| ObjectiveGestational diabetes mellitus (GDM) is a multifactorial genetic disorder characterized by the inability of reduced pancreatic β-cell function to overcome progressive insulin resistance, and GDM shares same genetic factors with type 2 diabetes (T2DM). miRNAs, as a class of newly identified gene expression regulators, have been implicated in a wide variety of biological processes. Growing evidence suggests that common single nucleotide polymorphisms within miRNA-binding site (miRSNPs) contribute to the development of various diseases. However, the roles of miRSNPs in GDM have not been fully elucidated. The study aims to first extensively investigate the association between miRSNPs in candidate genes of T2DM and GDM susceptibility and quantitative metabolism traits in Chinese Han pregnant women.MethodsWe screened SNPs in the 3’UTR of all T2DM candidate genes that was found by GWAS and confirmed in Chinese population. We identified the SNPs with a minor allele frequency>5% in the Chinese population based on the NCBI dbSNP database and 1000 genomes browser. Four miRNA target prediction software including PolymiRTS, TargetScan, miRDB and MicroRNA.org were used to investigate SNPs in target binding sites for miRNAs. Genotyping of SNPs was performed with TaqMan allelic discrimination assays in 839 GDM patients and 900 controls. Direct sequencing was performed to confirm the accuracy of genotyping results. Multivariable logistic regression analysis and multiple linear regression adjusted for age were used to analyze the association between miRSNPs in candidate genes of T2DM and GDM susceptibility and quantitative metabolism traits. Dual-Luciferase Reporter Assay System was used to confirm the binding between miRSNPs and predicted miRNA.Results1. A total of 17 miRSNPs were selected from 49 T2DM candidate genes:SLC30A8 rs2466293, KCNB1 rs1051295, KCNQ1 rs8234, CDC123 rs12126, VPS26A rs1802295, LPIN1 rs1050800, KCNJ11 rs5210, INSR rs1366600, rs3745550, PAX4 rs712699, NLRP3 rs10802502, MFN2 rs1042842, HNF1B rs2688, GIPR rs73042335, CDKN2B rs 1063192, rs3217992 and CDKN2A rs3088440. The seventeen selected miRSNPs were predicted to interact with hsa-miR-660, hsa-miR-216a, hsa-miR-380, hsa-miR-361, hsa-miR-510, hsa-miR-96, hsa-miR-219, hsa-miR-106, hsa-miR-600, hsa-miR-223,hsa-miR-548,hsa-miR-183,hsa-miR-299, hsa-miR-135b, hsa-miR-323b, hsa-miR-138 and hsa-miR-328 respectively and all the miRSNPs were in Hardy-Weinberg equilibrium in control subjects (p>0.05).2. GDM patients were older, exhibited higher levels of fasting glucose and insulin, increased glucose and insulin levels at 1h,2h and 3h during an OGTT, higher insulin resistant (HOMA-IR) and AUC, and lower beta-cell function (HOMA-B) with respect to controls (p value<0.05).3. Comparison of genotype and allele frequencies:The genotype frequencies of six SNPs including rs 1063192 (CDKN2B), rs 1051295 (KCNB1), rs 1042842 (MFN2), rs2466293 (SLC30A8), rs1366600 (INSR) and rs712699(PAX4) were significantly different between GDM and controls (p value<0.05). The homozygous genotype of minor allele (CC,CC, AA, CC, CC and AA genotype, respectively) were the risk factors for GDM (OR= 1.455,1.579,1.398,1.480,2.191 and 1.366, respectively). The minor allelic frequencies of rs1063192, rs1051295, rs1042842 and rs2466293 was significantly higher in GDM group than in controls (p value<0.05).4. The correlation between SNPs and quantitative metabolism traits1) The risk allele C of rs2466293 was positively associated with glucose at OGTT 2h (b= 0.062), and negatively associated with HOMA-B (b=-0.057). The CC genotype exhibited lower fasting insulin than TT and TT+TC genotype (p value <0.05).2) The risk allele C of rs1051295 was positively associated with fasting glucose, glucose at 2h and 3h during OGTT, and HOMA-IR (b=0.006,0.056,0.058 and 0.065, respectively). The CC+TT genotype exhibited higher fasting insulin and insulin at 2h and 3h during OGTT than TT genotype (p value<0.05).3) The risk allele A of rs1042842 was positively associated with glucose at OGTT 3h (b=0.056, p value<0.05).4) The CC genotype of rs1063192 was positively associated with glucose at 1h,2h and 3h during OGTT (b=0.062,0.048 and 0.062,respectively). The CC genotype exhibited higher insulin levels at 3h and 3h during OGTT than TT and TT+CT genotype (p value<0.05).5) The risk allele A of rs712699 was positively associated with glucose at OGTT 2h (b=0.051, p value<0.05).6) The minor allele T of rs 1802295 was negatively associated with fasting glucose and HOMA-IR (b=-0.050 and -0.056,respectively; p value<0.05).7) The minor allele T of rs 1050800 was positively associated with glucose at 2h and 3h during OGTT, and negatively associated with HOMA-B (b= 0.047, 0.061and -0.059, respectively; p value<0.05).8) The minor allele C of rs2688 was negatively associated with HOMA-B and HOMA-IR (b=-0.070 and-0.065, respectively). The CC genotype exhibited lower fasting insulin than AA genotype (p value<0.05).9) The minor allele C of rs 10802502 was negatively associated with glucose at OGTT 1h (b=-0.058). The CC genotype exhibited lower insulin level at OGTT 2h than TT and TT+CT genotype (p value<0.05).10) The minor allele A of rs73042335 was positively associated with glucose at OGTT 1h (b= 0.049, p value<0.05).11) The AA genotype of rs3217992 exhibited lower insulin level at OGTT 2h and 3h than TT genotype (p value<0.05).5. The results of Dual-Luciferase Reporter Assay System showed that miR-223 inhibited the expression of luciferase gene in which 3’UTR harbored the AA genotype of rs712699 in PAX4 gene.Conclutions1. The study first extensively investigated the association between miRSNPs in candidate genes of T2DM and GDM susceptibility, and found that minor allele homozygous genotype of miRSNPs rs2466293, rs1051295, rs1042842, rs1063192, rs 1366600 and rs712699 were associated with higher risk for GDM.2. The genotypes of rs2466293, rs1051295, rs1042842, rs1063192, rs1366600, rs712699, rs1802295, rs1050800, rs2688, rs10802502 and rs73042335 were associated with various glucose metabolism traits.3. The AA genotype of rs712699 in PAX4 gene 3’UTR creates a novel miR-223 binding site. |
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