Fatty Acid Metabolism-related Gene Polymorphisms And Their Association With Fatty Acids In Serum And Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)refers to a clinical and pathological syndromethe which main feature is diffuse bullous fatty liver cells,in addition of ethanol-induced liver damage and other specific factors.NAFLD can be manifested as simple fatty liver to fatty liver cirrhosis,and is believed to be closely correlated with insulin resistance(IR)and genetic predisposition.Since there’s currently no effective therapy,prevention of NAFLD would be very important.And it would be of great significance to investigate the susceptibility genes,so as to predict NAFLD risk of an individual and to guide high-risk individuals to develop a proper diet.Polymorphisms of the susceptibility genes and abnormality in fatty acid metabolism have attracted the most attention.Fatty acids play an important role in development and progression of NAFLD-each type of fatty acid has its specific physiological roles and exerts different cytotoxicity to liver cells.How the ratio of polyunsaturated fatty acids(PUFAs)changed during NAFLD,especially the biologically essential n-3 and n-6 PUFAs?Which fatty acids are protective factors for NAFLD,and which are risk factors?All these questions require in-depth studies.Single nucleotide polymorphisms(SNPs),as the third generation of genetic polymorphism markers,widely present in the human genome.SNPs may affect gene expression,whether it’s in the coding region or the non-coding region.Abnormality in lipid metabolism and biosynthesis are closely related to β-oxidation impairment,so the key enzymes involved in these two pathways would be the key factors in NAFLD susceptibility.So,are there SNPs in these key enzymes that predispose affected individuals to NAFLD?Whether these genes affect expression of corresponding enzymes and fatty acid profile in NAFLD patients or not?In this study,we sought answers to these questions in the northern Han Chinese population.First,changes in serum fatty acid profile of the NAFLD patients were analyzed by capillary gas chromatography;then,by referring to literatures and searching the Hapmap database,key enzymes involved in biosynthesis and oxidation of fatty acids were identified;at last,mRNA and protein expression of the key genes in individuals of different genotypes were determined by real-time PCR and Western blot.Basing on these data,we analyzed the correlations between SNPs of the key genes in fatty acid metabolism,NAFLD susceptibility,and changes in fatty acid profile in NAFLD patients,and investigated the role of gene polymorphism and abnormal fatty acid metabolism in develop and progression of NAFLDs,so as to provide theoretical and experimental basis to identify individuals at risk of NAFLD,to predict risk of an individual to develop NAFLD,and to develop a proper diet for these individuals.Part One Profile of serum fatty acids and the activities of desaturase in non-alcoholic fatty liver diseaseObjective:Studying the profile fatty acids and the activities of desaturase in the patients with NAFLD,to find the target for research the mechanism in fatty acid metabolism.Methods:A case-control study was employed in this study.866 subjects were collected and divided into 2 groups,controls(421 cases)and the patients with NAFLD(445 cases).waist circumference,systolic blood pressure,diastolic blood pressure,fasting serum insulin,fasting blood glucose,triglyceride,total cholesterol,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,free fatty acid,alanine aminotransferase,aspartate aminotransferase,gamma-glutamyl transpeptidase and were determined and BMI was calculated to evaluate lipid metabolism in the patients with NAFLD.Fasting serum fatty acids with carbon atoms 16-24 were determinated by gas chromatography,and analysed according to the category.Logistic regression was applied to case-control comparisons for the NAFLD outcome for co-dominant,dominant and recessive genetic models,with adjustment or without adjustment.Results:1 The general biochemical indexes:Compared with Con group,there was no difference in ages and the male sex ratio of NAFLD group was,significantly higher(42.7%vs 33.0%,P=0.003);body mass index,waist circumference,systolic blood pressure and diastolic blood pressure of NAFLD group also increased significantly,P<0.001.Compared with Con group,fasting blood glucose,fasting insulin,total cholesterol,triglyceride,low-density lipoprotein cholesterol,free fatty acids,alanine aminotransferase,aspartate aminotransferase and gamma-glutamyl transpeptidase in NAFLD group,were