The Effects Of FADS1 Gene Variants On The Composition Of Polyunsaturated Fatty Acids In Maternal Plasma And Breast Milk

Breast milk is the perfect nutrition for infants,a result of millions of years of evolution,finely attuning it to the requirements of the infants.Breast milk is an extremely complex and highly variable biofluid that has evolved over millennia to nourish infants and protect them from disease whilst their own immune system matures.The composition of human breast milk changes in response to many factors,matching the infant's requirements according to its age and other characteristics.Fatty acids are a vital component of breast milk.They influence infant neurodevelopment and immune function,and they provide about 50%energy of breast milk.Fatty acids in human milk are derived from maternal body stores,endogenous synthesis in the mammary gland,and uptake from maternal plasma.Variants in the?-5fatty acid desaturase genes(FADS1)have been shown to influence long-chain PUFAs(LC-PUFAs)in human milk.However,many functional SNPs in FADS1 gene have not been identified,and how SNPs affect PUFA in breast milk is still unclear.Therefore,this study searched domestic and foreign studies on FADS1 gene variation and PUFA levels for meta-analysis;and we collected blood samples from pregnant women in Changchun to verify the relationship between FADS1 gene variation and plasma phospholipid PUFA levels;the potential mechanisms about the effects of genetic variants on fatty acid were explored through DHA intervention;by bioinformatics,we predicted the mi RNAs that might bind to FADS1 gene.Double luciferase reporter assay was used to verify whether the mi RNA targeted to FADS1 gene.We explored the effect of gene variation and mi RNA on PUFA in breast milk of postpartum healthy mother.This study includes the following three parts:Part one:The association between FADS1 gene variation and long-chainpolyunsaturated fatty acid levelObjectives:A meta-analysis was carried out to analysis FADS1 gene variants and PUFA levels.Furthermore,we analyzed the relationship between FADS1 gene variants and plasma PUFA levels of pregnant women,and clarify the effect of SNP on PUFA levels.Methods:Four databases(Pub Med,web of science,CNKI and Wanfang database)were used to search for important keywords such as fatty acid,single nucleotide polymorphism,fatty acid desaturase 1 gene,(English:fatty acid,SNP,FADS1 gene),rs174546/rs174547,etc.to search for Chinese and English literatures published before October 5,2020.Stata12.0 software was used for meta-analysis.One hundred and ten pregnant women who delivered in a 3A grade hospital in Changchun were included,informed consent was signed and basic information and blood samples were collected.The plasma phospholipid fatty acid level was detected by gas chromatography.The rs174546 and rs174547 of FADS1 gene were genotyped by Sequenom Mass Array system.The effect of gene variation on PUFA level was analyzed by IBM SPSS 24.0software.Results:1.Meta-analysis of rs174546 and PUFA levels showed that compared with CC genotype,the levels of linoleic acid(LA),dihomo-gamma-linolenic acid(DGLA)and alpha-linolenic acid(ALA)in CT+TT genotype carriers were significantly increased(P<0.05),while the levels of arachidonic acid(AA),eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)were significantly decreased(P<0.05).2.Meta-analysis of rs174546 and enzyme activity showed that compared with CC genotype,?-5 fatty acid desaturase(D5D)activity(AA/DGLA),n-6 pathway(AA/LA),and n-3 pathway(EPA/ALA)level of CT+TT genotype carriers decreased(P<0.05).3.Meta-analysis of rs174547 and PUFA levels showed that compared with TT genotype,the levels of LA and ALA in TC+CC genotype carriers were significantly increased(P<0.05),while the levels of gamma-linolenic acid(GLA),AA,EPA and DHA were significantly decreased(P<0.05).4.Meta-analysis of rs174547 and enzyme activity showed that compared with TT genotype,D5D and?-6 fatty acid desaturase(D6D)activity(GLA/LA)and n-6 pathway level of TC+CC genotype carriers decreased(P<0.05).5.The percentage of LA in maternal plasma was the highest.6.Complete linkage disequilibrium between rs174546 and rs174547(R~2=1).7.In n-6 PUFA,compared with the major allele,the levels of LA(P=0.002)and DGLA(P=0.047)in the minor allele carriers were significantly higher,while the level of AA(P<0.001)was significantly lower.8.In n-3 PUFA,the levels of EPA(P=0.031)and DHA(P=0.004)of the minor allele carriers decreased compared with the major allele.9.Compared with the major allele,the D5D activity(P<0.001)and D6D activity(P=0.050)of the minor allele carriers were significantly lower,the n-3 pathway(P=0.001)and the n-6 pathway(P<0.001)level were significantly lower.Conclusions:1.Meta-analysis showed that the levels of LA,DGLA and ALA in the minor allele carriers of rs174546 increased,while the levels of AA,EPA,DHA,the activities of D5D,n-3 and n-6 pathways decreased.Meta-analysis showed that the levels of LA and ALA in the minor allele carriers of rs174547 increased,while the levels of GLA,AA,EPA,DHA,the activities of D5D,D6D and n-6 pathway decreased.2.The levels of plasma LA and DGLA in rs174546 and rs174547 allele carriers were increased,while the levels of AA,EPA and DHA and the activities of D5D,D6D,n-3 and n-6 pathways level were significantly decreased.Part two:The effect of DHA intervention on polyunsaturated fatty acid levels oflactating mothers with different FADS1 genotypes in breast milkObjectives:Objective to investigate the effect of DHA intake on PUFA level and gene expression level with different SNPs of FADS1 gene.Methods:A total of 73 healthy lactating mothers were included in this study.They were given 420 mg/d DHA supplement for 30 days after delivery for 60±5 days.The breast milk was collected before and after the intervention.The levels of PUFA in breast milk were detected by gas chromatography.The SNP of FADS1 rs174546,rs174547,rs174553 and rs174556 were genotyped by Sequenom Mass Array system.The m RNA relative expression levels of FADS1/2/3 gene,transcription factors sterol-regulatory element binding protein 1c(SREBP-1c)and peroxisome proliferator activated receptors?