the active vitamin D (1, 25-dihydroy vitamin D3), play an important role in the regulation of immune cells proliferation and differentiations, lymphocytes activation, cytokines production ^[3], and insulin secretion ^[4]. Vitamin D bioactivity is through the vitamin D receptor (VDR), which is belonging to the nuclear hormone receptors superfamily^[5].

The VDR gene is located on the long arm of chromosome 12 (12q12) and 14 (q14), and four common single nucleotide polymorphisms (SNPs) have been identified ^[6,7,8,9] namely Bsm1, Fok1, Apa1 and Taq1polymorphisms (designated as rs1544410, rs10735810, rs7975232, and rs731236 SNP, respectively). VDR polymorphism was found to be associated with many autoimmune disorders in human, such as celiac disease ^[10], multiple sclerosis ^[11,12], systemic lupus erythematosus ^[13,14,15], rheumatoid arthritis ^[16,17], and hashimoto, s throiditis ^[18]. The role of VDR polymorphisms in the pathogenesis of T1DM was not clear, large number of studies ^{[5,7,19,20,21,22,23,24,25]} has been investigated the association between the aforementioned SNPs and the risk of T1DM but the results were inconsistent.

This study was conducted to investigate the association between VDR polymorphisms (at position Bsm1, Taq1 and Apa1) and susceptibility to T1DM in Sudanese population.

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2. Materials and Methods

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2.1. Patients and controls

The study encompasses 174 T1DM patients [mean onset age 7.68±3.59 (range, 1-16) years old; 90 female and 84 male] refer to the diabetic clinic at Wad Medani Pediatric hospital which was founded in 1987 to serve children at Gezira state and the out skirt provinces. The patients group was diagnosed according the American DiabetesAssociation for type 1 diabetes ^[2]. Fifty six unrelated healthy children [9.50±4.23 (range 4-16) years old; 32 female and 24 male]. The patients were classified into metabolically poor glycemic control with HbA_1c > 8% and well glycemic control with HbA_1c≤8%. HbA_1c was assessed by chromatographic-spectrophotometer ion exchange (BioSystems). The demographic and the clinical information were obtained from each subject through well structured questionnaire. Informed consent was obtained from the children guardians or relatives, and the Faculty of Medicine Institutional committee approval was obtained.

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2.2. VDR gene analysis

DNA was extracted from the whole blood by using the QIAamp DNA Blood Mini Kit protocol (Qiagen, USA). The target gene of the VDR was extracted from whole gene sequence available at the Pubmed (NCBI Reference Sequence: NG_008731.1), and amplified by the PCR based on primers specific for Bsm1, Apa1 and Taq1polymorphisms (designated as rs1544410, rs7975232, and rs731236 SNP, respectively) (Table 1). The PCR condition and digestion used for amplification of Bsm1 as follows; 94 °C for 5 min, and 30 cycles using the following temperature profile: 94 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min, and final elongation for 5 min. The PCR products were 825-bp long (B allele), and were digested with Mva12691 (BsmI) at 37 °C for 3 hrs, and then subjected to electrophoresis in 2% agarose gel stained with 5 μL ethidium bromide. The lengths of the restriction fragments were 649 and 176 bp (mutant b allele). The PCR condition and digestion used for amplification of Apa1 and Taq1 as follows; 94 °C for 10 min, and 30 cycles using the following temperature profile: 94 °C for 1 min, 62 °C for 1 min, 72 °C for 1 min, and final elongation for 5 min. The PCR products were 716-bp long (A or T alleles) and were digested with ApaI and TaqI at 37 °C and 65 °C for 2 hrs respectively, and then subjected to electrophoresis in 2% agarose gel stained with 5 μL ethidium bromide. The lengths of the ApaIrestriction fragments were 481 and 235 bp (mutant “a” allele), and for TaqI were 419 and 297 bp. Five percent of the samples were checked twice for results accuracy.

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2.3. Statistical analysis

A chi-squared test (χ² test) was used to evaluate the associations between different genotypes of Bsm1, Taq1, and Apa1 variants and the disease (T1DM vs controls). The comparisons of the genotype frequencies between groups was analysed using the t-test. Odd ratios (ORs) and 95% confidence intervals (CIs) were calculated for each allele and genotype using logistic regression. The differences were considered significant if the P-value less than 0.05.

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3. Results

The base line characteristics of the study subjects are shown in (Table 2). The male to female ratio in diabetic and control groups was 1:1.1 and 4.3 respectively. Among diabetic patients 59.2% were having family history of diabetes, the mean HbA_1c among diabetic group was significantly high (P < 0.001) as compared with the control group, on the contrary, BMI which was significantly low among diabetic group, 85.6% of T1DM patients characterized by metabolically poor diabetic control.