significantly increased,and high-density lipoprotein cholesterol in NAFLD group decreased significantly P<0.001;insulin resistance index(HOMA-IR)were significantly increased,P<0.001.And in NAFLD group,serum total free fatty acids also increased significantly(2.09±0.46 mmol/L vs 0.82±0.21 mmol/L),P<0.001.2 Association of the profiles of serum fatty acids with the risk of NAFLDTo analysis the different types of fatty acids,we found that,compared with Con group,the relative content of C16:0,C16:ln-7,C18:ln-9,C18:3n-3(a-linolenic acid,ALA),total monounsaturated fatty acids and total long-chain fatty acids were increased in NAFLD group,and they were risk factors for NAFLD,P<0.01;but the relative content of C18:0,C20:0,C22:0,C24:0,C18:2n-6(linoleic acid,LA),C20:4n-6(arachidonic acid,AA),C22:6n-3(docosahexenoic acid,DHA),total n-3 and n-6 polyunsaturated fatty acids(PUFAs)and total very long-chain fatty acids as well as AA/LA(the overall desaturation activity of the n-6 PUFAs metabolic pathway)and EPA/ALA(the overall desaturation activity of the n-3 PUFAs metabolic pathway)were decreased,and they were protective factors for NAFLD,P<0.01;and C20:5n-3(eicosapentaenoic acid,EPA),C22:4n-6,C22:5n-3(docosapentaenoic acid,DP A),total saturated fatty acid and total unsuaturated fatty acids did not change significantly.3 Association of the profiles of serum fatty acids with lipidemia(TG,TC)as well as HOMA-IRC16:0,C18:ln-9,ALA,total monounsaturated fatty acids,total long-chain fatty acids were positively related with TG,C18:0,LA,C20:0,DTA,DHA,C24:0,total n-6 PUFAs,total n-3 PUFAs,total very long-chain fatty acids and EPA/ALA were negatively related with TG,P<0.01.C16:ln-7 was positively related with TC,C18:0,DTA,DHA and total very long-chain fatty acids were negatively related with TC,P<0.05.C16:0,C18:ln-9 and total monounsaturated fatty acids were positively related with HOMA-IR,C18:0,LA and total n-6 PUFAs were negatively related with HOMA-IR,P<0.01.Summary:1 C16:0,C16:ln-7,C18:ln-9 and C18:3n-3(α-linolenic acid,ALA)were risk factors for NAFLD,C18:0,C20:0,C22:0,C24:0,C18:2n-6(linoleic acid,LA),C20:4n-6(arachidonic acid,AA)and C22:6n-3(docosahexenoic acid,DHA)were protective factors for NAFLD.2 A lower levels of AA and DHA in NAFLD patients may be related to the reduce of overall desaturation activity of the n-6 and n-3 PUFAs metabolic pathway.Part Two Study of correlation between SNPs in FADS1-FADS2 gene cluster and serum PUFAs changes and the risk of NAFLDObjective:To investigate the correlation between the mutation of SNP in FADS1-FADS2 gene cluster and the risk of NAFLD and the PUFAs in serum of patients with NAFLD.Methods:A case-control study was employed in this study.866 subjects were collected and divided into 2 groups,controls(421 cases)and the patients with NAFLD(445 cases)(the inclusion and exclusion criteria as same as part one of this study).Peripheral blood was collected to extract DNA for SNP classification by Matrix Assisted Laser Desorption/ionization Time of Flight Mass Spectrometry(MALDI-TOF MS).Logistic regression was applied to case-control comparisons for the different phenotypic outcomes for co-dominant,dominant and recessive genetic models,with adjustment or without adjustment,to analyes the correlation between SNP mutation in rs174545,rs2072114,rs174602 and rs174616 loci and serum PUFAs in the patients with NAFLD and the risk of NAFLD.Results:1 The genotyping results of SNPs in FADS1-FADS2 gene clusterIn Hebei Han Chinese population,FADS 1 gene had rs 174545,and FADS2 gene had rs2072114,rs 174602 and rs 174616.2 Allele frequency analysis of the SNPs in FADS1-FADS2 clusterThe frequency of allele G of FADS2 gene rs174602 was 0.184 in the NAFLD group,which was obviously lower than that in the control group(0.23 5).Allele G was associated with NAFLD,and was one of its protective factors(OR=0.735,95%CI =0.582-0.927,P=0.009).3 Association of the SNPs in FADS1-FADS2 gene cluster with the risk of NAFLDThe results showed that rs174602 A>G and the risk of NAFLD had negative correlation.In codominant model,individuals carrying rs 174602 AG and rs174602 GG were not susceptible of NAFLD,the OR values were 0.728(95%CI=0.537-0.986,P=0.041)and 0.389(95%CI=0.171-0.885,P=0.024);the same results as well as dominant(OR=0.657,95%CI=0.492-0.878,P=0.013)and recessive models(OR=0.317,95%CI=0.131-0.767,P=0.021).In the 3 models above,174545,rs2072114 and rs174616 were not related with the risk of NAFLD.4 Construction and analysis of haplotypes composed by the SNPs in FADS1-FADS2 gene clusterrs174545,rs2072114 and rs174602 of FADS1-FADS2 gene cluster were in the same haplotype