| ObjectiveDyslipidemia during pregnancy women is a risk factor for several diseases.Association of blood lipid-related SNPs in normal population and serum lipid levels during pregnancy have not been reported.The aim of the study was to investigate the correlation between lipid-related SNPs and levels of blood lipids in pregnancy,including triglyceride(TG),total cholesterol(TC),high density lipoprotein-cholesterol(HLD-C)and low density lipoprotein-cholesterol(LDL-C),to identify the risk alleles.And to restructure the plasmid containing the target gene sequence with miRNA-binding sites which were related with blood lipids levels in pregnancy.MethodsSNP selection:? We screened SNPs in the genome-wide association study(GWAS)that was related with TG?TC?HDL-C and LDL-C levels and then selected SNPs with larger effect size as well as smaller p values as candidate genes.We identified the SNPs with a minor allele frequency>5%in the Chinese population based on the NCBI dbSNP database and 1000 genomes browser.? Then we searched for 3'UTR as well as 5'UTR of the genes which were related with more than three kinds of lipids.Software were used to investigate SNPs in target binding sites for miRNAs and transcription factor binding sites.Genotyping of SNPs was performed with Sequenom MassArray system in 2018 pregnant women.Direct sequencing was performed to confirm the accuracy of genotyping results.Pre-pregnancy weight,age and birth weight of all subjects were recorded,and levels of blood lipid,blood glucose,insulin and glycated hemoglobin were tested in 24-28 weeks during pregnacy.Multiple linear regression adjusted for age,gestational week and fasting plasma glucose were used to analyze the association between these SNPs and blood lipid levels in pregnancy.We select SNPs in 3'UTR of the genes which were significantly related with serum lipid levels in pregnant women and restructure the plasmid containing the target gene sequence with these miRNA-binding sites.Results1.A total of 29 SNPs were selected from 157 candidate genes:CILP2 rs10401969?APOB rs1042034.rs1367117?LPLrs1059611?ANGPTL3 rs12565895?rs2131925?HMGCR rs12916?LIPC rs 1532085?GALANT2 rs16851339?MLXIPL rs17145738?FADS1/2 rs174561?ABCA1 rs1883025?HPR rs2000999?ABCG5 rs2278356?TRIB1 rs2280826?rs2954029?rs3201475?PCSK9 rs2479409?CETP rs3764261?LIPG rs3813082?rs3813083?rs3829632?rs7241918?rs8088283?APOC3 rs4225?APOE-C1-C2 rs439401?PCSK9 rs45448095?TIMD4 rs6882076 and ABO rs9411489.The genotyping success rate of all the SNPs was over 95%except fot three loci,including rs17145738?rs1042034 and rs8088283,and the accuracy rate was 100%.2.The correlation between SNPs and quantitative metABOlism traits1)SNPs associated with TC levels in late-second pregnancy The risk allele A of rs3764261 was positively associated with TC level(B=0.139 mmol/L,P<0.05),the risk allele T of rs174561 was positively associated with TC level(B=0.120 mmol/L,P<0.01),and the risk allele G of rs3813083 was positively associated with TC level(B=0.157 mmol/L,P<0.01).Rs174561 and rs3813083 were located in 5'utr of the genes respectively.2)SNPs associated with TG levels in late-second pregnancy The risk allele G of rs4225 was positively associated with TG level(B=0.168 mmol/L,P<0.01)and the risk allele T of rs1059611 was positively associated with TG level(B=0.187 mmol/L,P<0.05),and they were both located in 3'utrof the genes.The risk allele T of rs2280826 was positively associated with TG level(B = 0.102mmol/L,P<0.05)and the risk allele T of rs3201475 was positively associated with TG level(B = 0.117 mmol/L,P<0.05),and they were both located in 5' utr of the genes.3)SNPs associated with HDL-C levels in late-second pregnancy The risk allele T of rs4225 was negatively associated with HDL-C level(B=-0.071 mmol/L,P<0.01),the risk allele C of rs3764261 was negatively associated with HDL-C level(B=-0.055 mmol/L,P<0.05),the risk allele T of rs1883025 was negatively associated with HDL-C level(B=-0.054 mmol/L,P<0.05)and the risk allele T of rs16851339 was negatively associated with HDL-C level(B =-0.064 mmol/L,p<0.05).Rs4225 and rs16851339 were located in 3'utr of the genes respectively.4)SNPs associated with LDL-C levels in late-second pregnancy The risk allele A of rs 13 67117 was positively associated with LDL-C level(B=0.110 mmol/L,P<0.05),the risk allele T of rs174561 was positively associated with LDL-C level(B=0.081 mmol/L,P<0.05),the risk allele A of rs2278356 was positively associated with LDL-C level(B=0.136 mmol/L,P<0.01),the risk allele C of rs2280826 was positively associated with LDL-C level(B=0.087 mmol/L,P<0.05).Rs2278356 was located in 3'utr and rs174561 as well as rs2280826 were located in 5'utr of the genes respectively.3.To investigate the combinability of miRNA predicted by different softwares with these four SNPs,we successfully made the DNA fragments containing each SNP cloned into the 3'UTR of the firefly luciferase report gene in the pmir-GLO carrier.There we get four kinds of recombinant plasmid,including pmir-GLO-rs4225?pmir-GLO-rs1059611.pmir-GLO-rsl6851339 and pmir-GLO-rs2278356.ConclusionsThe study first extensively investigated the association between lipid-related SNPs indentified in GWAS and the lipid levels in pregnant population.And we found several risk alleles significantly associated with blood lipid levels.Some miRNA-binding sites and transcription factor binding sites related with lipid levels during pregnancy were firstly found in our study.It may provide evidence to the genetic mechanism of blood lipids changes in pregnancy and the early intervention and treatment of hyperlipidemia in pregnancy.And four kinds of recombinant pmir-GLO plasmid were successfully made which is ready for further study. |
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