Association Of Lipid Metabolism Related MicroRNA Binding Sites Polymorphisms With The Risk Of Alzheimer’s Disease

Objective Alzheimer’s disease(AD)happens in the old age and is characterized by cognitive impairments and behavioral disorders.AD has a 50%~70% incidence in the old suffered from dementia,which can be classified into early-onset AD(EOAD)and late-onset AD(LOAD)according to the age 65.These two types of AD are more or less proved to be related with heredity and the latter is affected by the gene,environment and metabolism in vitro comprehensively.APOE ε4 gene carriers are considered to be sporadic AD high-risk population.Although altered lipid metabolism has been extensively implicated in the pathogenesis of LOAD through epidemiological studies,genetic studies linking lipid metabolism and LOAD are still not well understood.Mutated APOE gene accounts for 50% susceptible individuals,which catalyses the study of epigenetic in AD.The features of epigenetic are including DNA methylation,histone modification and microRNA(miRNA)regulation.Imbalanced miRNAs induce the emerging toxics of Aβ and Tau.Unlike the traditional concepts of heredity,epigenetic often plays its own roles depending on the suitable time,locations and expression manners.MiRNAs have previously been implicated in expression regulation of lipid related genes,such as Clusterin gene(CLU),lipoprotein lipase gene(LPL)and low-density lipoprotein receptor-related protein 6 gene(LRP6).MiRNAs have previously been implicated in expression regulation of lipid related genes mentioned above,and exert post-transcriptional downregulation and their target sequence on the 3’UTR may be altered by single nucleotide polymorphisms.CLU,also known as apolipoprotein J(ApoJ),is currently the third most associated risk gene according to Alzgene database(http://www.alzgene.org/).It is located in chromosome 8p21–p12 that is a chromosomal region of interest in AD and it may explain around 9% of the LOAD attributable risk.LPL is a key enzyme in triglyceride(TG)metabolism.LPL gene is located in chromosome 8p22,whose single nucleotide polymorphisms(SNPs)are strongly associated with AD pathology.LPL binds to Aβ and promotes cell-surface association and uptake of Aβ in astrocytes.Low-density lipoprotein receptor-related protein 6(LRP6)gene is located in chromosome12p11.2-p13.3.Previous researches found that single nucleotide polymorphisms in the LRP6 gene were associated with AD.The LRP6Δ3 isoform is a novel splice variant,which might have a functional role in individuals with AD.We therefore explore whether the six loci in CLU(rs9331949),LPL(rs1059507,rs3200218,rs3208305,rs3735964 and LRP6(rs2160525)could modulate LOAD risk through alteration of miRNA bindingsites.Finding out susceptible AD gene from the aspect of epigenetic could be useful to diagnosis and therapy.Methods We performed a case–control study of 2338 unrelated subjects(984 cases and1354 age-and gender-matched controls)in Northern Han Chinese from Qingdao municipal hospital,the affiliated hospital of Qingdao university,Qingdao hiser hospital,and other hospitals of districts in and around Qingdao.We collected the subjects spanning from 2011 to 2015,with the informed consent obtained from each subject,either directly or from the subject’s guardian,and the protocol of this study was approved by the Ethical Committee of Qingdao Municipal Hospital.Probable AD was diagnosed clinically according to the criteria of National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association(NINCDS-ADRDA).No LOAD patients reported a family history of neurodegenerative disorders or other dementias.The control group underwent medical history,general examinations,laboratory examinations,and Mini Mental State Examination score by physicians,which showed that they were free of any symptoms suggestive of cognitive decline.All of the participants were more than 65 years old,and had been excluded from serious internal medical disease,which could lead to central nervous dysfunction.We performed sequencing of the CLU,LPL and LRP6 by using genomic DNA extracted from peripheral venous blood of participates with Wizard genomic DNA purification kit.SNPs genotyping of rs3208305,rs2160525,rs1059507 and rs9331949 were performed according to double ligation and multiplex fluorescent polymerase chain reaction(PCR).In addition,the rs3735964,rs3200218 and polymorphisms in APOE gene were genotyped by polymerase chain reaction ligase detection reaction(PCR-LDR).APOE genotypes were detected based on the methods represented by Donohoe.Goodness-of-fit to Hardy-Weinberg equilibrium(HWE)in the controls and genotype were calculated.Differences in genotype and allele distributions between cases and controls were analyzed by χ2 tests,and the P value,odds ratios(ORs),and 95%confidence intervals(CIs)were also calculated.Statistical analysis of the AD case and control subjects which divided into the APOE ε4-positive and APOE ε4-negative subgroup was also carried out.Multivariate logistic regression analyses,adjusting for gender,APOE ε4 status and age at onset or age at examination,were used to estimate ORs and the 95% CIs for assessing genotypic and allelic associations with AD under three genetic models that were defined as 1(aa+Aa)versus 0(AA)for dominant,1(aa)versus 