block(D’>0.5),the Mi A haplotype GGG(rs174545-rs2072114-rs1746020)of FADS1-FADS2 gene cluster was negatively associated to the incidence of NAFLD,which was protective factor for NAFLD(OR=0.702,95%CI=0.519-0.949,P＝0.021).But the other 5 haplotypes of FADS1-FADS2 gene cluster were not related with the risk of NAFLD.5 The effect of rs 174602 A>G mutation on serum PUFAs according to the dominant model in healthy people and NAFLD patientsThe results showed that the content of serum LA increased,AA and ratio of AA/LA decreased in subjects carrying rs174602(AG+GG)both healthy people and patients with NAFLD;in addition,the content of TC declined and ratio of EPA/ALA increased in subjects carrying rs174602(AG+GG)of NAFLD patients.No changes were found in n-3 PUFAs.6 The effect of MiA haplotype GGG on serum PUFAs in healthy people and NAFLD patientsThe results showed that the content of serum LA increased,AA and ratio of AA/LA decreased in subjects carrying MiA haplotype GGG both healthy people and patients with NAFLD;in addition,the content of TC also declined in subjects carrying MiA haplotype GGG of NAFLD patients.Summary:1 In the Han population in northern China,the SNP of FADS2 gene in rs 174602 was negatively associated to the incidence of NAFLD,which was protective factor for NAFLD.2 In the Han population in northern China,the MiA haplotype GGG(rs 174545-rs2072114-rs 1746020)of FADS 1-FADS2 gene cluster was negatively associated to the incidence of NAFLD,which was protective factor for NAFLD.Part Three Study of correlation between SNPs in peroxisomal β-oxidation key enzyme gene polymorphism and serum VLCFAs changes and the risk of NAFLDObjective:To investigate the correlation between the mutation of SNP in peroxisomal β-oxidation key enzyme gene and the risk of NAFLD and the VLCFAs in serum of patients with NAFLD.Methods:A case-control study was employed in this study.866 subjects were collected and divided into 2 groups,controls(421 cases)and the patients with NAFLD(445 cases)(the inclusion and exclusion criteria as same as part one of this study).Peripheral blood was collected to extract DNA for SNP classification by Matrix Assisted Laser Desorption/ionization Time of Flight Mass Spectrometry(MALDI-TOF MS).Logistic regression was applied to case-control comparisons for the different phenotypic outcomes for co-dominant,dominant and recessive genetic models,with adjustment or without adjustment,to analyes the correlation between 17 SNPs mutation in 6 peroxisomal(3-oxidation key enzyme genes(ACOX1,EHHADH,ACAA1,ACOX2,HSD17B4,SCP2)and serum VLCFAs in the patients with NAFLD and the risk of NAFLD.Results:1 The genotyping results of SNPs in peroxisomal(3-oxidation key enzyme genesIn Hebei Han Chinese population,ACOX1 gene had rs 12430,rs7219716 and rs8065144;EHHADH gene had rs4687136,rs2160815,rs1509501 and rs2284994;ACAA1 gene had rs 156265 and rs2239621,ACOX2 gene had rs4317122,rs4681689 and rs4387990;HSD17B4 gene had rs2075698,rs3756513,rs10064000 and rs3797371.The genotyping of rs3766765 failed in SCP2 gene locus.2 Allele frequency analysis of the SNPs in peroxisomal β-oxidation key enzyme genesThe frequency of allele T of ACOX1 gene rs12430 was 0.08 in the NAFLD group,which was obviously lower than that in the control group(0.11).Allele T was associated with NAFLD,and was one of its protective factors(OR=0.698,95%CI=0.505-0.966,P＝0.029).3 Association of the SNPs in peroxisomal β-oxidation key enzyme genes with the risk of NAFLDThe results showed that rs 12430 C>T and the risk of NAFLD had negative correlation.In codominant model,individuals carrying rs 12430 CT was not susceptible of NAFLD,the OR values were 0.696(95%CI=0.489-0.990,P=0.044);the same results as well as dominant model(OR=0.575,95%CI= 0.340-0.972,P=0.039).The results showed that rs 15 626 5 C>G and the risk of NAFLD had positive correlation.In codominant model,individuals carrying rs 156265 GG was susceptible of NAFLD,the OR values were 1.634(95%CI=1.051-2.541,P=0.029);the same results as well as recessive model(OR=1.809,95%CI=1.191-2.748,P=0.005).In the 3 models above,rs7219716 and rs8065144 in ACOXl,rs4687136,rs2160815,rs1509501 and rs2284994 in EHHADH,rs2239621 in ACAA1,rs4317122,rs4681689 and rs4387990 in ACOX2,rs2075698,rs3756513,rs 10064000 and rs3797371 in HSD17B4 were not related with the risk of NAFLD.4 Construction and analysis of haplotypes composed by the SNPs in peroxisomal(3-oxidation key enzyme genesrs 12430,rs7219716 and rs8065144 of ACOX1 gene were in the same haplotype block(D’>0.5),the haplotype CCG(rs12430-rs7219716-rs8065144)was positively associated to the incidence of