Effect Of DHA Intake And ELOVL2/5 Genetic Variants On Maternal And Neonatal Fatty Acid Levels And Its Mechanism

Early life is a critical period for human physiology,immune system,metabolism and structural development.During this period,maternal dietary intake and infant feeding can have long-term effects on infant growth and metabolic health.Nutritional supply in early life,such as fatty acid supply plays a key role in the development of the fetus and infant and may regulate the development of the immune system and the composition of intestinal bacteria that cause autoimmune disorders or allergic diseases.During pregnancy,the supply of fatty acids in fetus is mainly transported from mother through placenta.As ideal food source for infants,breast milk and its nutritional status is directly related to the growth and development of infants.Fatty acids in breast milk vary greatly according to the nutritional status of maternal lipids.It is well-known that nutrients may modulate molecular mechanisms of physiological functions by interacting with genes.Fatty acid levels in the body are regulated by both dietary intake and metabolic mechanisms in vivo.Fatty acid elongases(ELOVL)encoded by the ELOVL2/5 gene,as key enzymes in the process of PUFA synthesis in the body,are regulated by complex biological mechanisms,and these enzymes change the levels of different fatty acids by participating in the diversity of synthetic pathways.Single nucleotide polymorphisms(SNPs)of ELOVL gene are associated with human fatty acid composition and affect fatty acid metabolism in different tissues.Carriers of different genotypes have different levels of human fatty acid.At present,the results of mechanism studies on the association between elongases and PUFA levels are still inconsistent,but most involved in ELOVL2/5 gene variants and docosahexaenoic acid(DHA)intake.In this study,paired samples of maternal blood and cord blood were collected to investigate the combined effects of dietary DHA intake and genetic variants on maternal and infant plasma fatty acid levels;on the other hand,breast milk was collected from postpartum healthy nursing mothers to analyze the effects of DHA and ELOVL2/5 polymorphisms on breast milk fatty acids.Finally,the potential mechanisms of genetic variants on fatty acid effects were explored through DHA supplement intervention studies.This study includes the following three parts.Part Ⅰ: Effect of DHA intake and ELOVL2/5 gene variation on plasma fatty acid levels of pregnant women and newbornsObjective:The aim of the study was to observe maternal and cord blood plasma phospholipid fatty acid levels and to identify the combined effects of dietary DHA intake and genetic variants on maternal and infant fatty acid levels.Methods:Total 116 pairs of healthy,Han Chinese pregnant women and their newborns registered in the hospital in Changchun were included into the study.All signed informed consent and received face to face investigation of basic information and dietary intake and provided blood sample.Plasma phospholipid fatty acid levels were measured by gas chromatography,and a total of 10 SNPs on the ELOVL2/5 genes were selected and genotyped using the Sequenom Mass Array system.SPSS 24.0 software was used to analyze the plasma phospholipid fatty acid composition and correlation of maternal and cord blood fatty acid.SNPstats online analysis software was used to analyze the association between gene variants and fatty acid levels.Results:1.The corresponding kind of fatty acids in the plasma of pregnant women and newborns showed a significant positive correlation overall.C18:2n-6cis(LA)level was highest in pregnant women plasma,while C20:4n-6(ARA)level was the highest in neonatal cord blood.2.Compared with the major allele homozygous carriers,the plasma LA level of the minor allele carriers or minor allele homozygous carriers of rs2281591(P=0.006),rs9468304(P=0.012)of ELOVL2 gene and rs209512(P=0.024)of ELOVL5 decreased,while plasma ARA levels(P<0.05)increased in heterozygous carriers.3.Plasma phospholipids C18:3n-6(GLA)(P=0.045),C18:3n-3(ALA)(P=0.016)levels in pregnant women who were rs3778166 homozygous of ELOVL2 gene are significantly higher than those carrying the major allele,while ARA(P=0.035)levels were significantly lower than them.4.Compared with carriers of homozygous major alleles,plasma phospholipid C20:2 n-6(P=0.005)levels of the heterozygous carriers of rs209512 among pregnant women were reduced;while C20:5n-3(EPA)(P=0.023)level were higher among carriers of minor allele homozygous of rs2397142.5.Compared with carriers of major allele homozygous,women with heterozygous of rs2281591(P=0.011)and minor alleles of rs9468304(P=0.018)of ELOVL2 had higher EPA.6.We found that the pregnant women who carry minor allele of rs209512 in ELOVL5 were related to the increase of GLA(P=0.013)in neonatal plasma,but the minor allele of rs209512(P=0.023)was associated with low C18:2n-6(LA)levels adjusted the neonatal genotype.Carry minor allele of rs9468304 in ELOVL2 were related to the increase of C20:2n-6(P=0.012)in neonatal plasma.For n-3 PUFA,minor allele of rs12332786 and rs3798713 in ELOVL2 were associated with increased levels of C20:3n-3 and DHA in neonatal plasma,significantly.However,EPA and DHA in minor allele carriers of rs3778166 and rs12207094 were significantly lower(P<0.05).7.Adjusted the genotypes of pregnant women,we also found that infants who carry minor alleles of most SNPs within the ELOVL2/5 had higher levels of LA,C20:2n-6,ALA and C20:3n-3(P<0.05).but minor alleles