Assessment of vitamin D status and vitamin D receptor polymorphism in Egyptian children with Type 1 diabetes

Abstract

Background

The endocrine system of vitamin D regulates about 3 % of the human genome. Vitamin D exerts its actions via a nuclear vitamin D receptor (VDR) which in turn regulates insulin secretion from the pancreas. VDR gene polymorphisms could have an impact on how autoimmune illnesses like Type 1 diabetes mellitus (T1DM) develop. We aimed to explore the relation between T1DM and VDR gene polymorphisms in Egyptian diabetic children and their siblings.

Methods

Enzyme-linked immunosorbent assay was used to quantify 25(OH) vitamin D in the study, which had 179 participants (group 1 = 85 diabetic children, group 2 = 57 siblings of the patients, group 3 = 37 healthy controls). Real-time polymerase chain reaction (RT-PCR) was used to analyze the genotyping of the VDR gene polymorphisms Apa-I (rs7975232), Fok-I (rs2228570), Taq-I (rs731236) and Bsm-I (rs1544410).

Results

The mean serum 25(OH) vitamin D levels was significantly lower in T1DM patients (14.99 ± 9.24 ng/mL) and siblings (16.31 ± 7.96 ng/mL) compared to the controls (19.48 ± 7.42 ng/mL) (p = 0.031). The genotypes distribution of VDR Fok-I (rs2228570) and Bsm-I (rs1544410) polymorphisms showed a significant difference between patients, siblings and controls as P = 0.001 and 0.026 respectively, while the VDR ApaI and TaqI polymorphisms did not. FokI-A allele frequency was significantly lower in T1DM patients and siblings than in controls (p < 0.001). FokI-AA genotype had a statistical significant higher vitamin D levels than other genotypes with p value of 0.024.

Conclusion

Our study found that T1DM children had lower vitamin D levels, and VDR FokI and BsmI gene polymorphisms were linked to T1DM in Egyptian children. Determining the relationship between vitamin D levels and VDR polymorphisms, particularly the FokI and other genetic analyses may aid in the early diagnosis of T1DM in children.

1. Background

Type 1 diabetes mellitus is characterized by the death of pancreatic islet cells due to dysregulation of both humoral and cell-mediated immune systems.¹ Resulting in deficiency of insulin, which makes the affected individual dependent on exogenous insulin. T1DM can occur at any age group, but usually affects children or adolescents younger than 20 years. Compared to high-income countries, incidence has been rising more quickly in low- and middle-income countries.² In Egypt, 1.5 % of 1,000 are newly diagnosed with T1DM in people under the age of 20.³

Genetics play a key role in the onset of T1DM and show a significant clustering in affected families as the average risk prevalence in siblings is 6 % while the general population risk is only 0.6 %.⁴ The human leukocyte antigen region (HLA) on the short arm of chromosome 6 has been the genetic factor most reliably linked to T1DM risk. However, a number of additional non-HLA areas have been recognized as risk factors.⁵ The VDR gene is one of these susceptibility genes that has received the most attention, and mounting data suggests that vitamin D and its receptor may be connected to T-cell-mediated autoimmune illness, which in turn affects susceptibility to T1DM.⁶

25 (OH) Vitamin D has an immune modulatory role in T1DM protection, as it reduces the expression of pro-inflammatory cytokines included in T1DM pathogenesis,⁷ Vitamin D deficiency (VDD) is a serious health problem in different populations even the ones with abundant sun exposure such as Egypt⁸ and India.⁹ The Vitamin D endocrine system controls about 3 % of the human genome.¹⁰

Vitamin D does its action through nuclear vitamin D receptors (VDR).¹¹ VDR have been shown to be expressed in a variety of tissues as pancreatic β cells, making Vitamin D a potential direct modulator of insulin response to high blood glucose as well as an indirect modulator via calcium homeostasis.¹² Four frequently found single nucleotide polymorphisms (SNPs) have been identified in the VDR gene, which is located on chromosomes 12 and 14. As Apa-I (rs7975232), Taq-I (rs731236), Fok-I (rs2228570) and Bsm-I (rs1544410).¹³ Despite extensive investigation, it is still unclear how the polymorphisms of the VDR gene, which are associated with altered gene function or expression, connect to the aetiology of T1DM.¹⁴

The aim of the current study was to examine the significance of Vitamin D receptor gene polymorphisms and Vitamin D levels in Egyptian type 1 diabetic children and their siblings.

