The Genetic Variation Influence The Serum Alanine Aminotransferase Activities In Adult Male Population From Fangchenggang Area And Replication In Hepatic Cell Lines

CHAPTER IPART ICombinative analysis of factors influence serum alanineaminotransferase activity in adult male population from southernChinaObjective: Abnormal alanine aminotransferase (ALT) activity isindicative of liver disease even a burden of overall health. Therefore, based onthe investigation of Fangchenggang Area Male Healthy and ExaminationSurvey (FAMHES), We assessed the factors associated with ALT activity andtheir internal relationships in a male population from southern China.Methods: Data of physical examinations, laboratory tests, hepaticultrasounds and standardized questionnaire were collected from2119malesparticipating in a population-based survey from September2009to December2009.Results: Nonalcoholic fatty liver disease (NAFLD) and metabolicsyndrome (MetS) were associated with the elevation of ALT levels (P<0.05).Prevalence of NAFLD was correlated to MetS (r=0.991, P=0.009). The levels of abnormal metabolic syndrome components increased in proportion with theALT elevation (P<0.01). Obesity and hyperlipidemia were associated with theALT levels in following multivariate regression analysis (P<0.01). There was nosynergic effect of hepatitis B virus surface antigen (HBsAg) and MetS on theALT levels (synergy index [SI]=0.74,95%confidence interval [CI]:0.71–0.80).Conclusions: NAFLD and MetS were associated with ALT levels in amale population from Fangchenggang area, southern China. Obesity andhyperlipidemia were independent MetS components contributing to elevatedALT (e-ALT). This finding might suggest necessity on justification of theseconfounding factors when detecting ALT levels among this population. CHAPTER IPART IIElevated Alanine Aminotransferase Is Strongly Associated withIncident Metabolic Syndrome: A MetaAnalysis of ProspectiveStudiesObjective: The incidence of metabolic syndrome (MetS) is rapidlyincreasing worldwide and associated with alanine aminotransferase (ALT)activity. However, the impact of ALT activity on MetS incidence is inconsistentin published literature. We therefore estimated the association between elevatedALT activity and incident MetS through a meta-analysis of prospective cohortstudies.Methods: All published prospective cohort studies on the associationbetween elevated ALT activity and incident MetS were retrieved from Pubmed,Embase, and the Institute for Scientific Information (ISI)(literatures publishedbefore January10th,2014). Nationality, gender, age of the enrolled population,follow-up duration, definition of MetS, adjusted covariates, relative risk (RR)are retrieved or calculated directly or indirectly from enrolled reference. We willevaluated the pooled results of dichotomous RR and their95%confidenceinterval(CI)(presented as the incidence of MetS in population with highestALTvalue/the incidence of MetS in population with lowest ALTvalue).Meanwhile, the continuous RR and95%CI was also evaluated (presented as theRR of MetS per5U/l of ALT increment)。If there was no significantheterogeneity (P-value>0.05and I2<50%), the fixed-effect model will be chosento estimate the overall RR and95%CI. Otherwise, the random-effect model willbe used. Results: In all, seven prospective cohort studies, with31545participantsand2873cases of incident MetS were recruited. The calculated RR was1.81(95%CI:1.49–2.14) when the incidence of MetS was compared between thehighest versus the lowest classification of ALT activities. The pooled RR was1.13(95%CI:1.11–1.16) in dose-response analysis with5U/l of ALTincrement. Subgroup analysis suggested that gender disparity might be the mainorigin of heterogeneity in overall analysis (P=0.007between RRs ofgender-specific subgroups evaluated with5U/l increments of ALT). Women hada higher dose-response risk of MetS incidence (1.38,95%CI:1.20–1.55per5U/l of ALT elevation) than men. Furthermore, sensitivity analysis confirmed thestability of results. No publication bias was found in our meta-analysisConclusion: Current evidence from prospective studies supports theassociation between ALT elevation and increasing MetS incidence. Thisassociation is closer and more consistent in female population. Further studiesare needed to confirm this association and to investigate the potentialmechanism of ALT activity on MetS