NAFLD,which was risk factor for NAFLD(OR= 1.639,95%CI=1.086-2.473,P=0.018).But the other 4 haplotypes of ACOX1 gene were not related with the risk of NAFLD.All 5 haplotypes composed by rs4687136,rs2160815,rs1509501 and rs2284994 of EHHADH gene,3 haplotypes composed by rs 15 6265 and rs2239621 of ACAA1 gene,6 haplotypes composed by rs4317122,rs4681689 and rs4387990 of ACOX2 gene,and 4 haplotypes composed by rs2075698,rs3756513 and rs 10064000 of HSD17B4 gene were not related with the risk of NAFLD.5 The effect of rs 12430 C>T mutation on serum VLCFAs according to the dominant model in healthy people and NAFLD patientsCompared with subjects carrying rs 12430 CC,the content of serum VLCFAs and lipidemia(TG and TC)did not change significantly in subjects carrying rs12430(CT+TT)of healthy people;in addition,the content of TC declined in subjects carrying rs12430(CT+TT)of NAFLD patients.No changes were found in VLCFAs.6 The effect of rs 156265 C>G mutation on serum VLCFAs according to the codominant model in healthy people and NAFLD patientsCompared with subjects carrying rs 156265 CC,the content of serum VLCFAs and lipidemia(TG and TC)did not change significantly in subjects carrying rs156265 CG and GG of healthy people;in addition,the content of C22:0 and DPA declined in subjects carrying rs 156265 GG of NAFLD patients.No changes were found in TG and TC.7 The effect of haplotype CCG on serum VLCFAs in healthy people and NAFLD patientsThe results showed that the content of serum DTA decreased in subjects carrying haplotype CCG both healthy people and patients with NAFLD;in addition,the content of DHA and total VLCFAs also declined in subjects carrying haplotype CCG of NAFLD patients.No changes were found in in TG and TC.Summary:1 In the Han population in northern China,the SNP of ACOX1 gene in rs 12430 was negatively associated to the incidence of NAFLD,which was protective factor for NAFLD.2 In the Han population in northern China,the haplotype CCG(rs12430-rs7219716-rs8065144)of ACOX1 gene was positively associated to the incidence of NAFLD,which was risk factor for NAFLD.3 In the Han population in northern China,the SNP of ACAA1 gene in rs 156265 was positively associated to the incidence of NAFLD,which was risk factor for NAFLD.Part Four The influence of single nucleotide polymorphisms on peroxisomal β-oxidation key enzyme gene expressionObjective:To study the mechanism of affect of rs156265 C>G on NAFLD risk from gene expression.Methods:The human whole blood total RNA and peripheral blood leukocytes were isolated,and the expression of ACAA1 in mRNA and protein levels.was detected according to rs 156265 carrying different genotypes.Results:1 The relative expression of ACAA1 mRNA in people carrying different rs 156265 genotypeACAA1 mRNA of white blood cells express low levels in human peripheral blood.Compared with subjects carrying rs 156265 CC,the relative expression of ACAA1 mRNA in peripheral blood leukocytes showed no significant difference in subjects carrying rs156265 CG and GG both healthy people and patients with NAFLD.2 The relative expression of ACAA1 protein(PTH1)in people carrying different rs156265 genotypeCompared with subjects carrying rs156265 CC,Western blot analysis showed that PTH1 protein content significantly decreased in the peripheral blood leukocytes of subjects carrying rs156265 CG and GG genotype.Summary:1 The expression of ACAA1 protein decreased in people carrying rs156265(CG + GG)genotype.2 The reduce of ACAA1 protein levels may be not related with the reduce of C22:0 in subjects carrying rs156265 GG of NAFLD patients.Conclusions:1 C16:0,C16:ln-7,C18:ln-9 and C18:3n-3(a-linolenic acid,ALA)were risk factors for NAFLD,C18:0,C20:0,C22:0,C24:0,C18:2n-6(linoleic acid,LA),C20:4n-6(arachidonic acid,AA)and C22:6n-3(docosahexenoic acid,DHA)were protective factors for NAFLD.2 In the Han population in northern China,the SNPs of FADS1-FADS2 gene cluster in rs174602 as well as ACOX1 gene in rs12430 were negatively associated to the incidence of NAFLD,which were protective factors for NAFLD;the SNP of ACAA1 gene in rs156265 was positively associated to the incidence of NAFLD,which was risk factor for NAFLD.3 In the Han population in northern China,the MiA haplotype GGG(rs174545-rs2072114-rs1746020)of FADS1-FADS2 gene cluster was negatively associated to the incidence of NAFLD,which was protective factor for NAFLD;the haplotype CCG(rs12430-rs7219716-rs8065144)of ACOX1 gene was positively associated to the incidence of NAFLD,which was risk factor for NAFLD.4 The expression of ACAA1 protein decreased in people carrying rs 156265(CG + GG)genotype.