| Type 2 diabetes mellitus(T2DM)is a chronic metabolic disease characterized by hyperglycemia and caused by insufficient insulin secretion,insulin resistance,or both.More and more experimental evidence and epidemiological data showed that vitamin D deficiency was a risk factor of T2 DM.CYP2R1,CYP27B1,CYP24A1,GC and VDR are the key genes for vitamin D metabolism,translocation and functioning.Their abnormal expression will affect the level of vitamin D in vivo and its biological function,which may increase the risk of T2 DM.In view of the role of genetic and epigenetic variations of single nucleotide polymorphism(SNP),copy number variation(CNV)and DNA methylation in gene expression,investigating the associations of SNPs,CNVs,methylation levels of the key genes in vitamin D metabolic pathway with vitamin D deficiency,the level of insulin,insulin resistance and T2 DM is of great significance in understanding the molecular mechanisms associated with vitamin D in the pathogenesis of T2 DM,as well as in developing prevention strategies for vitamin D deficiency and T2 DM.AimTo compare the differences of SNPs,CNVs,methylation levels of CYP2R1,CYP27B1,CYP24A1,GC and VDR between case group and control group and to investigate the the associations of SNPs,CNVs,methylation levels of CYP2R1,CYP27B1,CYP24A1,GC and VDR with vitamin D deficiency,insulin resistance and T2 DM by case-control study.Then the associations will be verified by a family-based study.Methods272 patients with T2 DM were selected as the case group from the cross-sectional survey of chronic diseases in both Houzhai Town,Erqi District,Zhengzhou City,Henan Province and Qiaomiao Town,Wuzhi County,Jiaozuo City,Henan Province.And 272 people were selected as control groups for 1:1 case-control matching with the principles of the same region,same gender,similar age(no more than 3 years older or younger),same exposure to tobacco and alcohol and no blood relationship.Meanwhile,232 T2 DM patients were selected as probands.The probands and their parents,siblings were included for family-based study.Taqman fluorescent probe was applied for SNP genotyping.The variation of copy number was analyzed by TaqMan fluorescence probe relative quantitative PCR method.DNA methylation level was determined by high resolution melt curve method.Then logistic regression model,FBAT and PBAT software were applied to investigate the the associations of SNPs,CNVs,methylation levels of CYP2R1,CYP27B1,CYP24A1,GC and VDR with T2 DM.Results 1.Epidemiological characteristicsEpidemiological data showed that lack of vegetable intake,decreased physical activity,increased resting time and high BMI were risk factors for T2 DM.2.Association between SNPs in vitamin D metabolic pathway genes and T2DM: A case-control study(1)The results of associations between SNPs and vitamin D deficiency indicated that rs12794714 and rs10766197 in CYP2R1 were significantly associated with vitamin D deficiency in the control group(P<0.05).(2)The results of associations between SNPs and the level of insulin indicated that rs10877012 and rs4646536 in CYP27B1 were significantly associated with the level of insulin in the control group(P<0.05).(3)The results of associations between SNPs and HOMA-IR index indicated that rs10766197 in CYP2R1 and rs739837 in VDR were significantly associated with HOMA-IR index in the control group(P<0.05).(4)The results of single factor logistic model showed that rs12794714 and rs10766197 in CYP2R1,rs2248359 and rs6068816 in CYP24A1,rs7975232 and rs2189480 in VDR were significantly associated with T2 DM.The risk of T2 DM for AG genotype of rs12794714 was 1.699 times higher than GG genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR,1.732,95% CI 1.148-2.613,P=0.009).The risk of T2 DM for AG genotype of rs10766197 was 1.565 times higher than GG genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR,1.552,95% CI 1.027-2.344,P=0.037).The risk of T2 DM for TT genotype of rs2248359 was 1.916 times higher than CC genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR,2.261,95% CI 1.167-4.382,P=0.016).The the risk of T2 DM for TT genotype of rs6068816 was 0.497 times lower than that of CC genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR 0.480,95% CI 0.258-0.892,P=0.020).The risk of T2 DM for TT genotype of rs7975232 was 1.426 times higher than GG genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR 1.489,95% CI 1.025-2.165,P=0.037).Compared with CC genotype of rs2189480,CA genotype increased the risk of T2 DM for 2.239 times(Adjusted OR 2.207,95% CI 1.207-4.035,P=0.010)and AA genotype increased the risk of T2 DM for 2.336 times(Adjusted OR 2.598,95% CI 1.386-4.870,P=0.003).