occurrence. CHAPTER IIA genome-wide association study identified two novel loci onalanine aminotransferase level in a male population fromFangchenggang area and their bioinformatic analysisObsjective: Alanine aminotransferase (ALT) plays an important role inliver disease detection and health evaluation. ALT level is influenced by geneticand environmental factors. Therefore, a genome-wide association study (GWAS)was performed in2018healthy Chinese men, combining with the previousepidemiology data from the Fangchenggang Area Male Healthy andExamination Survey (FAMHES), we are trying to find out the common geneticvariation that influence the ALT level in male population from Fangchenggangarea and their interaction with environmental covariates.Methods: Venous blood of enrolled subjects were sampled to extract theDNA. A genome-wide association study (GWAS) was performed in1999healthy male subjects enrolled in FAMHES to check the genotype ofgenome-wide Single Nucleotide Polymorphisms (SNPs), platform illuminaomini one。GWAS was adjusted the body mass index (BMI), triglycerides ascovariates in multi-variate regression model. Further, GWAS was performed inthe subgroup with positive hepatitis B surface antigen (HBsAg). The statisticalsignificance in GWAS of the whole participants was P<1*10-5. The statisticalsignificance in GWAS of the positive HBsAg participants was P<0.05. We thenapplied the following QC criteria to filter SNPs: P <0.001for theHardy-Weinberg equilibrium (HWE) test, minor allele frequency(MAF)<0.01and genotype call rate <95%. The ALT level was compared between positive HBsAg or MetS subgroups classified by variousSNPs with non-parametric analysis (Mann-Whitney Test). Finally, we adoprtedthe synergic effect model to assess the interaction between genetic variation andenvironmental covariates.Results: As results,7SNPs in5genetic loci showed statisticalsignificance after GWAS on the level of P<1*10-5, including the rs10157061(P=7.46*10-7),rs10752781(P=1.29*10-6), rs1878544(P=2.16*10-6) at1q42.2ofSLC35F3gene； rs4766453(P=1.11*10-6) at12q24.12of CUX2gene；rs4801847(P=3.26*10-6)at19q13.3of SHANK1gene；rs2010020(P=7.73*10-6)at16q23.3of WWOX gene；rs3743484(P=8.20*10-6) at15q24.1of CYP1A2gene. Among them,4SNPs in2gene loci (rs10157061(P=7.46*10-7),rs10752781(P=1.29*10-6), rs1878544(P=2.16*10-6) at1q42.2ofSLC35F3gene；rs4766453(P=1.11*10-6) at12q24.12of CUX2gene)showedmore significance with ALT level (P<3*10-6).In population with positiveHbsAg, the rs10157061,rs10752781, rs1878544at1q42.2of SLC35F3gene,rs4801847at19q13.3of SHANK1gene；rs2010020at16q23.3of WWOXgene；rs3743484at15q24.1of CYP1A2gene still reached the statisticalsignificance (P<0.05). Combining with the potential SNPs and MetS or HbsAgstatus, we found the ALT level is significantly different followed with thechange of SNP genotype in different subgroups(P<0.05). We found significantsynergic effects on ALT variation between candidate SNPs(rs10157061,rs10752781, rs1878544at1q42.2of SLC35F3gene,andrs4766453at12q24.12of CUX2gene) and positive HbsAg, the synergic indexsand their95%confidence interval (CI)>1.Conclusions: In conclusion a GWAS found new genetic variation that influence the variation of ALT level in adult males from Fangchenggang area.Among them, SLC35F3and CUX2might be the susceptible gene that influencethe ALTlevel in adult male of this area. However, these effects might be distichtfor the background of HBVinfection. The influence might be amplified on thebackground of HBVinfection. The candidate SNPs and their located loci providepotential genetic biomarker for the diagnosis and treatment of individualizedmedicine. Meanwhile, it provide a significant a meaningful target for thevalication of genetic foundation of ALTlevel on the next step. And themechanism of these influence needs investigation in future. CHAPTER IIIPART IThe replication in the hepatic cell lines on the new findingsthrough genome-wide association study that significantlyinfluenced the alanine amniotransferase level in adult malepopulation from Fangchenggang areaObsjective: Based on the results of genome-wide association study(GWAS) in the previous Fangchenggang Area Male Healthy and ExaminationSurvey (FAMHES), we found new genetic variation that influence the serumalanine aminotransferase (ALT) level in adult male population fromFangchenggang area. However, false connection exsists in the SingleNucleotide