Type 1 diabetes (T1D) is generally considered a chronic autoimmune disease caused by the destruction of insulin-producing pancreatic beta cells (1, 2), although T1D can occur at any age, it is one of the most common chronic diseases in childhood. Peak clinical presentation occurs between 5 and 7 years of age, at or near adolescence (3, 4). Globally, both the incidence and prevalence of T1D are increasing, however the greatest increase in incidence occurs in children under 15 years of age, especially in children under 5 years of age (5), These increases cannot be fully explained by genetic changes, involving environmental or behavioral factors, or both. T1D, once considered a single autoimmune disease, is now increasingly recognized as caused by complex interactions between environmental factors and the microbiome, genome, metabolism, and immune system (6). Many environmental factors have been associated with T1D, including diet, vitamin D deficiency, viruses associated with islet inflammation (e.g., enteroviruses), and decreased gut—microbiome diversity. There is growing evidence that low vitamin D concentrations are strongly associated with a variety of adverse outcomes, such as asthma, diabetes, cardiovascular disease, and certain cancers (7–10). Common genetic variants affecting circulating 25(OH) D levels may be important in enhancing our understanding of the observed associations between vitamin D status and several diseases.

Vitamin D is a fat-soluble steroid, ergocalciferol (vitamin D2) is produced by ultraviolet irradiation of the plant sterol ergosterol, and cholecalciferol (vitamin D3) is produced from its precursor 7-dehydrocholesterol (7-DHC) synthesized in human skin under ultraviolet light. Epidemiological evidence suggests that vitamin D deficiency is associated with the pathogenesis of T1D (11, 12). as 25(OH) D synthesis is genetically regulated, single nucleotide polymorphisms (SNPs) may alter bioavailability and targeting effects of vitamin D metabolites, polymorphisms in genes critical for vitamin D metabolism have also been shown to modulate T1D risk (13). Genome-wide association studies(GWAS) have confirmed that distinct polymorphisms in genes involved in vitamin D metabolism may affect islet autoimmunity and risk of T1D, SNPs of important genes involved in synthesis (7-dehydrocholesterol reductase, DHCR7), hydroxylation (cytochrome P450 family 2 subfamily R member 1, CYP2R1), degradation (cytochrome P450 family 24 subfamily A member 1, CYP24A1) and transcription (vitamin D receptor, VDR) (14, 15). The purpose of this study was to evaluate the relationship between CYP2R1(rs1993116, rs12794714), DHCR7(rs12785878), CYP24A1 (rs17216707), VDR (rs1544410), T1D and vitamin D levels in Chinese children.

We designed this case-control study to determine the role of five polymorphisms and their influence on 25(OH)D levels and susceptibility to T1D in Chinese children. we have identified that CYP2R1 (rs1993116) polymorphism was significantly correlated with T1D in Chinese children. In addition, GMDR analysis showed that there was significant gene-gene interaction among rs12794714 and rs1993116, which was the model with the highest prediction accuracy of the risk of T1D in the five SNPs.

Genome wide association studies (GWAS) meta-analysis of serum 25-hydroxyvitamin D suggests that SNPs in CYP2R1, DHCR7, CYP24A1, and VDR genes are associated with circulating 25(OH) D levels (14, 19). However, no evidence of this association was observed in our study. Yan Wang et al. also did not find correlation between CYP2R1 (rs12794714, rs1993116) and 25(OH) D concentration levels in the Chinese rural population (20). The possible reasons are as follows: (i) There are genetic background and ethnic differences in circulating vitamin D (21), GWAS-related study populations are mostly of European ancestry, and Geographic differences in diet, sun exposure, and genetic background may alter the susceptibility caused by these genetic variants (22). (ii) Each SNP has a small undetectable effect, and combinations of several SNPs may result in an additive effect, leading to a reduction in vitamin D levels. (iii) The sample size of our study is not large enough, and Vitamin D levels are not controlled for seasonal effects.

