Association Of Vitamin D And Its Pathway Genes' Polymorphisms With Gestational Diabetes Mellitus

Backgrounds and objectivesGestational diabetes mellitus?GDM?is one of the most common complications during pregnancy,the prevalence of GDM had been increased by over 30%globally in the past two decades.Some common risk factors of GDM have been revealed,such as advanced age,high prepregnancy body mass index?BMI?,and family history of diabetes.Besides,a lot of attention has paid to the role of vitamin D?VD?in the progress of GDM,as accumulative evidence has shown an association between VD deficiency and GDM.Furthermore,animal studies also have demonstrated the beneficial effect of VD on insulin sensitivity and glucose homeostasis.Serum 25-hydroxyvitamin D?25?OH?D?is currently the most commonly used index to evaluate the nutritional status of VD of an individual.The level of 25?OH?D are not only affected by seasons and VD supplementation,but also by genetic background.There were some studies,conducting among healthy adults,aiming at revealing the association of VD metabolic pathway genes and 25?OH?D level.However,large sample studies on those genes and 25?OH?D levels throughout the pregnancy are still lacking,as several existing studies of a small sample have only focused on the association of the two during a certain period of pregnancy.Two functional single nucleotide polymorphism?SNP?on the GC gene,rs4588 and rs7041,which encoding the vitamin D binding protein?DBP,also known as Gc globulin?can form six different Gc isoforms,and different Gc isoforms have differences both in DBP levels and 25?OH?D binding affinity.According to the free hormone hypothesis,only a small proportion of 25?OH?D and 1,25?OH?₂D without binding to DBP,namely,free 25?OH?D and 1,25?OH?₂D,can be directly used by most cells to exert the biological function of VD.Therefore,combining individual 25?OH?D levels and their Gc isoforms can better reflect their VD nutritional status.However,most of the studies on VD and GDM focus on either 25?OH?D or the SNPs of its metabolic pathway genes,are inconsistent.Currently,there is no research to test the combined effect or interaction between 25?OH?D levels and the SNPs.Therefore,we utilized a prospective cohort study design to explore the association between the VD metabolic pathway genes SNPs and 25?OH?D levels in the first,second,and third trimester of pregnancy,respectively.The associations of VD,VD metabolic pathway genes SNPs,and Gc isoforms with each time-point blood glucose level of the oral glucose tolerance test?OGTT?,as well as GDM and GDM subtypes were explored.Materials and methodsZhoushan Maternal and Child Health Hospital of Zhejiang Province was selected as the research site and 2658 pregnant women were selected.The questionnaire was used to collect demographic characteristics and health behaviors of pregnant women during pregnancy.Plasma 25?OH?D₂ and 25?OH?D₃ levels in the first,second,and third trimester were measured.A total of 15 SNPs that were found to be related to the first-trimester 25?OH?D levels in our previous study,were selected and genotyped.Multivariate linear regression model and Logistic model were used to explore the association of VD metabolic pathway gene SNPs with VD levels,as well as VD deficiency,in the first,second,and third trimester,respectively.Stratified analyses were used to explore whether the season,VD supplementation will modify the associations.OGTT results were extracted from the electronic medical record system?fasting blood glucose:FBG;postprandial 1-hour glucose:PG1H;postprandial 2-hour glucose:PG2H?in the same cohort,and 3318 pregnant women with both available OGTT data and blood samples in either trimester were selected.Gestational VD levels were measured;VD pathway gene SNPs and GDM-related SNPs were genotyped.GDM was divided into three subtypes according to the OGTT.Multivariate linear regression model and Logistic regression model were used to explore the associations of prepregnancy BMI,gestational VD levels,VD pathway gene SNPs,GDM-related SNPs,Gc isoforms and the risk of GDM and GDM subtypes.Stratified analyses were used to explore the modifying effect of prepregnancy BMI and gestational VD nutrition status on the above-mentioned associations,and the possible interactions between SNPs.ResultsThe study of the VD pathway genes SNPs and gestational VD levels showed that rs2282679,rs3755967,rs2298850,rs1155563,and rs7041 