Vitamin D receptor rs7975232, rs731236 and rs1544410 single nucleotide polymorphisms, and 25-hydroxyvitamin D levels in Egyptian children with type 1 diabetes mellitus: effect of vitamin D co-therapy

Abstract

Purpose: We aimed to examine the possible association role of vitamin D and vitamin D receptor (VDR) single nucleotide polymorphisms (SNPs) in type 1 diabetes mellitus (T1DM) development, glycemic control and complications among a cohort of Egyptian children. Subjects and methods: A prospective case-control study has been conducted on 50 Egyptian children with T1DM who were comparable with 50 controls. Vitamin D and HbA1c were measured. VDR-SNPs [ApaI (rs7975232), TaqI (rs731236) and BsmI (rs1544410)] detection was done by polymerase chain reaction through restriction fragment length polymorphism (PCR-RFLP) technique. Vitamin D supplements were given to the included T1DM children with low vitamin D and reassessments of both HbA1c% and 25(OH)D serum levels were performed in those children three months later. Results: Eighty percent of the included diabetic patients have poor glycemic control. Vitamin D was deficient in 68% and insufficient in 16% of diabetic patients. Significant improvements in both vitamin D and glycemic status among T1DM children, who have low vitamin D and received vitamin D supplementations. There were significantly negative correlations between serum levels of vitamin D with both HbA1c % (r= -0.358, P˂0.05) and daily insulin dose (r=-0.473, P˂0.05). Compared with controls, T1DM children presented more commonly with ApaI a allele (OR: 2.87; 95%CI: 1.39-5.91, P˂0.05) and BsmI b allele (OR: 4.38; 95%CI: 2.30-8.33, P˂0.05). TaqI t allele wasn't significantly differing among patients and controls (P˃0.05). Aa+aa and Bb+bb genotypes were significantly higher among T1DM vs the controls (OR: 3.08;, 95%CI: 1.33-7.15, P˂0.05 and OR: 9.33; 95%CI: 3.61-24.17, P˂0.05respectively). Conclusion: ApaI and BsmI were associated with risk of T1DM development among Egyptian children. Low vitamin D status was frequently occurring among T1DM with significant improvement in the glycemic control of such children when adding vitamin D supplements to the standard insulin therapy.

Keywords: Egyptian children; genomic DNA; type 1 diabetes mellitus; vitamin D; vitamin D receptor single nucleotide polymorphisms; vitamin D therapy.

Figure 1

Flowchart of the study participants and methodology.

Figure 2

Gel electrophoresis of the PCR products (A) Numbers refer to lanes. Lane 1 shows 50 bp DNA ladder. Lanes 2–5 showed amplified DNA segments of length 716 bp: Lanes 2 and 3 for ApaI alleles, Lanes 4 and 5 for Taq1 alleles (both have the same size 716 bp); Lanes 6, 7 and 8 for BsmI alleles (with DNA fragment size of 825 bp). Detection of ApaI polymorphism using PCR-RFLP method (B) Lane 1: 50 bp DNA ladder, Lane 2 mutant (235 bp and 481 bp) homozygote (aa), Lanes 3, 4, 6, and 8 are mutant heterozygote (Aa), Lanes 5, 7 are wild type homozygote (AA), nonmutant. Detection of Taq1 polymorphism using PCR-RFLP method (C) Lane 1: 50 bp DNA ladder, Lanes 5, 9: mutant (297 bp and 419 bp) homoozygote (tt), Lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12 are mutant heterozygote (Tt). Detection of BsmI polymorphism using PCR-RFLP method (D) Lane 1: 50 bp DNA ladder, Lane 2: mutant (649 bp and 176 bp) homozygote (bb), Lane 3 is mutant heterozygote (Bb), Lane 5 is wild type homozygote (BB), nonmutant.

Figure 3

Negative correlations between serum vitamin D with both HbA1c (A), and insulin dose requirements (B).

Figure 4

Distribution of vitamin D receptor ApaI and alleles (A and a) genotypes (A); TaqI (B); BsmI (C) in patients with T1DM in comparison to the controls. Abbreviation: T1DM, type 1 diabetes mellitus.