2. Methods

A case-control study was carried out in children aged (3–18 years). The study included(179) subjects divided into three groups, group 1 = 85 children with type 1 diabetes mellitus (41 boys and 44 girls; their mean age was 12.28 ± 3.32 years), group2 = 57 siblings of the patients (29 boys and 28 girls; their mean age was 9.70 ± 3.86 years) and group 3 = 37 healthy controls (11 boys and 26 girls; their mean age was10.86 ± 3.32 years).

Patients were collected from our Pediatrics Endocrine Clinic in the Centre of Excellence in the National Research Centre.

All patients that participated were diagnosed with T1DM according to American Diabetes Association guidelines.¹⁵

We excluded children with T2DM or secondary DM and any condition that affects VD state as diuretic treatment, malignancy and liver or kidney disease.

2.1. Every group that was being researched underwent:

Data including the age and gender of patients, history comprising of type and dose of insulin therapy, the onset of the diabetes, duration of the disease, VD or calcium intake, rickets, and family history of diabetes. Any past history of symptoms that might be indicative of cardiac, neurological, autonomic dysfunction, or renal disease. Clinical evaluations that include regional and general exams. Each participant had their blood pressure checked, as well as anthropometric data like height, weight, waist circumference, and hip circumference. Body mass index (BMI), which was computed as weight divided by height squared (kg/m²), was measured as the waist-to-hip ratio (cm/cm).

2.2. Laboratory investigation

After a 12-hour overnight fast, blood samples were taken from all individuals using Ethylenediamine tetra-acetic acid (EDTA) and plain vacutainer tubes. EDTA blood was used for complete blood picture (CBC), hemoglobin A1C (HbA1C) and genotyping analysis, while plain vacutainer was left to clot and serum was separated and partially utilized in routine laboratory workup and the rest was stored at −80 °C for assessment of vitamin D levels.

2.2.1. Assessment of CBC

Utilising a Sysmex xs-500i Haematology Analyzer (Sysmex, Kobe, Japan), a CBC was performed.

2.2.2. Evaluation of HbA1c

HbA1c was assessed using Labona check™ HbA1c analyzer (CERAGEM Medisys, Cheonan, South Korea Korea).

2.2.3. Calcium and lipid evaluation

Calcium and lipid profile were assessed using XL 300 - ERBA Mannheim autoanalyzer (ERBA Diagnostics Mannheim, Germany).

2.2.4. Assessment of vitamin D₃

ELISA, or enzyme-linked immunosorbent assay, was used to measure vitamin D levels. Kit catalog number E1981Hu (Bioassay technology laboratory, Zhejiang, China).

When a subject's serum 25(OH) vitamin D level at least 30 ng/mL, was considered sufficient and between 21 and 29 ng/mL or less than 20, it was considered to have insufficient and deficient vitamin D status respectively.¹⁶

2.3. Genotyping analysis for VDR gene polymorphisms

Following the instructions in the user's manual, the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) extracted genomic DNA from the entire blood samples. The extracted DNA samples were kept at 20 °C. Following the manufacturer's instructions, the TaqMan allelic discrimination method was used to genotype the VDR SNPs rs1544410, rs7975232, rs731236, and rs2228570 on the LightCycler® 480 Real-Time PCR System (- Roche Diagnostics, Basel, Switzerland). TaqManTM SNP Genotyping Assay by Applied Biosystems, which produced all primers and probes, Cat no. 4351379) of Foster City, California. In the PCR procedure, there were 40 cycles of denaturation for 15 s at 95 °C and annealing/extension for 1 min at 60 °C. First, a 10-minute activation step at 95 °C was performed. Florescence information was gathered during the extension process, and the finished products were examined using software that was pre-programmed for the LightCycler® 480 Real-Time PCR System. To assure quality control, 10 % of samples were randomly chosen and measured in duplicates with a concordance rate of 100 %. Additionally, positive and negative controls were co-genotyped with each assay.