Polymorphisms (SNPs) and phenotypic traits found by GWAS.Therefore, we selected the hepatic cell lines as observation to evaluate theassociation between candidate SNPs and ALT level. Meanwhile, we checked thegene that the SNPs located, the protein expression that the candidate geneencoded. We are trying to investigate the association between candidate SNPs,the expression of their located gene, encoded protein by the candidated gene andthe phenotype of various hepatic cell lines.Methods:7hepatic cell lines including BEL-7402, BEL-7404,HL-7702,SMMC-7721, Hep-G2, MHCC-97H, MHCC-97L were selected for observation.The hepatic cell lines were cultured for the following experiments:1.deoxyribonucleic acid (DNA) extraction, design the SNP-specific primer usingthe Polymerase Chain Reaction (PCR) method ti amplify the products.And thePCR products were tested by Sanger’s methods to get the genotype of potentialSNPs;2. Supernate of hepatic cell lines was extracted, the ALT level of various hepatic cell lines was tested by Enzyme Linked Immunosorbent Assay (elisa);3.design the gene-specific primer for real-time PCR(RT-PCR) to test theexpression of candidate gene in various hepatic cell lines;4. the total proteinextraction, design the protein-specific antibody to test the specific proteinexpression by using the western-blot;5. the digested cell was planted in thegerm-free disinfected glass slides, test the specific protein expression withprotein-specific antibody using the Immunological Histological Chemistrymethod.All the cell experiments was performed three times, measurement datawas expressed by mean±standard deviation (SD). The ALT level in varioushepatic cell lines, expression of potential gene was compared with thenon-parametric test (Mann-Whitney U test). The correlation between expressioncovariates was compared by using pearson index. P<0.05was considered assignificance unless special illustration.Results: In the7hepatic cell lines, polymorphisms were presented in5SNPs located in3loci, all the candidate SNPs in the hepatic cell lines ispresented as homozygote.7hepatic cell lines were divded into3groupsaccording to the genotype distinction among different genotypes: A groupincluded the cell line MHCC-97H and MHCC-97L take allele associated withhigher ALT value in rs10157061,rs10752781,rs1878544in SLC35F3,rs4766453in CUX2, rs4801847in SHANK-1; B group included theSMMC-7721take the allele associated with higher ALT level at rs4801847inSHANK-1; C group included the BEL-7402, BEL-7404, Hep-G2, and HL-7702take allele associated with lower ALT level in all the7SNPs located in5geneloci. After comparison, the ALT value in supernate of A group was higher than the ALT value in groups B and C, and no significance was found between groupB and C. RT-PCR indicated that the GPT expression was higher than group Band C, but the SLC35F3expression is negative correlated to the GPT expression(r=-0.672, P=0.001). The CUX2expression had no significant difference indifferent groups (P>0.05). The results from western-blot and IHC confirmedthese results. In western-blot experiments, the SLC35F3encoded proteinexpression in group A was much lower than the group B and C statistically(P<0.05). The GPT protein expression was higher than group B and Cstatistically (P<0.05). No significant difference between CUX2proteinexpression among various hepatic cell lines (P>0.05). The protein expression ofSLC35F3was negative correlated to GPT expression (r=-0.527, P=0.002). TheIHC results confirmed the western-blot results.Conclusions: Based on the SNPs that influence the serum ALT level inadult male population from previous GWAS, the hepatic cell line experiment invitro confirmed:1. The candidate SNPs in1q42.2of SLC35F3(includingrs10157061, rs10752781, rs1878544) significantly influenced the expression ofSLC35F3. Alleles associated with higher ALT level was associated with lowerSLC35F3expression.2. the SLC35F3gene might be a susceptible gene thatinfluence the ALT secrection. The upgrade of SLC35F3expression might becorrelated to the low GPT expression. The quantitative trait loci (QTL) withstrong Linkage disequilibrium (LD) composing by rs10157061, rs10752781,rs1878544influence and regulate the ALT secrection of ALT through regulatingthe expression of SLC35F3. The inner mechanism investigation is waiting to be confiemed. CHAPTER IIIPART IIExpression of SLC35F3gene in