(PPAR?)were detected by q PCR.IBM SPSS 24.0 was used to analyze the effects of DHA intake and gene variation on PUFA and m RNA expression level in breast milk.Results:1.The levels of DGLA(P=0.002)were significantly decreased and DHA(P<0.001)were significantly increased after DHA supplementation.2.Compared with before DHA supplementation,the m RNA expression level of PPAR?decreased significantly after DHA supplementation(P=0.031).3.Before DHA supplementation,compared with the major allele,the levels of GLA,AA and D5D activity of the minor allele carriers of FADS1 were significantly lower(P<0.05);the decrease of D6D activity of the minor allele carriers of FADS1 was only found in rs174556 genotype(P=0.036).4.After DHA supplementation,compared with the major allele,AA,EPA,D5D activity and n-3 pathway and n-6 pathway levels of the minor allele carriers were significantly decreased(P<0.05).The decrease of D6D activity level of the minor allele carriers was only found in rs174556 genotype(P=0.040).5.In different genotype groups,compared with before DHA supplementation,DGLA levels were significantly decreased(P<0.05),while DHA levels were significantly increased(P<0.05).6.Before DHA supplementation,the m RNA expression levels of FADS2 and FADS3 in the minor allele carriers of rs174546 were lower than those in major allele carriers(P<0.05);the m RNA expression levels of FADS1 in minor allele carriers of rs174556 were lower than those in major allele carriers(P=0.020).7.Compared with the major allele,the m RNA expression levels of FADS1,FADS2and FADS3 in the minor allele carriers decreased after DHA supplementation(P<0.05).8.In the minor allele carriers group,the m RNA expression levels of FADS1 and PPAR?were significantly lower after DHA supplementation than before(P<0.05).Conclusions:1.DHA supplementation reduced DGLA level but increased DHA level,decreased PPAR?m RNA expression level.DHA supplementation reduced the expression level of FADS1 and PPAR?of the minor allele carriers of FADS gene in breast milk.2.Before and after DHA intervention,the AA and D5D activities of the minor allele carriers of FADS1 in breast milk decreased,indicating that the same dose of DHA supplementation did not change the difference in fatty acid levels caused by genotype.The expression level of FADS2 and FADS3 in the minor allele carriers of rs174546decreased,while the expression level of FADS1 in the minor allele carriers of rs174556was significantly lower.Part three:The effect of mi R-149-5p targeting FADS1 gene on PUFAs in breast milkObjectives:To investigate the effects of FADS1 gene variation and mi R-149-5p on PUFA level and gene expression level.Methods:Bioinformatics method were used to predict the function of SNP sites and their linked imbalance sites in FADS1 gene.The dual luciferase reporter system was used to verify the targeting effect of mi RNA on FADS1 gene in HEK293T cells.Twenty-four healthy lactating mothers were included,and breast milk was collected 40-65 days after delivery.The levels of PUFA in breast milk were detected by gas chromatography,FADS1 rs174546 were genotyped by Sequenom Mass Array system,and the m RNA relative expression levels of FADS1/2/3 gene and transcription factors SREBP-1c,PPAR?and mi R-149-5p were detected by q PCR method.IBM SPSS 24.0 was used to analyze the effects of rs174546 and mi R-149-5p on the level of PUFA and m RNA expression level in breast milk.Results:1.Bioinformatics methods predicted that rs174546 may bind to mi R-149-5p.2.Dual luciferase report assay confirmed that mi R-149-5p can target bind to FADS1 rs174546 allele T.3.Compared with wild-type CC genotype,the D6D activity(P=0.044)and n-3pathway(P=0.014)of mutant TT genotype in breast milk was decreased.4.Compared with wild-type CC genotype,the m RNA expression levels of FADS1(P<0.001)and FADS3(P=0.007)of mutant TT genotype in breast milk were decreased.5.Compared with wild-type CC genotype,the expression level of mi R-149-5p of mutant TT genotype in breast milk was increased(P=0.012).Conclusions:1.The dual luciferase reporting system verified the specific binding between mi R-149-5p and the T site of rs174546 allele of FADS1 gene.2.The level of mi R-149-5p in the breast milk of lactating mothers with rs174546TT genotype was significantly higher.The m RNA expression levels of FADS1 and FADS3 genes in the breast milk of lactating mothers with rs174546 TT genotype were significantly decreased.The activity level of D6D and n-3 pathway in breast milk of lactating mothers with rs174546 TT genotype was significantly decreased.Summary1.The minor allele carriers of FADS1 gene have higher levels of PUFA substrates and lower levels of PUFA products.2.The minor allele carriers of FADS1 gene were associated with the fatty acid levels in breast milk and plasma;the same dose of DHA supplementation did not improve the difference in fatty acid levels caused by genotype.3.Dual luciferase report assay confirmed that mi R-149-5p can target bind to FADS1 rs174546 allele T.