2.4. Statistical analysis

Statistical analysis was conducted using commercial SPSS software (SPSS 17.0; IBM Inc., Chicago, IL, USA). The frequency and percentage of the descriptive properties of the qualitative variables were provided. Results for continuous variables were shown as mean ± standard deviation (SD). The ANOVA test, post HOC test, and LSD test were each used to evaluate the normally distributed quantitative variables. Using Pearson and Spearman's correlations, respectively, associations between the normally distributed and abnormally distributed continuous variables were investigated. Using the Chi-square test, categorical variables in study groups were compared. A p-value of 0.05 or lower was considered statistically significant.

3. Results

The study included 179 subjects divide into three groups, group 1 = 85 children with type 1 diabetes mellitus (41 boys and 44 girls; their mean age was 12.28 ± 3.32 years), group 2 = 57 siblings of the patients (29 boys and 28 girls; their mean age was 9.70 ± 3.86 years) and group 3 = 37 healthy controls (11 boys and 26 girls; their mean age was 10.86 ± 3.32 years). Positive family history of T1DM was noticed in 80 % of cases.

Table 1 illustrated clinical and laboratory data of the studied groups which showed statistical significant difference in age, systolic blood pressure, diastolic blood pressure, HbA1c, MCV, HDL, LDL and Ca as P value <0.001, <0.001, 0.02, <0.001, 0.002, <0.001, 0.003 and 0.002 respectively.

Table 1.

Data on the study groups' demographics and laboratory conditions.

Parameter	Group 1 (T1DM) N = 85 Mean ± SD	Group 2 (Siblings) N = 57 Mean ± SD	Group 3 (controls) N = 37 Mean ± SD	ANOVA Sig
Age (Years)	12.28 ± 3.32	9.70 ± 3.86	10.86 ± 3.32	0.000*
Males no (%)	41 (48.2 %)	29 (51.0 %)	11 (51.8 %)	0.10
Females no (%)	44 (51.8 %)	28 (49.0 %)	26 (51.8 %)
BMI (kg/m2)	20.25 ± 4.18	19.00 ± 5.14	19.03 ± 3.85	0.136
Systolic BP (mmHg)	107.18 ± 11.81	68.77 ± 17.498	94.70 ± 10.27	0.000*
Diastolic BP (mmHg)	69.29 ± 8.09	73.80 ± 13.96	70.78 ± 10.29	0.028*
WC/HC	0.86 ± 0.07	0.86 ± 0.047	0 0.84 ± 0.034	0.248
HbA1c%	8.11 ± 1.67	4.72 ± 0.608	4.61 ± 0.44	0.000*
MCV	76.88 ± 6.42	75.93 ± 6.01	80.67 ± 4.45	0.002*
T. C (mg/dl)	174.30 ± 31.24	163.12 ± 26.50	174.47 ± 33.35	0.184
Triglyceride (mg/dl)	85.40 ± 35.62	78.03 ± 23.12	74.75 ± 31.32	0.212
HDL (mg/dl)	51.62 ± 14.22	49.69 ± 11.91	63.05 ± 14.42	0.000*
LDL(mg/dl)	80.06 ± 28.66	75.36 ± 23.62	96.44 ± 28.03	0.003*
Ca (mg/dl)	9.14 ± 0.736	9.02 ± 0.86	9.70 ± 0.78	0.002*
25(OH) D (ng/ml)	14.99 ± 9.24	16.31 ± 7.96	19.48 ± 7.42	0.031*

T1DM = Type 1 diabetes mellitus Data are expressed as means ± SD, *P < 0.05 is significant, (BMI) Body mass index, (BP) Blood pressure, (WC) Waist circumference, (HC) Hip circumference, (HbA1C%) Glycosylated hemoglobin, (MCV) Mean corpuscular volume, 25(OH)D = 25 hydroxy vitamin D, (T.C) (LDL) Total Cholesterol High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) (Ca) Calcium.

Table 2 showed that the frequency of complications in T1DM group was 74.1 % with the highest frequency was recorded for neuropathy 41.2 % (Fig. 1).

Table 2.

Frequency of complications in T1DM group.