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3.1. Genetic set

There was a difference in the VDR genotype and allele frequencies between T1DM patients and the control group. Bsm1 BB and B allele were significantly (P=0.004 and p=0.001 respectively) higher frequency in patients with T1DM (Table 3). The complete cleavage of Bsm1 to the target gene into homozygous (wild type BB=825 bp or mutant bb=649 bp) (Figure 1). On the other hand, Taq1 TT genotype (p=0.012) and T allele (p=0.05) were more frequent among study patients (Table 4), where as its complete cleavage of the target gene into homozygous (wild type TT=712 bp or mutant tt=419 bp) (Figure 2). Apa1 AA genotype (p=0.932) and A allele (p=0.862) were less frequent in TIDM patients without significant difference (Table 5), its complete cleavage to the target gene generates homozygous (wild type AA=716 bp or mutant aa=481 bp) (Figure 3). Bms1 BB genotype frequency was significantly (p < 0.02) high among patients with poor metabolic control group of T1DM as compared with well metabolic control group (Table 6).

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4. Discussion

Vitamin D could play a role in the pathogenesis of autoimmune diseases particularly T1DM, with its metabolites could inhibit T cell proliferation and suppress the production of certain pro-inflammatory cytokine such as TNF-α and IL-1 ^{[26,27,28,29]}. Our study showed high frequency of patients with family history of diabetes. An early family study has supported the association between first degree relatives with T1DM and the risk of developing T1DM ^[30], while other study indicated that, this association depends on which parent has diabetes ^[31]. Several studies investigated the association between VDR gene polymorphisms and T1DM in different populations like Greece ^[5], Taiwanese ^[7], Germany ^[19], Finnish ^[20], Portuguese ^[21], Japanese ^[22], Iranian ^[23], Dalmatian^[24], and Egyptians ^[25]. Most of the aforementioned studies reported controversial findings, which were most likely due to ethnic variations in addition to the role of the environmental and genetic factors. Thus far, no study indicated the association between VDR gene polymorphisms and susceptibility to T1DM in Sudanese population. Sudanese regions, particularly central Sudan, is inhabited by ethnically, linguistically and culturally diverse populations which makes the comparison of our results with previous studies from different regions is comparably useful, previous studies combines the genotype data of Sudanese populations with other subordinate countries such as Somalia, Egypt and Uganda ^[32], in addition to Ethiopia and Fulani ^[33]. Our study analysed three well characterized VDR polymorphisms (Bsm1, Taq1, and Apa1) among Sudanese T1DM children. We were not being able to investigate the fourth Fok1 polymorphism owing to difficulties associated with the enzyme itself.

Bsm1 and Apa1 are located in the intron between exon 8-9, which seems not affecting VDR protein structure, however, Bsm1 may influence VDR mRNA stability because it was found strongly linked with the 3 polyA microsatellite repeat in 3’untranslated region ^[3,38]. Our results showed both Bsm1 BB genotype and B allele were significantly high in patients than controls and is associated with increased risk of T1DM. This result was consistent with findings from other populations ^{[13,16,35,36]}. On the contrary, population study in Iran ^[14], showed no significant association between Bsm1 and susceptibility to T1DM, and was found to have protective role in Greece population ^[5].

Taq1 is a silent SNP within the 3’-noncoding sequences in exon 9 ^[37], its exact role in the pathogenesis of T1DM is not well defined. In this study and similar to Bsm1, Taq1 TT genotype and T allele exerted susceptibility action to T1DM, which is agreed with other study populations from Greece and Uruguayan ^[5,29]. This data was not in agreement with other populations such as Portuguese, Iranian, and Korean, where there was no association found ^[21,23,39], or there was low frequency in Egyptian population ^[25]. Our data showed no significant difference in Apa1 between patients and control. In agree with our result, Apa1 genotype and alleles frequencies showed no significant differences between T1DM and the control among Portuguese ^[21]. In contrary, Apa1 gene polymorphism was found associated with T1DM in Greece and Taiwanese populations ^[5,7].

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5. Conclusion

In conclusion, the VDR Bsm1 and Taq1 were associated with susceptibility to T1DM among Sudanese population. Our sample size was not large enough to represent the whole population, although it enrols the majority of the patients from central Sudan, nevertheless, the strength of an association between SNPS and T1DM in this study is measured by the odds ratio (OR). Hardy-Weinberg equilibrium for the genotypes was not used in this study because of small sample size and to override violation. Since Sudan is known as small continent and characterized by different ethnic groups, further studies from different regions is necessary to correlate between the genetic and environmental factors, VDR polymorphisms, and T1DM.

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Acknowledgment

Thanks to Dr. Mohammed Osman Abdelwahid, Department of Molecular Biology, National Cancer Institute, University of Gezira for his contribution.

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Conflict of interest

The author declares no conflict of interest.

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