of rs9468304 and rs2397142 were related to the decreased ALA and ARA,respectively(P<0.05).Conclusion:1.Plasma phospholipid fatty acid in pregnant women and newborns showed significant correlations.2.Dietary DHA intake was positive correlated with DHA and EPA levels of pregnant women plasma.3.ELOVL2/5 gene polymorphism among pregnant women affects their plasma PUFA level.4.The level of PUFA in neonatal plasma was jointly regulated by maternal and infant genetic variation.XPart Ⅱ: Effect of DHA intake and Genetic Variants of ELOVL2/5 on Breast Milk PUFA LevelsObjective:To investigate the effect of DHA intake during pregnancy and Genetic Variants of ELOVL2/5 on LC-PUFA levels in lactating mothers.Methods:A total of 422 healthy Han Chinese lactating mothers were included.Breast milk was collected and dietary and DHA supplement intake during pregnancy was investigated,PUFA levels breast milk were measured by gas chromatography,and 10 SNPs in the ELOVL2 and ELOVL5 genes were genotyped.Statistical analysis of basic information and dietary intake was performed using SPSS 24.0.Association analysis between individual SNP loci and PUFA levels was performed using SNPstats online analysis software,and analysis of the interaction between DHA intake and haplotypes on PUFA levels was performed using haplo.stats package of R software.Results:1.Carriers of the minor allele at rs3798713 in the ELOVL2 gene had lower LA concentrations than homozygotes for the major allele(P=0.019).2.Lactating mothers homozygous for the minor allele of rs2294867 in the ELOVL5 gene had higher EPA concentrations than those carrying the major allele(P=0.036).3.Carriers of the minor allele at rs9357760 in the ELOVL5 gene had higher concentrations of GLA,DGLA,ARA and DTA in breast milk than those homozygous for the major allele(P<0.05).4.Minor allele carriers of rs2397142 had higher DTA levels(P=0.027)compared to major allele homozygotes.5.Carriers homozygous for the minor allele of rs209512 had lower milk GLA(P=0.042)and DGLA concentrations(P=0.039)than those carrying the major allele(P=0.039).6.A significant association was also observed between the minor allele of rs12207094 and higher levels of GLA in breast milk(P=0.029).7.The haplotype of SNP(A-G-G)in ELOVL2 was associated with lower levels of LA,EPA,and DHA(P<0.05);the interaction between this haplotype and DHA intake affected LA,EPA,DHA,and ARA levels in breast milk(P<0.05).8.Higher levels of DHA intake interacted with the ELOVL5 haplotype(A-A-C-A-A)to affect DGLA,ALA,and EPA levels(P<0.05).9.The interaction between the ELOVL5 haplotype(C-A-C-A-A)and DHA intake increased EPA levels(P<0.05).Conclusion:1.There was a significant association between ELOVL2/5 gene variants and PUFA levels.2.There is an interaction between DHA intake and specific haplotypes of the ELOVL2/5 genes regulating PUFA levels(especially EPA)levels of breast milk.Part Ⅲ: Effect of DHA intervention and ELOVL2/5 genetic variants on PUFA levels of breast milk and its mechanismObjective:To explore the interaction between DHA intake and ELOVL2 and ELOVL5 gene variants in lactating mothers,and the gene transcription mechanism activated by DHA intake.Methods:A total of 67 healthy lactating mothers were included and DHA supplement intervention was started at 60 ± 5 days postpartum with an intervention period of 30 days.Intervention dose was 420mg/d.Breast milk was collected before and after the intervention and subjects were investigated for a dietary survey.We combined with the positive sites in the second part of our study and selected 6 SNPs of ELOVL2/5 gene for genotype.The detection of breast milk PUFA levels used gas chromatography and the relative expression levels of the target genes ELOVL2,ELOVL5 mRNA and the transcription factors SREBP-1c,and PPARγ mRNA were detected by real-time PCR.The relationship among gene variants,fatty acid levels and mRNA expression levels was analyzed using SPSS 24.0 statistical software.Results:1.The level of breast milk DGLA decreased significantly after DHA(P=0.006)intervention compared with initial point of the study,and the DHA in breast milk increased significantly(P<0.001).2.Carriers of rs3798713 minor allele had higher levels of DGLA at the end point of the intervention than major allele homozygotes(P=0.041).3.DHA intervention increased the level of DHA(P<0.001)in breast milk of carrying rs12207094 major allele homozygous of ELVOL5,and also reduced the level of DGLA(P=0.001)significantly.4.DHA intervention reduced the level of DGLA in breast milk that carries the minor allele of rs3798713(P=0.016)in ELOVL2 and rs209512(P=0.004),rs2294867(P=0.009)in ELOVL5.5.DHA intervention reduced the level of DGLA in breast milk that carries rs9357760(P=0.029)and rs2397142(P=0.001)major alleles of the ELOVL5 gene.6.DHA intervention increased the magnitude of LA level in breast milk carrying rs3798713 minor allele of ELOVL2 greater than those with major allele homozygotes(P=0.041).7.Rs12207094 minor allele carrier of ELOVL5 had a higher change of DGLA level than major allele homozygous carriers after DHA intervention(P=0.035).Conclusion:1.N-6 PUFA levels decreased and DHA levels increased after DHA intervention.2.Gene variation play a weak role in DHA intervention to increase DHA levels in breast milk,but related to the reduced n-6 PUFA level.3.DHA intervention has different effects on PUFA levels for subjects with different genotypes.4.DHA intervention had a weak effect on the mRNA expression of related genes levels in breast milk of subjects with different genotypes.