		N (%)
Complications	Positive Negative	63(74.1) 22(25.9)
Type of complications	Neuropathy Myopathy Renal Cardiac Ocular	35(41.2) 35(41.2) 17(20.0) 13(15.3) 24(28.2)

Fig. 1.

Frequency of complication in T1DM group.

Fig. 1 showed the frequency of complications among diabetic children with highest frequency was recorded for neuropathy (41.2 %).

3.1. Vitamin D status in the studied groups

As illustrated in Table 1 and Fig. 2, mean serum 25(OH) Vitamin D levels were significantly reduced in T1DM children (14.99 ± 9.24 ng/ml) and siblings (16.31 ± 7.96 ng/ml) compared to the controls (19.48 ± 7.42 ng/ml) with p value = 0.031.

Fig. 2.

Serum 25(OH) Vitamin D levels (ng/mL) in the studied groups.

Fig. 2 showed that serum 25(OH) vitamin D levels were significantly reduced in T1DM children and their siblings.

By comparison between the three studied groups regarding to the history of exercise performance, Vitamin D supplementation and rickets affection, there were no significant difference as P value = 0.632, 0.505 and 0.356 respectively.

In our study we found significant correlation between serum 25(OH) Vitamin D and Ca (r = 0.218, p = 0.007), meanwhile it was not correlated with HbA1c in the studied groups.

3.2. VDR polymorphisms in the studied groups

Table 3 showed the distribution of genotype and allele frequencies of VDR polymorphism ApaI (rs7975232), FokI (rs2228570), TaqI (rs731236) and BsmI (rs1544410) in the studied groups. We found high significant difference in FokI and BsmI genotypes as p value = 0.001 and 0.026 respectively. Meanwhile, there was no significant difference between them in ApaI and TaqI as p value = 0.474 and 0.444.

Table 3.

Distribution of genotypes and alleles frequencies of ApaI, FokI, TaqI and BsmI among studied groups.

SNP	Group 1 (T1DM) N = 75(%)	Group 2 (Siblings) N = 54(%)	Group 3 (Controls) N = 36(%)	P value
ApaI (rs7975232)
AA	47(62.7)	36(66.7)	24(66.7)	0.474
AC	22(29.3)	12(22.2)	6(16.7)
CC	6(8.0)	6(11.1)	6(16.7)
ApaI allele
A	116(77.4)	84(77.8)	54(75)	0.914
C	34(22.6)	24(22.2)	18(25)
FokI (rs2228570)
AA	4(5.3)a	2(3.7) b	10(27.8)a,b	0.001
AG	28(37.3)	20(37.0)	12(33.3)
GG	43(57.3)	32(59.3)	14(38.9)
FokI allele
A	36(24)	24(22.2)	32(44.4)*	<0.001
G	114(76)	84(77.8)	40(55.6)
TaqI (rs731236)
AA	45(60.0)	24(44.4)	17(47.2)	0.444
AG	23(30.7)	22(40.7)	15(41.7)
GG	7(9.3)	8(14.8)	4(11.1)
TaqI allele
A	113(75.3)	70(64.8)	49(68.1)	0.164
G	37(24.7)	38(35.2)	23(31.9)
BsmI (rs1544410)
CC	8(10.7)	4(7.4) c	3(8.3) c	0.026
CT	49(65.3)	42(77.8)	17(47.2)
TT	18(24.0)	8(14.8)	16(44.4)
BsmI allele
C	65(43.3)	50(46.3)	23(31.9)	0.141
T	85(56.7)	58(53.7)	49(68.1)

Similar lower case letters are statistically significant when P < 0.05 is used in an analysis via the Chi square test.

^*The control group differs statistically from the other two groups.

The genotype frequency of FokI-GG was higher in T1DM patients (57.3 %) and siblings (59.3 %) than controls (38.9 %) but didn’t reach statistical significance. While, FokI-AA genotype frequency (p = 0.001) was significantly lower in T1DM patients (5.3 %) and siblings (3.7 %) compared with controls (27. 8 %). Regarding FokI-A allele frequency, it was significantly lower in T1DM patients (24 %) and siblings (22.2 %) than in controls (44. 4 %) as p value < 0.001. However, FokI-G allele frequency increased in T1DM patients (76 %) and siblings (77.8 %) than in controls (55.6 %) but didn’t reach statistical significance. This might explain that (GG genotypes and G allele) in the FokI polymorphism increased the risk for T1DM, meanwhile (AA genotypes and A allele) in the FokI polymorphism decreased the risk for T1DM.

