| Objective:?-synuclein is a 140-amino acid protein produced predominantly by neurons in the brain which plays a role in the regulation of neurotransmitter release,synaptic function and plasticity,thus making it the focus in understanding the etiology of a group of neurodegenerative diseases.Endophenotypes approach could be a complementary to GWAS case-control association studies.Here,on the basis of adequate evidence on the role of CSF?-synuclein in neurodegenerative disorders,we conducted a GWAS of CSF?-synuclein from ADNI database.Further examinations of the variants that we have identified in different datasets may lead to a deeper understanding of?-synuclein regulation and provide important insights into its effects on?-synuclein-related function and disorders.Methods:Data used in this study were obtained from the ADNI database?http://adni.loni.usc.edu?.The most recent information from the ADNI is available online?http://www.adni-info.org?.The ADNI is a large,multicenter,longitudinal neuroimaging study,launched in 2003 by the National Institute on Aging,the National Institute of Biomedical Imaging and Bioengineering,the Food and Drug Administration,private pharmaceutical companies,and nonprofit organizations.The study gathered and analyzed thousands of brain scans,genetic profiles,and biomarkers in blood and cerebrospinal fluid.This study was approved by institutional review boards of all participating institutions and written informed consent was obtained from all participants or authorized representatives.Our study population consisted of all cognitively normal?CN?,mild cognitive impairment?MCI?,and AD dementia group participants from the ADNI-1.In this study,686?CN=194,MCI=330,AD=162 at baseline?non-Hispanic Caucasian individuals from the ADNI cohort whose data met all quality control criteria were included.Levels of CSF-synuclein concentration were measured using Luminex MicroPlex?Luminex Corp,Austin,TX?.The?-synuclein Luminex assay demonstrated low day-to-day as well as plate-to-plate signal variability.The accuracy for the assay,as determined by recovery of spiked-synuclein,was93%.The ADNI samples were genotyped with the Illumina 610 chip.Only SNPs with a minor allele frequency?MAF?>5%,call rates>98%,and Hardy-Weinberg equilibrium P>0.001 were retained for analysis.Finally,a maximum of 519,442 SNPs were retained after these procedures.On the basis of data for all of these SNPs,we excluded 151individuals who had more than 5%missing genotypes within 757 samples.This more stringent threshold was chosen to reduce the likelihood of false-positive results in the context of modest sample size.In order to decrease CSF contamination by RBC,a human hemoglobin ELISA quantitation kit was used,which has sensitivity well beyond the cut-off value of 1000ng/ml For this reason,326 samples were removed.In addition,we excluded 71samples that were Hispanic Caucasians.Finally,209 individuals with CSF?-synuclein were retained at last.The distribution of?-synuclein levels were approximately considered as normal distribution after log transformation.One-way ANOVA models were used for quantitative normally distributed variables.Rank-based two-way methods were used for non-normally distributed quantitative variables.Chi-square text was applied to categorical data.To examine the main effect of each SNP on the CSF?-synuclein biomarker,GWAS was performed with additive genetic model.We used a multiple linear regression model to estimate possible correlation between genotypes and CSF?-synuclein concentration?e.g.,dose-dependent effect of the minor allele?.Covariates such as age,gender,APOE status,educational level and baseline disease status were considered and retaind in the final models if P<0.05.We focused on SNPs with uncorrected P<5×10-8?or Bonferroni correction P<0.01?as genome-wide significant and secondarily examined SNPs with p values less than 1×10-5to identify potential candidates.All statistical analyses were performed by R 3.4.0 and PLINK.Results:There were 209?CN=59,MCI=101,AD=49 at baseline?non-Hispanic Caucasian participants at baseline diagnosis.No difference was found across the diagnostic groups for age,education and sex?P>0.05?.Compared to CN and MCI subjects,AD individuals have higher CSF?-synuclein concentration,higher frequency of APOE?4 allele,and worst cognitive function displayed by neuropsychological scales?MMSE and CDRSB??P<0.05?.In addition,associations were detected between baseline demographics?e.g.,APOE status,disease status and educational years?and CSF?-synuclein level?P<0.05?,which were considered as the evidence of covariates.Six SNPs in the regions of long intergenic non-protein coding RNA 1515?LINC01515?and clusterin associated protein 1?CLUAP1?reached genome-wide significance(unadjusted P<10-7,adjusted P<0.01).In addition,SNPs near APP?rs1394839?(P=2.31×10-7),RAPGEF1?rs10901091?(P=8.07×10-7),and two intergenic loci on chromosome 2 and 14(rs11687064 P=2.50×10-7and rs7147386 P=4.05×10-7)were identified as suggestive loci associated with CSF?-synuclein levels.Among all the SNPs,rs7072338,which is located in intron region of LINC01515 on chromosome 10,showed the strongest association with CSF?-synuclein(Uncorrected P=4.167×10-9,Bonferroni corrected P=2.164×10-3).Another four SNPs located near rs7072338 also reached a GWAS significant P value(Uncorrected P=1.909×10-8,Bonferroni corrected P=9.917×10-3).Besides that,three SNPs around rs7072338 showed a P value lower than 10-5.We confirmed the most significant SNP in this locus and other seven SNPs in linkage disequilibrium?LD,r2>0.8?.However,after controlling for rs7072338 genotype,no SNPs in this region showed association with CSF?-synuclein levels indicating that all the association in this locus was driven by rs7072338).In addition,the linkage disequilibrium?LD?pattern between rs7072338 and nearby SNPs was almost identical in the ADNI cohort compared with 1000 Genomes European subjects,suggesting that the SNP genotypes from this study were accurate.The minor allele?T?of rs7072338 was associated with higher CSF?-synuclein levels in a dose-dependent effect within both combined groups and each diagnostic group(normal group,P=5.14×10-5;pMCI group,P=3.77×10-3;sMCI group,P=0.034 and AD group,P=8.56×10-4).Moreover,rs17794023,located in CLUAP1,also showed genome-wide significant association with CSF?-synuclein levels(P=9.56×10-9).This locus survived even after Bonferroni corrections for multiple testing(Bonferroni corrected P=4.964×10-3).The minor allele?T?of rs17794023 was associated with higher CSF?-synuclein levels in a dose-dependent effect within both combined groups and each diagnostic group(normal group,P=2.81×10-3;pMCI group,P=3.32×10-3;sMCI group,P=0.56 and AD group,P=3.14×10-5).Conclusion:In summary,we report for the first time that CSF?-synuclein could be a suitable endophenotype for genetic research of AD.We identified two new SNPs associated with high CSF?-synuclein with statistical evidence which had never been detected by traditional case-control GWAS and candidate gene association studies.In addition,SNPs near APP?rs1394839??P=2.31×10-7?,RAPGEF1?rs10901091??P=8.07×10-7?,and two intergenic loci on chromosome 2 and 14?rs11687064 P=2.50×10-7 and rs7147386 P=4.05×10-7?were identified as suggestive loci associated with CSF?-synuclein levels. |
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