Patent Application: US-201414252153-A

Abstract:
the invention relates to novel variants that associate with alzheimer &# 39 ; s disease ad and their use in kits as a means for diagnosing ad ; and also their use in nucleic acid molecules or cells / cell lines for identifying novel therapeutic , label of identification means .

Description:
the study comprised a stage 1 ‘ discovery sample ’ of 4 , 957 ad cases and 9 , 682 controls and a stage 2 ‘ follow - up sample ’ of 2 , 023 ad cases and 2 , 340 controls . individuals included in this study were drawn from europe and the united states . all individuals were of caucasian ancestry . all ad cases met criteria for either probable ( nincds - adrda , dsm - iv ) or definite ( cerad ) ad . this study used ‘ elderly screened controls ’ and ‘ population controls ’. elderly controls were screened for cognitive decline or neuropathological signs of ad . ‘ population controls ’ were drawn from large existing cohorts with available genome - wide association data . stage 1 discovery sample : the discovery sample included 4 , 113 cases and 1 , 602 elderly screened controls genotyped at the sanger institute on the illumina 610 - quad chip , referred to collectively hereafter as the 610 group . these samples were recruited by the medical research council ( mrc ) genetic resource for ad ( cardiff university ; institute of psychiatry , london ; cambridge university ; trinity college dublin ), the alzheimer &# 39 ; s research trust ( art ) collaboration ( university of nottingham ; university of manchester ; university of southampton ; university of bristol ; queen &# 39 ; s university belfast ; the oxford project to investigate memory and ageing ( optima ), oxford university ); washington university , st louis , united states ; mrc prion unit , university college london ; london and the south east region ad project ( laser - ad ), university college london ; university of bonn , germany and the national institute of mental health ( nimh ) ad genetics initiative . these data were combined with data from 844 ad cases and 1 , 255 elderly screened controls ascertained by the mayo clinic , jacksonville , fla . ; mayo clinic , rochester , minn . ; and the mayo brain bank , which were genotyped using the illumina humanhap300 beadchip . these samples were used in a previous gwas of ad 4 . a total of 6 , 825 population controls were included in stage 1 . these were drawn from large existing cohorts with available gwas data , including the 1958 british birth cohort ( 1958bc ) ( http :// www . b58cgene . sgul . ac . uk ), ninds funded neurogenetics collection at coriell cell repositories ( coriell ) ( see http :// ccr . coriell . org /), the kora study 11 , heinz nixdorf recall study 12 , 13 and als controls . the als controls were genotyped using the illumina humanhap300 beadchip . all other population controls were genotyped using the illumina humanhap550 beadchip . clinical characteristics of the discovery sample can be found in table s1 . stage 2 follow - up sample : the follow - up sample comprised 2 , 023 ad cases and 2 , 340 controls . samples were drawn from the mrc genetic resource for ad , the art collaboration , university of bonn , aristotle university of thessaloniki , a belgian sample derived from a prospective clinical study at the memory clinic and department of neurology , zna middelheim , antwerpen 14 and the university of munich . clinical characteristics of the follow - up sample can be found in table s2 . additional stage 2 samples ; the additional stage2 sample comprised 1 , 239 ad cases and 2 , 724 controls which were used for genotyping of the abca7 and ms4a loci . samples were drawn from the mrc genetic resource for ad , the art collaboration , university of bonn , and aristotle university of thessaloniki . clinical characteristics of the full stage 2 follow - up sample can be found in table s3 . dna was obtained from blood samples from each participant , by phenol / chloroform extraction , followed by precipitation in ethanol and storage in te buffer ( some dna was extracted using qiagen kits ). initial dna concentrations were determined by uv spectrophotometry ( μquant microplate spectrophotometer , bio - tek ®, beds , uk ) or nanodrop ™ ( thermo scientific , de , usa ). the concentration of each sample was then determined using the picogreen ® dsdna quantitation reagent ( molecular probes ®, eugene , ore .) in a labsystems ascent fluoroskan ® ( lifesciences int ., basingstoke , uk ). each sample was then diluted to 50 ng / ul and allowed to equilibrate at 4 ° c . for 48 h . dna quality was assessed by agarose gel electrophoresis under standard conditions . samples showing no evidence of degradation were then genotyped in a panel of 30 snps using the massarray ® and iplex ® systems ( sequenom ®, san diego , calif .) following manufacturer &# 39 ; s protocols . this allowed gender to be checked and permitted sample identity checks after re - arraying samples for gwas . genotyping was performed at the sanger institute , uk . all normalised samples passing quality control ( qc ) were re - arrayed for gwas using a biomek ® fx laboratory automation workstation ( beckman coulter ®, inc ., fullerton , calif .) into 96 well plate formats . 