The genotype frequency of BsmI-TT was 24 %, 14.8 % and 44.4 % in diabetic children, siblings and controls respectively without reaching statistical significance. But the genotype frequency of BsmI-CC was observed to be significantly higher in T1DMpatients than siblings and controls (p = 0.026), which could make the children at risk for development of T1DM.

In Table 4, by studying the association of metabolic phenotypes with FokI genotypes polymorphism in the studied parameters, there were no significant difference with BMI, DBP, SBP, lipid profile and HbA1c, meanwhile there was statistically significant difference between FokI genotypes and Vitamin D, with an overall p value = 0.024. After doing a pairwise comparison significant difference between AA and GG adjusted p value = 0.04, AA and AG p value = 0.014, however, there was no statistically significant difference between GG and AG p-value = 0. 088. Also AA genotype associated with higher Vitamin D levels this might explain that, AA could be a protective gene in our study.

Table 4.

The association of metabolic phenotypes with FokI Genotype polymorphism in Vitamin D levels.

	FokI Genotype	Mean	Std. Deviation	Minimum	Maximum	ANOVA Sig
25(OH)D (ng/ml)	GG	16.8879	7.73346	5.00	34.00	0.024*
	AG	13.9000	7.06836	3.50	28.10
	AA	20.5000	11.87939	12.10	28.90

25 hydroxy vitamin D = 25(OH)D.

^*P < 0.05 is statistically significant.

Regarding the association of metabolic phenotypes with BsmI genotypes polymorphism in the studied parameters, there was no statistically significant difference with BMI, DBP, lipid profile, HbA1c, or vitamin D, but there was a difference between BsmI genotypes and SBP, with an overall p value of 0.009, as shown in Table 5., After doing a pairwise comparison no significant difference between CT and CC adjusted p value = 0.925, TT and CC p value = 0.258, however, there was statistical significant difference between CT and TT p-value = 0.002.

Table 5.

The association of metabolic phenotypes with BsmI Genotype polymorphism in systolic blood pressure.

	Bsm1 Genotype	Mean	Std. Deviation	Minimum	Maximum	ANOVA Sig
SBP(mmHg)	CT	88.53	22.402	47	130	0.009**
	TT	108.57	13.363	75	120
	CC	90.00	42.426	60	120

SBP: systolic blood pressure.

^**p < 0.001 is highly statistically significant.

4. Discussion

Type 1 diabetes mellitus contributes about 5–15 % of diabetic cases, it is one of the most predominant chronic global public health issues with significant economic burden. Genetics are now likely the most prevalent and successful approach of identifying people who are at high risk of having T1DM before it is caused by environmental factors.¹⁷

We conducted this study to assess serum vitamin D levels and VDR polymorphism; ApaI (rs7975232), FokI (rs2228570), TaqI (rs731236), and BsmI (rs1544410) among Egyptian diabetic children and their apparently healthy siblings compared with unrelated healthy subjects, analyzing VDR polymorphisms in siblings of diabetic children can provide insights into the genetic basis of diabetes within families and contribute to better risk assessment and potentially lead to better prevention and management strategies.

Positive family history is considered one of the main features of TIDM,18, 19 and we noticed that positive family history of T1DM accounted for 80 % of our results.

According to Kirac et al. (2018),¹⁴ vitamin D plays a significant role in controlling the release of insulin by pancreatic beta-cells. Also, vitamin D has an immune-modulatory effect, as it decreases the expression of proinflammatory cytokines included in the pathogenesis of T1DM which might delay the onset of T1DM.⁷