Several human observational studies have provided evidence for an association between serum 25 (OH) D concentration and the risk of T1D (23, 24), our study also confirmed a higher prevalence of vitamin D deficiency and insufficiency in T1D. As 25(OH)D synthesis is genetically regulated, single nucleotide polymorphisms (SNPs) may alter bioavailability and targeting effects of vitamin D metabolites (25). Previous correlation studies have shown the association of common variant polymorphisms of DHCR7, CYP2R1, CYP24A1, and VDR with T1D, providing early support for the causal role of 25(OH) D in the pathogenesis of T1D (8, 26–29). However, some of the results are controversial, Evidence from a Mendelian randomization study and several systematic reviews and meta-analyses suggests an invalid association between selected variants affecting serum 25 (OH) D concentration and T1D (25, 30). The association of CYP2R1 (rs1993116) with T1D susceptibility in Chinese children was confirmed in our case-control study, We found genotypes “CC” (OR = 2.968, 95% CI = 1.313–6.713, p = 0.009,) and “CT” (OR = 2.271, 95% CI = 1.066–4.838, p = 0.033,) in CYP2R1 (rs1993116) is significantly related to an increased T1D risk, Carriers of the CC, CT genotypes of rs1993116 have higher T1D risk than those carrying the TT genotype, and the C allele carriers of rs1993116 had a higher risk of T1D than the T allele carriers.

The CYP2R1 gene is localized on chromosome 11p15.2 and has five exons, spanning across a region of approximately 15.5 kb, In humans, CYP2R1 is mainly distributed in the pancreas, liver, and kidney (31). It encodes microsomal vitamin D 25 hydroxylase, which is generally considered to be the most important enzyme in vitamin D metabolism. CYP2R1 catalyzes the 25 hydroxylation of vitamin D3 and vitamin D2 at comparable rates, In mice, knockout of CYP2R1 reduced 25(OH) D levels by only 50% and did not affect circulating 1,25(OH)2D (32), Which proves that there is a compensatory mechanism for CYP2R1 deletion, This may explain the lack of association between CYP2R1 and 25(OH)D concentration levels in our study. We confirmed that CYP2R1 (rs1993116) increases the risk of T1D, but CYP2R1 (rs12794714) does not, The probable reason is that Since rs12794714 is located in an intronic region, these synonymous variants do not alter the protein sequence and therefore have minimal impact on 25-hydroxylase activity and function (33). We originally hypothesized that genetic polymorphisms associated with vitamin D deficiency would increase the risk of T1D, however, our results indicated that CYP2R1 (rs1993116) was not related to 25(OH)D levels, but was significantly associated with T1D. Whether vitamin D deficiency is a cause or secondary to T1D is still controversial. Due to the relatively limited sample size, these results should be interpreted with caution and further research is required.

Cooper et al. obtained evidence that DHCR7 and CYP2R1 are associated with T1D (26), A recent study showed that CYP2R1 (rs12794714) is closely associated with T1D in Korean children (8). However, we did not observe an effective association between DHCR7 (rs12785878), CYP24A1 (rs17216707), CYP2R1 (rs12794714), and VDR (rs1544410) polymorphism and T1D in Chinese children, Further research is needed in the future.

Growing evidence suggests that T1D is a polygenic metabolic disease with multiple etiologies, and it is necessary to evaluate gene interactions when studying the genetic etiology of T1D. In our study, we found gene interaction between CYP2R1 (rs12794714) and CYP2R1 (rs1993116) was significantly associated with the risk of T1D. Carrying the CYP2R1 (rs1993116) genotype CC + CT in conjunction with the CYP2R1 (rs12794714) genotype CC or genotype TT + CT increases the risk of T1D. Although the effect of rs12794714 on T1D is insignificant, the additive interaction between rs12794714 and rs1993116 will increase the risk of T1D.

So far, there are few studies on the relationship between vitamin D, vitamin D-related genes and T1D in Chinese children. We have studied five SNPs of four genes, which can better understand the impact of vitamin D on the pathogenesis of T1D. Also, our study had some limitations, Firstly, the selection bias in this case-control study was its hospital-based design, and it may not be representative of the general population. Secondly, we did not study all the genes involved in the vitamin D metabolic pathway. Finally, our sample size is relatively small, Further expansion of the sample size is required in future studies.

In summary, We studied the triangulation between five SNPs in CYP2R1, DHCR7, CYP24A1, VDR genes, serum 25(OH) D levels and T1D in Chinese children. Our study confirms that vitamin D deficiency and insufficiency are more prevalent in T1D, and that CYP2R1 (rs1993116) polymorphism is a candidate gene for susceptibility to T1D in Chinese children, There was a synergistic effect of gene-gene interactions between CYP2R1 (rs12794714) and CYP2R1 (rs1993116) on the risk for T1D, Vitamin D metabolic pathway gene polymorphism, and gene-gene synergy can better explain the effect of vitamin D on the pathogenesis of T1D.