hepatic cell lines MHCC-97H andBEL-7402in low exposure of ethanolBackground and Obsjective: Based on the validation in previous study,we found SLC35F3gene influced the alanine aminotransferase (ALT)secrection, GPT gene expression in hepatic cell lines in vitro. The lowexpression of SLC35F3gene and its encoded protein was correlated to thehigher GPT expression and higher ALT secrection. However, the SLC35F3geneexpression in different kinds of hepatic cell lines on the press outside isunknown. Therefore, we designed interaction experiment on low exposure ofethanol, observed the SLC35F3and GPT gene expression of hepatic cell lines,and assess their interaction.Methods: The hepatic cell line BEL-7402and MHCC-97H was selectedfor observation. The expression of SLC35F3, GPT gene and encoded proteinwas tested in different ethanol concentration (50mmol/l,100mmol/l, and200mmol/l) at3hours,6hours,12hours,24hours. The procedure of experimentwas described as follows:1. extracting the supernate of hepatic cell lines andtested the ALT value with Enzyme Linked Immunosorbent Assay (elisa);2.aiming at the SLC35F3and GPT gene, design the specific primer to test theexpression level of SLC35F3and GPT level on different time-concentration ofethanol exposure in various hepatic cell lines with real-time PCR;3. extractingthe total protein of hepatic cell lines in different time-concentration of ethanolexposure,designing the SLC35F3and GPT specific antibody, to check theprotein expression by western-blot method;4. digest the cell in different time-concentration of ethanol exposure, to plant on the germ-free glass slides,the protein expression was checked by protein specific anti-body withImmunological Histological Chemistry (IHC) method.The cell experiments were duplicated for three times. Measurement datawas presented as mean±standard deviation (SD). The statistic comparison wasfollowed the criteria as below: one-way ANOVA was used in comparisonbetween data with normal distribution; non-parametric test (Mann-Whitney Utest) was used in comparison data with un normal distribution. P<0.05wasconsidered as statistical significance unless specific illustration.Results: We have checked the ALT secrection, SLC35F3, GPT gene andencoded protein expression level in different time-concentration ethanolexposure of BEL-7402and MHCC-97H. The specific results is as follows:1.the time-to-peak ALT level in BEL-7402was6hours, while the time-to-peakALT level was shorten from12hours on the exposure of100mmol/l ethanolconcentration to3hours on the exposure of200mmol/l ethanol concentration.In different intervention of various ethanol concentration, no significantdifference for the peak ALT value was observed between different ethanolconcentration exposure (P>0.05), while in MHCC-97H, the peak ALT value wasincreased significantly with the increased ethanol interactive concentrationstatistically (P<0.05);2. significant difference of time-concentration ethanolinteraction between BEL-7402and MHCC-97H on SLC35F3and GPTexpression. The time-to-peak of SLC35F3and GPT in BEL-7402is12hoursand6hours. No significant variation was observed between peak expressionlevel on different ethanol interaction concentration (P>0.05). While inMHCC-97H, the time-to-peak and peak level of SLC35F3and GPT expression varied with different ethanol concentration (P<0.05). The time-to-peak isshorten from24hours on the exposure of50mmol/l to3hours on the exposureof200mmol/l. Significant increasing GPT and SLC35F3expression level wereobserved followed with increasing exposure of ethanol concentration (P<0.05).3. the western-blot showed: the SLC35F3and GPT protein expression werestabilized at12hours and6hours respectively, while the time-to-peak ofSLC35F3and GPT in MHCC-97H was shorten followed with the increasingconcentration of ethanol exposure, the SLC35F3and GPT protein expression inMHCC-97H is higher on200mmol/l of ethanol exposure(P<0.05). Thewestern-blot results were confirmed by IHC results.Conclusions: In the stress environment, the BEL-7402with highexpression of SLC35F3is more susceptible to the ethanol exposure than theMHCC-97H with low expression of SLC35F3. The SLC35F3expression mightbe associated with the self-regulation of hepatic cell and the ALT secrectionmechanism on the ethanol exposure. The low expression of SLC35F3isfacilitated to decrease the susceptibility and injury on the exposure of ethanol.