It is believed that Vitamin D deficiency is linked to diabetic problems because it has a significant impact on beta cell dysfunction and insulin resistance.²⁰ In this study, serum 25 (OH) Vitamin D level was lower in diabetic children and siblings compared to healthy controls p-value 0. 031. About 91.7 % of diabetic children showed abnormally low serum vitamin D (either deficiency or insufficiency), which agreed with Abd-Allah et al. (2014) and Bener et al. (2009)8, 21 who reported low levels of Vitamin D in both healthy and diabetic children but it was more deficient in diabetic children. Much the same results were shown by Ahmed et al. (2019) and Alshawi et al. (2018)22, 23 stating lower levels of 25(OH) Vitamin D in diabetic children than in healthy controls. According to a meta-analysis study ²⁴ children with T1DM have considerably higher rates of vitamin D deficiency. Moreover, supplementation of Vitamin D during pregnancy for women and infants has a protective role against T1DM development.²⁵ Similarly, Giri et al. (2017)²⁶ reported that glycemic control in diabetic children and adolescents improved potentially by vitamin D intake. Although the status of vitamin D in diabetic patients is well documented, the definit impact of vitamin D insufficiency on the occurrence of T1DM is still unclear.²⁷

Our study found significant correlations between serum 25(OH) Vitamin D and Ca because it regulates the synthesis of calcium, increases intestinal absorption, and decreases fatty acid absorption from the gut.²⁸

The genes of the Vitamin D pathway, specifically the VDR, regulate the levels of cytokines in autoimmune illnesses like T1DM in addition to controlling the production and transport of vitamin D.29, 30

VDR gene polymorphisms; ApaI (rs7975232), FokI (rs2228570), TaqI (rs731236), and BsmI (rs1544410) SNPs, are the most frequently investigated VDR polymorphisms in non-skeletal disorders, such as T1DM.³¹

Different studies have looked at both the individual and combined impact of each VDR gene variant and the development of T1DM in various ethnic communities over the past few years with conflicting results.³² Also, few studies are detecting the VDR genotypes and alleles in the families of T1DM-affected children.³³

The current study showed statistically significant differences regarding the VDR genes (FokI-rs2228570 and BsmI-rs1544410) polymorphisms between the studied groups, stating significant association with T1DM. The same results were found in an Egyptian study that examined 120 diabetic children in comparison to 120 healthy subjects aged 7–17 years old, the genotype analysis was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).⁸ Matching results were found in Greek³⁴ and subgroup analysis of the American and African populations.³² In disagreement with our finding, some studies revealed that (FokI-rs2228570 and BsmI-rs1544410) polymorphisms were not associated with T1DM as in Egyptian study³⁵ which included 50 children with T1DM and 50 nondiabetics, age range was 6–12 years old and method for genotype analysis was Real-time polymerase chain reaction (PCR-RT), in addition to another study in the Portuguese population.¹¹ The variations in the results of similar studies in Egypt might be due to small and different sample sizes, using different methods for genotype analysis, and variations in the environmental factors affecting these populations as diet and ultraviolet ray exposure.

In agreement with our study, FokI-rs2228570 polymorphism significantly differed between diabetics and nondiabetics and it was significantly associated with the onset of T1DM as in a Brazilian study that examined 180 diabetic children divided into 2 groups according to the presence or absence of thyroid autoantibodies, aging from 9 to 16 years old and method for genotype analysis was PCR-RFLP,³⁶ the same association was shown in two Egyptian studies.18, 37

Meanwhile, another Brazilian study³⁸ included 189 diabetic patients and 194 controls, and genotype analysis done by PCR-RT showed that FokI-rs2228570 polymorphism was not associated with T1DM, which agreed with numerous studies in various populations as in Danish³³ and Pakistan.³⁹

Following our findings, BsmI-rs1544410 polymorphism had a significant association with T1DM development in Saudi⁴⁰ and Egyptian patients.²² But on the contrary, in Kuwaiti children⁴¹; there was no association between it and T1DM.

Other studies have found that BsmI polymorphism is more common in Korean controls⁴² and Brazilian nondiabetic populations,³⁶ indicating that BsmI polymorphism may be protective against the development of T1DM.