on the GC gene were in high linkage disequilibrium?r²>0.96?.The rs2298849 and rs7041 mutations were associated with higher 25?OH?D levels in the first and third trimester but were not associated with the second-trimester 25?OH?D levels.Compare to women with AA genotype at GC-rs2298849,the 25?OH?D levels of the GA and GG genotypes increased by 0.967 ng/m L?P=0.0043?and 1.657 ng/m L?P=0.0007?in the first trimester,and increased by 1.751ng/m L?P=0.0237?and 1.030 ng/m L?P=0.3531?in the second trimester,respectively;Compare to women with AA genotype at GC-rs7041,the 25?OH?D levels in CA and CC genotypes increased by 1.160 ng/m L?P=0.0004?and 4.092 ng/m L?P<0.0001?in the first trimester,and increased by 1.467 ng/m L?P=0.0488?and 4.788 ng/m L?P=0.0012?in the third trimester.The mutations of the remaining 7 SNPs?rs1155563,rs16846876,rs17467825,rs2282679,rs2298850,rs3755967,rs4588?on the GC gene are all related with lower 25?OH?D levels in the first,as well as the second trimester,of which except for rs1155563,are also associated with lower 25?OH?D levels in the second trimester;heterozygous mutations at these GC site SNPs reduce 25?OH?D levels in the first,second,and third trimester by 1.278-2.118,2.217-2.367,and 2.297-4.190 ng/m L,respectively.And homozygous mutations reduce 25?OH?D levels by 1.889-3.807,2.199-2.777,and3.469-5.929 ng/m L,respectively.Besides,pregnant women with CC genotype at CYP24A1-rs2209314 loci were 1.878 ng/m L higher in the second-trimester 25?OH?D than those with TT genotype?P=0.0270?,but were not associated with the first-or third-trimester 25?OH?D levels.Compare with Gc2/2 with the lowest DBP levels,the 25?OH?D levels in 2/1f,2/1s,1 f/1 f,1 f/1s,and 1s/1s in the first trimester increased by 1.396 ng/m L?P=0.0147?,1.715ng/m L?P=0.0048?,2.801 ng/m L?P<0.0001?,3.774 ng/m L?P<0.0001?,and 5.829ng/m L?P<0.0001?,respectively;however,the difference in levels during the second trimester became smaller,only the 25?OH?D levels in 1f/1s increased by 3.230 ng/m L?P=0.0020?;The difference became bigger in the third trimester,compare with Gc2/2the 25?OH?D levels in 1f/1f,1f/1s,and 1s/1s increased by 5.441 ng/m L?P<0.0001?,5.845 ng/m L?P<0.0001?,and 7.478 ng/m L?P<0.0001?,respectively.Mutations at the CYP24A1-rs2209314 are associated with a higher increment of25?OH?D levels from the first to second trimester;while mutations at the rs1155563,rs16846876,rs17467825,rs2282679,rs2298850,rs3755967,and rs4588 on the GC gene are associated with lower changes in 25?OH?D levels from the second to third trimester,and the mutation of rs7041 is related to a higher 25?OH?D level increase in the second to third trimester.Compared with pregnant women with Gc isoform of 2/2,1f/1f,1f/1s,and 1s/1s are associated with higher increments in 25?OH?D levels in the second to third trimester.GC-rs1155563 and GC-rs2298849 are associated with first-trimester25?OH?D levels only in summer and autumn,while LRP2-rs10210408 is associated with first-trimester 25?OH?D levels only in winter and spring.The association of GC-rs7041,as well as its highly linked loci?rs2282679,rs3755967,rs2298850,rs1155563,r²>0.96?,Gc isoforms and the first-trimester 25?OH?D levels were more evident in pregnant women with a higher frequency of VD supplementation?>3 times/week?.The study of VD and its pathway genes and GDM found that there was a negative association between changes in 25?OH?D levels during pregnancy and gestational weight gain,and it was more obvious in pregnant women with VD deficiency at the first trimester??=-0.035 kg,P=0.0008?.Prepregnancy BMI was significantly positively correlated with FBG??=0.030?,PG1H??=0.063?,and PG2H??=0.025??all P<0.005?,respectively.Prepregnancy BMI was also associated with a higher risk for GDM subtype 1?OR=1.18,95%CI:1.12-1.24?and subtype 3?OR=1.26,95%CI:1.18-1.34?,respectively.The levels of VD in the first and second trimester,as well as the VD level change during the pregnancy,are negatively associated with FBG?first trimester:?=-0.003;second trimester:?=-0.0004;first to second trimester:?