(5)The results of multifactor factors logistic model showed that rs12794714 in CYP2R1,rs6068816 in CYP24A1,and rs2189480 in VDR were significantly associated with T2 DM.The risk of T2 DM for AG genotype of rs12794714 was 1.78 times higher than that of GG genotype.And it was still statistically significant after adjusting confounding factors(Adjusted OR,1.833,95% CI 1.197-2.806,P=0.005).Compared with CC genotype of rs2189480,CA genotype increased the risk of T2 DM for 2.208 times(Adjusted OR 2.189,95% CI 1.175-4.077,P=0.014)and AA genotype increased the risk of T2 DM for 2.302 times(Adjusted OR 2.599,95% CI 1.356-4.982,P=0.004).The the risk of T2 DM for TT genotype of rs6068816 was 0.481 times lower than that of CC genotype(Adjusted OR 0.477,95% CI 0.252-0.904,P=0.023).It was a protective factor for T2 DM.(6)The statistical results of the simple additive genetic risk scoring model showed that the risk of T2 DM were increased in the 2nd,3rd and 4th groups with higher risk score when compared with the 1st as reference(P<0.05).The adjusted OR were 1.644(95%CI 1.046-2.584),2.418(95%CI 1.455-4.020)and 2.305(95%CI 1.312-4.051),respectively.The statistical results of the weighted genetic risk scoring model indicated that the risk of T2 DM were increased in the 3rd and 4th groups with higher risk score when compared with the 1st group as reference(P<0.05).The adjusted OR were 2.10(95%CI 1.156-3.493)and 2.951(95%CI 1.749-4.987),respectively.3.Association between copy-number variation in vitamin D metabolic pathway genes and T2DM: A case-control studyThe results of associations of CNVs with vitamin D deficiency,HOMA-IR index and T2 DM indicated that the CNV of CYP2R1 may be associated with vitamin D deficiency in the case group(P=0.053).The CNV of CYP24A1 was significantly correlated with HOMA-IR index in the case group(P<0.001).The CNV of CYP27B1 was significantly associated with T2 DM.The risk of T2 DM were increased in the 2nd and 3rd groups with higher CNV when compared with the 1st group as reference(P<0.05).The OR were 1.635(95%CI 1.005-2.661)and 1.961(95%CI 1.180-3.262),respectively.It was still significant after adjusting confounding factors(OR 2.008,95%CI 1.176-3.427,P=0.011).4.Association between methylation status in vitamin D metabolic pathway genes and T2DM: A case-control studyThe results of associations of DNA methylation with vitamin D deficiency,HOMA-IR index and T2 DM indicated that there was no significant association for methylation level of CYP2R1,CYP27B1,CYP24A1,GC,and VDR with vitamin D deficiency in both case and control groups.Methylation in VDR was correlated with HOMA-IR index inversely(r=-0.119,P=0.05).High methylation level of GC may increase the risk of T2DM(OR 1.179,95% CI 0.999-1.391,P=0.05),even adjusting confounding factors(Adjusted OR 1.222,95% CI 1.025-1.458,P=0.026).5.Associations of SNPs,copy-number variation and methylation status in vitamin D metabolic pathway genes with T2DM: A family-based study(1)The FBAT results of SNP and T2 DM indicated that rs2248359 and rs4809957 in CYP24A1 were associated with T2 DM in all the Additive,Dominant and Recessive models(P<0.05).Association between rs2248359 and T2 DM in case-control study was verified in this family-based study.Besides,there was genotype incompatibility for rs2248359 in T2 DM pedigree.The probability of CT genotype in offspring increased if the paternal and maternal genotypes were CC and CT(OR 6.245,95%CI 1.868-20.883).(2)The PBAT results of T2 DM with CNV and DNA methylation indicated that there was no significant association of CNV and methylation level of CYP2R1,CYP27B1,CYP24A1,GC,and VDR with T2DM(P>0.05).And there was no difference for CNV and methylation level of CYP2R1,CYP27B1,CYP24A1,GC,and VDR between parents and offspring(P>0.05).Conclusion1.The associations of CYP2R1 with vitamin D deficiency,HOMA-IR index and T2 DM indicated that CYP2R1 may play a role in regulations of vitamin D status and insulin sensitivity,and development of T2 DM.2.The associations of CYP27B1 with the level of insulin and T2 DM indicated that CYP27B1 may play a role in insulin secretion.Then CYP27B1 may take part in the development of T2 DM.3.The associations of CYP24A1 with the level of insulin,HOMA-IR index and T2 DM indicated that CYP24A1 may participate in regulation of the bioactivity of vitamin D,insulin secretion,and insulin sensitivity.Then CYP27B1 may take part in the development of T2 DM.4.The association between the methylation level of GC and T2 DM indicated that GC may play a role in regulation of vitamin D bioactivity.Then GC may take part in the development of T2 DM.5.The association between VDR with and T2 DM indicated that regulation of vitamin D bioactivity may play a role in the development of T2 DM.6.Cumulative mutations in vitamin D metabolic pathway genes can increase the risk of T2 DM.Therefore,the results of this study provide new evidence for the association between vitamin D and T2 DM,and provide data for establishment and improvement of the prevention strategy for T2 DM. 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