Regarding the VDR genes ApaI (rs7975232) and TaqI (rs731236) polymorphisms, our study found no statistically significant difference between the three studied groups with a p-value of 0.474 and 0.444 respectively. In agreement, the genotype and allele frequency in ApaI and TaqI gene polymorphisms in Saudi patients showed no difference between diabetic patients and healthy controls.⁴⁰ In contrast to us, an Egyptian study has shown that T1DM is linked to VDR (ApaI and TaqI) gene polymorphisms.⁴³ A Saudi study also discovered that ApaI was present in low frequency in T1DM.⁴⁴

It is valuable to know that the VDR polymorphisms distribution and their association with the T1DM development were not only debatable in the various populations but even in the same one.³⁵ For example; in our Egyptian population, results regarding FokI polymorphism were conflicting where it was associated with the susceptibility of T1DM in two studies8, 37 while it was not associated in a third one.⁴⁵ Another Asian study revealed that although the BsmI polymorphism was related with an increased risk of T1DM in East Asian patients, the FokI polymorphism was associated with a higher risk of T1DM in West Asian individuals.⁴⁶

The variations in the results of similar studies might be due to ethnic differences, small and different sample sizes, variable gene expressions, using different methods for genotype analysis, and variations in the environmental factors affecting these populations as diet, and ultraviolet ray exposure in addition to the multi genetic multifactorial nature of the disease itself.

When the relationship between risk factors and genotypes was examined, we discovered that people who carry the two A allele for the FokI-rs2228570 gene have statistically greater levels of vitamin D than people with other genotypes, which was in agreement with Ferraz et al. (2022)⁴⁷ results who documented that T1DM patients with TT (FokI-rs2228570) genotype (equivalent to AA in our study) had higher 25(OH) Vitamin D levels, suggesting this genotype may have protective effects. On the other hand, Morán-Auth et al. (2015)⁴⁸ found a link between this genotype and an increase in the fraction of CD4+ T cells in T1DM, indicating a potential risk effect of this genotype. The FokI-rs2228570 mutation, results in the production of a shorter protein affecting the translation start site, which might explain the discrepancy in results from different experiments.⁴⁹ This suggests that the serum vitamin D level may not be the only sign of vitamin D insufficiency. Consequently, VDR genotypes are crucial in determining biological outcomes of Vitamin D action in the body. To summarize, there is a link between the FokI genotypes and the vitamin D levels in the Egyptian children.

Another relation was noticed for BsmI-rs1544410 polymorphism between risk factors and mutations, according to the results of our study, those with the TT genotype have higher SBP than other genotypes this agreed with Lee et al. (2001).⁵⁰ On the contrary, in a Spanish Study, the (CC) genotype of BsmI-rs1544410 had elevated SBP than CT or TT genotypes in males but not in females.⁵¹ This may be due to vitamin D controlling genes as VDR is believed to be responsible for 30 -50 % of blood pressure variations,⁵² and a lack of vitamin D is associated with an increased risk of hypertension, chronic vascular inflammation.⁵³

Meanwhile, Vitamin D is linked to fatty acid oxidation, lipid synthesis inhibition, and regulation of lipid metabolism.⁵⁴ There was no association between VDR polymorphisms and lipid profile in our study which agreed with Gendy et al. (2019).⁵⁵

5. Conclusions

It is essential to monitor the status of Vitamin D in children with T1DM and supplement with it when necessary to help these diabetic children with low vitamin D levels benefit from vitamin D's therapeutic effects.

VDR polymorphisms may be a risk factor for the development of T1DM in Egyptian children as there is a link between the FokI genotypes and the vitamin D levels in the Egyptian children. Thus, establishing a link between vitamin D status and VDR polymorphisms with other genetic analyses of T1DM is crucial, as it has a significant impact on Egyptian children's health and primary care facilities, as well as their quality of life.

5.1. Recommendation

Identifying VDR polymorphisms in diabetic children and siblings can provide insights into the genetic basis of diabetes within families and leading to better risk assessment, counselling and management.

Ethics approval and consent to participate

Research protocol was approved by the medical ethics committee of National Research Centre, Cairo, Egypt, NO; 19207.All participants signed a written informed consent after the research protocols were carefully explained to them.

Consent for publication

All the authors approved the final version of this manuscript and consent for publication.

Availability of data and materials

Datasets used in this study are available in public databases as indicated in the paper citations. However, generated data and figures during this study are available on request to the corresponding author.

Funding

This study was a part of project (No. 12060148) funded by National Research Centre, Cairo, Egypt.

Author contributions

All authors participated in this study including data interpretation and manuscript preparation and revision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.