=-0.004;all P<0.0005?.In pregnant women who are overweight and obese before pregnancy,VD deficiency in the second trimester significantly increases the risk of GDM subtype1?OR=9.61,95%CI:3.30-28.0?,an increase in VD levels during the first and second trimester>6.7 ng/m L can also reduce the risk of GDM?OR=0.40,95%CI:0.17-0.92?and GDM subtype 1?OR=0.13,95%CI:0.03-0.52?.Compared with genotype CC of LRP2-rs10210408,PG1H and PG2H of genotype TC were reduced by 0.195 mmol/L?P=0.012?and 0.206 mmol/L?P=0.001?,and the risk of GDM was decreased by 21.6%?OR=0.784,95%CI:0.623-0.986?;compared with VDR-rs10783219 genotype AA,PG1H of genotype TT increased by 0.239 mmol/L?P=0.021?,and the risk of GDM of genotype TA/TT increased by 27.5%?OR=1.275,95%CI:1.030-1.577?.Compared with CDKAL1-rs7754840 genotype GC/GG,PG2H in genotype CC increased by 0.190 mmol/L?P=0.021?,and the corresponding GDM risk increased by42.5%?OR=1.425,95%CI:1.030-1.973?.Compared with MTNR1B-rs10830962genotype CC,PG1H and PG2H of genotype GC increased by 0.261 mmol/L?P=0.002?and 0.133 mmol/L?P=0.060?,and the blood glucose was more significant elevated in genotype GG?FBG:?=0.081,P=0.004;PG1H:?=0.613,P<0.001;PG2H:?=0.237,P=0.011?;accordingly,the risk of GDM in genotype GC and GG were increased by 52%?OR=1.523,95%CI:1.142-2.030?and 108%?OR=2.079,95%CI:1.457-2.967?,respectively.Compared with PRKCE-rs11682804 genotype GG,genotype AG had higher blood glucose levels at each time point of OGTT?FBG:?=0.044,P=0.025;PG1H:?=0.168,P=0.034;PG2H:?=0.132,P=0.044?,but the blood glucose level of OGTT of genotype AA was not different from that of genotype GG at all-time points?P>0.1?,and the mutation of this SNP did not increase the risk of GDM.In addition,VDR-rs10783219,CDKAL1-rs77548409 and MTNR1B-rs10830962 have significant interactions with each other on the risk of GDM?P<0.05 for all interaction terms?.Among pregnant women who were overweight or obese before pregnancy,compared with pregnant women with Gc isoform 1f/1f,those with 1f/2,2/2,1s/1f,1s/2,and 1s/1s showed both increasing trends on PG1H and PG2H?P for trend=0.031,0.037?,and the risk of GDM is 2.81?OR=2.81,95%CI:1.06-7.50?,3.09?OR=3.09?,95%CI:1.04-9.21),3.53?OR=3.53,95%CI:1.26-9.94?,4.18?OR=4.18,95%CI:1.46-11.9?,and 5.62 times?OR=5.62,95%CI:1.53-20.6??P for trend=0.005?than that of1 f/1 f.In pre-pregnant overweight and obese pregnant women,compared with women with Gc isoform of 1f/1f or 1f/2 and without VD deficiency in the second trimester,those of 1s/2 or 1s/1s and with VD deficiency in the second trimester had a profound risk for GDM?OR=11.5,95%CI:2.33-57.0,P=0.003?.However,in pregnant women who were not overweight before pregnancy,VD deficiency,and Gc isoforms were not associated with the risk of GDM.Conclusions1. This study found that the polymorphisms of GC,CYP24A1,and LRP2 of VD metabolic pathway genes can affect gestational 25?OH?D levels and the risk of VDdeficiency.As Gc isoform being represented as the level of DBP protein,the levelof 25?OH?D during pregnancy was positively associated with the level of DBP.Season and gestational VD supplementation will modify the associations of GC andLRP2 genes with the first-trimester 25?OH?D level.It is suggested that pregnantwomen with high-risk of VD deficiency can be early identified by detecting SNPsrelated to the VD metabolic pathway,and a personalized VD supplementationprogram can be conducted according to the season and the genetic background ofthe pregnant woman to prevent VD-related pregnancy complications.2. This study also suggests that GDM is a heterogeneous disease.Among pregnant women who are overweight and obese before pregnancy,VD deficiency in thesecond trimester and a relatively insufficient increase in gestational VD level are associated with a higher risk of elevated fasting blood glucose and GDM subtype 1.Polymorphisms at the VDR-rs10783219,CDKAL1-rs7754840,and MTNR1B-rs10830962 all increase the risk of GDM,and the three SNPs interact with each other on the risk of GDM.Among women who are overweight or obese before pregnancy,the lower percent of free 25?OH?D in the blood,the higher insulin resistance the pregnant women have,assuming the Gc isoform represents the percent of free 25?OH?D,which is manifested by elevated postprandial blood glucose.In addition,Gc isoform and second-trimester VD deficiency present a combined effect on the risk of GDM,implicating that VD may affect gluconeogenesis and Gc isoform affect the insulin resistance or insulin secretion.And the mechanisms need to be further studied.Therefore,the early detection of Gc isoform,25?OH?D levels or free 25?OH?D levels and insulin resistance in pregnant women may help clinicians to identify pregnant women with high risk for GDM at the early stage of pregnancy or even before pregnancy,thus conduct proper interventions to prevent the progress of GDM.