0(Aa+AA)for recessive,and 0(AA)versus 1(Aa)versus 2(aa)for additive(A represented major allele;a represented minor allele).The significance of an SNP-APOE interaction was also tested for each SNP in an optimal genetic model.All the analysis above was done by SPSS version 19.0 statistical software.P<0.05 was consideredstatistically significant.Linkage disequilibrium and haplotype analysis in total sample and subgroups were also conducted.Results The subjects recruited were age and gender matched.A total of 2338 ethnic Han Chinese subjects living in Shandong comprised of 984 LOAD patients and 1354 healthy controls were included in this study.No statistically significant differences were observed in age(age at onset for LOAD patients compared with age at examination for control subjects)(P=0.186)and gender(P=0.068)between cases and controls.As expected,MMSE scores were significantly lower in cases than in controls(P<0.001).In addition,the APOE ε4 allele status was also significantly different between patients with LOAD and control subjects(P<0.001).Carrying at least one ε4 allele increased LOAD risk by2.44-fold in our cohorts(OR=2.44;95% CI: 1.98~3.00;P<0.001).All these SNPs were in consistent with the HWE except rs3208305.Careful examination of the genotyping results did not reveal any genotyping errors.This might be associated with the sample size,the geographical distribution of the population,random genetic drift,or other uncertain factors.Based on the observed prevalence of the minor alleles in controls,our sample size had more than 80% power to detect these odds ratio at a significance level of0.05.This indicated a good representation of the Chinese Han population.In total sample,we only observed significant differences in genotype distributions of rs9331949(P<0.001,Pc=0.001)and rs3735964(P = 0.006,Pc=0.036)between cases and controls.After adjusting for age,gender and APOE ε4 allele status,the association of rs9331949 with LOAD still existed.The minor C allele in rs9331949 was significantly relative to the increased the risk of LOAD(P<0.001,OR=1.30,95% CI=1.14 ~ 1.50).Next,we stratified the subjects into APOE ε4 allele carrier and noncarrier subgroups according to APOE ε4 status.In the total sample,we detected an interaction between rs3200218,rs2160525 and APOE ε4 status.For rs9331949,genotype and allele frequencies differed significantly between LOAD patients and control subjects in APOE ε4 allele carriers and noncarriers,with the minor allele(C allele)significantly increasing the LOAD risk.After Bonferroni adjustment,genotype and allele frequencies differed significantly only in APOE ε4 allele noncarriers.For rs3200218,genotype differed significantly between LOAD patients and control subjects in APOE ε4 allele carriers,with the minor allele(G allele)significantly increasing the LOAD risk.Nevertheless,the significance disappeared after Bonferroni adjustment.The genotype and allele frequencies of the other four SNPs were not significantly associated with AD risk.Furthermore,we performed a multivariate logistic regression analysis to assess the effect of the six SNPs on LOAD risk(adjusting for gender,age at onset or at examination,APOE ε4 status in total sample,adjusting for only gender and age in APOE ε4 allele carriers and noncarriers)in total,APOE ε4 allele carriers and noncarriers.For rs9331949,additive model and recessive model showed positive association with LOAD after Bonferroni adjustment.Only recessive modelshowed positive association with LOAD in APOE ε4 noncarriers after Bonferroni adjustment(OR=2.09,95%CI=1.45~3.02,Pc=0.02).All the three genetic models of the other five SNPs showed negative association with LOAD in the total,APOE ε4 carriers and noncarriers after Bonferroni adjustment.In addition,we conducted linkage disequilibrium(LD)and haplotype analysis in total sample and subgroups.Four SNPs(rs1059507,rs3200218,rs3208305 and rs3735964)formed a haplotype block in all the three data set(total sample: D’= 1.0,r2 = 0.01).3 SNPs(rs1059507,rs3208305 and rs3735964)formed a haplotype block in APOEε4 carriers(APOEε4 allele carriers: D’ =1.0,r2 =0.011).Four SNPs(rs1059507,rs3200218,rs3208305 and rs3735964)formed a haplotype block in APOE ε4 noncarriers(APOE ε4 allele noncarriers: D’=1.0,r2= 0.376).Furthermore,we found 4 haplotype(TCCA,TCCG,ATCA,ACAA)derived from these 4SNPs in the total group and APOE ε4 noncarriers,but they showed no significant association with LOAD in any data set(P >0.05).Besides,3 haplotype(TCC,ATC,ACA)were derived from 3 SNPs in the APOE ε4 carriers(P >0.05).Conclusions Carrying at least one ε4 allele increased LOAD risk by 2.44 fold in our cohorts.The locus rs9331949 located in the binding site of 3’ UTR of CLU has a strong association with LOAD rather than loci in LPL and LRP6.The C allele of rs9331949 has an increased risk of 1.3-fold risk for AD.The polymorphism of rs9331949 is associated with LOAD risk in APOE ε4 allele noncarriers.Genotype and allele frequencies of the other four SNPs are not significantly associated with AD risk.Our study provide the preliminary theoretical basis for epigenetic about lipid metabolism related gene in AD.Although the mechanisms of this 3’ UTR of CLU gene lead to AD need further discussion,we have offered a new approach to comprehend LOAD.