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

Figures & data

Video abstract

Figure 1 Flowchart of the study participants and methodology.

Table 1 PCR primer sequences used to amplify VDR SNPs and length of DNA fragments of VDR gene polymorphism

SNP		PCR primer sequences (5‘-3‘)	Fragment size
ApaI	Forward	GGG ACG CTG AGG GAT GGC AGA GC	A:716 bp a:481 bp, 235 bp
	Reverse	GGA AAG GGG TTA GGT TGG ACA GGA
TaqI	Forward	GGG ACG CTG AGG GAT GGC AGA GC	T:716 bp t:419 bp, 297 bp
	Reverse	GGA AAG GGG TTA GGT TGGACAGGA
BsmI	Forward	CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA	B:825 bp b:649 bp, 176 bp
	Reverse	AAC CAG CGG AAG AGG TCA AGG G

Abbreviations: VDR, vitamin D receptor; SNPs, single nucleotide polymorphisms.

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.

Table 2 Demographic, clinical and biochemical characteristics of the study groups

	Cases (N=50)	Controls (N=50)	P-value
Sex N, (%)			0.841
•Male	24 (48.0%)	25 (50.0%)
•Female	26 (52.0%)	25 (50.0%)
Age (years, mean ±SD)	11.16±3.27	10.97±2.77	0.782
Age at onset: (years, mean ±SD)	7.00±3.09	–	–
Duration of T1DM (years, mean±SD)	4.20±3.14	–	–
Family history of T1DM, N, (%)			–
•Yes	16 (32.0%)	–
•No	34 (68.0%)	–
Weight (kg, mean ±SD)	31.70±13.56	35.07±12.00	0.105
Height (cm, mean ±SD)	132.16±19.91	139.92±14.61	0.060
BMI (kg/m^2mean ±SD)	17.50±3.24	17.32±2.92	0.694
Frequency of diabetic complications N, (%)			–
•Yes	25 (50.0%)	–
•No	25 (50.0%)	–
Type of complications N, (%)
•History of DKA	22 (44.0%)	–	–
•Neuropathy	8 (16.0%)	–	–
•Nephropathy	10 (20.0%)	–	–
•Retinopathy	4 (8.0%)	–	–
•Pubertal delay	7 (14.0%)	–	–
HbA1c, %, mean ±SD	10.72±2.22	4.20±0.94	0.000*
Glycemic control N., (%) •Good (HbA1c≤8)	10 (20.0%)	50 (100.0%)	0.000*
•Poor (HbA1c >8)	40 (80.0%)	0 (0.0%)
Serum 25(OH)D3 ng/ml, mean ±SD	13.46±10.50	35.03±2.46	0.000*
Serum vitamin D status N, (%) •Deficient <20	34 (68.0%)	0 (0.0%)	0.000*
•Insufficient 20 – <30 •Sufficient ≥30	8 (16.0%) 8 (16.0%)	0 (0.0%) 50 (100.0%)

Note: *Significant P-value (P˂0.05).

Abbreviations: T1DM, type 1 diabetes mellitus; BMI, body mass index; DKA, diabetic ketoacidosis.

Table 3 Mean ±SD of HbA1c and serum vitamin D among T1DM children in relation to glycemic control, vitamin D status, occurrence of diabetic complications and effect of vitamin D therapy

T1DM patients (total N=50)/variables	HbA1c (%, mean±SD)	Serum 25(OH)D3 (ng/ml, mean±SD)	P-value
Glycemic control
Good glycemic control (N=10) Poor glycemic control (N=40)	7.15±1.7 10.07±4.1	21.48±12.80 11.46±8.95	0.015*
Vitamin D status
Optimal (N=8) Decreased (N=42)	8.56±1.75 11.13±2.07	33.37±2.03 13.46±10.50	0.002*
T1DM complication
T1DM with complication (N=25) T1DM without complication (N=25)	12.20±1.19 9.25±2.02	7.97±5.82 18.95±11.34	0.000*
Effect of vitamin D therapy
Pretherapy levels (N=42) Post-therapy levels (N=42)	10.72±2.22 8.83±1.58	13.46±10.50 38.70±13.56	0.000*

Note: *Significant P-value (P˂0.05).

Abbreviation: T1DM, type 1 diabetes mellitus.

Figure 3 Negative correlations between serum vitamin D with both HbA1c (A), and insulin dose requirements (B).

Table 4 Genotypes and alleles frequencies of ApaI, TaqI and BsmI in T1DM patients in comparison to the controls

Study groups	Variables
	ApaI genotypes														ApaI alleles
	AA		Aa		aa		AA+Aa		aa		AA		Aa+aa		A		a
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
T1DM (n=50)	24	48	22	44	4	8	46	92	4	8	24	48	26	52	70	70	30	30
Controls (n=50)	37	74	13	26	0	0.0	50	100	0	0.0	37	74	13	26	87	87	13	13
P-value (χ ^2)	0.011*(9.09)						0.117(4.17)				0.008*(7.10)				0.003* (8.56)
OR (95%CI)	--						--				3.08 (1.33-7.15)				2.87 (1.39-5.91)
	TaqI genotypes														TaqI alleles
	TT		Tt		tt		TT+Tt		tt		TT		Tt+tt		T		t
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
T1DM (n=50)	0	0.0	42	84	8	16	42	84	8	16	0	0.0	50	100	42	42	58	58
Controls (n=50)	0	0.0	40	80	10	20	40	80	10	20	0	0.0	50	100	40	40	60	60
P-value (χ ^2)	0.603 (0.27)						0.603 (0.27)				--				0.774 (0.08)
OR (95%CI)	--						--				--				0.92 (0.52-1.62)
	BsmI Ggenotypes														BsmI alleles
	BB		Bb		bb		BB+Bb		bb		BB		Bb+bb		B		b
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
T1DM (n=50)	8	16	35	70	7	14	43	86	7	14	8	16	42	84	51	51	49	49
Controls (n=50)	32	64	18	36	0	0.0	50	100	0	0.0	32	64	18	36	82	82	18	18
P-value (χ ^2)	0.000*(26.85)						0.012* (7.53)				0.000* (24.00)				0.000* (21.57)
OR (95%CI)	--						--				9.33 (3.61-24.17)				4.38 (2.30-8.33)

Note: *Significant P-value (P˂0.05).

Abbreviations: T1DM, type 1 diabetes mellitus; χ², chi-squared.

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.