200 ng of input dna per sample were used and prepared for genotyping using the illumina infinium ™ system ( illumina ® inc ., san diego , calif ., usa ). manufacturer &# 39 ; s protocols were followed throughout . briefly , dna was isothermally amplified overnight then enzymatically fragmented , alcohol precipitated and resuspended . in this study we used illumina human 610 - quad beadchips ( illumina ® inc ., san diego , calif ., usa ) which were prepared for hybridization in a capillary flow - through chamber . the amplified and fragmented dna samples were hybridised to the bead chips using a tecan robot and an enzymatic base extension used to confer allele specificity . the chips were subsequently stained and scanned using an iscan reader ( illumina ® inc ., san diego , calif ., usa ) to detect fluorescence at each bead . data were loaded into beadstudio and final call reports containing x , y , x - raw and y - raw outputted . the illuminus algorithm for cluster analysis was used for genotype calling 15 . 4 , 113 ad cases and 1 , 602 controls were genotyped on the illumina 610 - quad chip as part of this study ( the 610 group ). in addition , 844 ad cases and 8 , 080 controls previously genotyped using either the illumina humanhap550 or illumina humanhap300 were included in the analysis . these genotypes were generated as part of 7 different studies , making 8 separate groups in total : 1 ) 610 ; 2 ) mayo ; 3 ) 1958 birth cohort ( sanger ); 4 ) 1958 birth cohort ( t1dgc ); 5 ) als control ; 6 ) coriell control ; 7 ) heinz nixdorf recall ( hnr ) study ; 8 ) kora . as we used genotype data from multiple sources , it was important to apply stringent qc filters , as differential genotyping error rates between groups could result in spurious associations when the data are combined 16 , 17 . these filters were applied separately to each of these 8 groups to remove poorly performing samples using tools implemented in plink v1 . 05 ( http :// pngu . mgh . harvard . edu - purcell / plink ) 18 . the specific qc thresholds applied and the breakdown of samples excluded by group are given in tables s4 and s5 , respectively . we removed 1 , 469 individuals with missing genotype rates & gt ; 0 . 01 . we also applied a filter based on mean autosomal heterozygosity , excluding 578 individuals with values above or below empirically determined thresholds . 71 individuals with inconsistencies between reported gender and genotype - determined gender and 22 individuals with ambiguous genotype - determined gender were removed . all individuals passing these qc filters were examined for potential genetic relatedness by calculating identity by descent ( ibd ) estimates for all possible pairs of individuals in plink , and removing one of each pair with an ibd estimate ≧ 0 . 125 ( the level expected for first cousins ). ibd estimates were calculated using snps that were common to the lumina 610 , 550 and 300 chips with a genotype missing data rate ≦ 0 . 01 , hardy - weinberg p ≧ 1 × 10 − 5 and a minor allele frequency ≧ 0 . 01 . as a result , 506 individuals were excluded ( note that this includes 311 individuals that were included in both the coriell and als control group ). we also sought to detect non - european ancestry . to this end , genotype data from snps typed in all cohorts was merged with genotypes at the same snps from 210 unrelated european ( ceu ), asian ( chb and jpt ) and yoruban ( yri ) samples from the hapmap project . subsequent to removing snps in extensive regions of linkage disequilibrium ( chr5 : 44 - 51 . 5 mb ; chr6 : 25 - 33 . 5 mb ; chr8 : 8 - 12 mb ; chr11 : 45 - 57 mb ) 19 , we further pruned snps if any pair within a 50 - snp window had r 2 & gt ; 0 . 2 . genome - wide average identity by state ( ibs ) distance was calculated in plink between each pair of individuals in the resulting dataset , based on 57 , 966 snps ( all with a genotype missing data rate ≦ 0 . 01 , hardy - weinberg p ≧ 1 × 10 − 5 and a minor allele frequency ≧ 0 . 01 ). the resulting matrix of ibs distances was used as input for classical multi - dimensional scaling ( mds ) in r v2 . 7 . 1 ( http :// www . r - project . org ). when the first two dimensions were extracted and plotted against each other , three clusters were observed corresponding to the european , asian and yoruban samples . sixteen samples appeared to be ethnic outliers from the european cluster and were excluded from further analysis . we assessed population structure within the data using principal components analysis ( pca ) as implemented in eigenstrat 20 to infer continuous axes of genetic variation . eigenvectors were calculated based on the previously described ld - pruned subset of 57 , 966 snps common to all arrays . the eigenstrat program also identifies genetic outliers , which are defined as individuals whose ancestry is at least 6 standard deviations from the mean on one of the top ten axes of variation . as a result , 188 outliers were identified and excluded . following sample qc 3 , 941 ad cases and 7 , 848 controls were included in the analysis . only autosomal snps were included in this analysis . individuals were genotyped on either the rumina 610 - quad as part of this project , or were previously genotyped on the illumina humanhap550 or the illumina humanhap300 array , and the genotypes made available to us . note that snps had already been filtered out of some groups prior to inclusion in this study . moreover , where different versions of the same array were used ( e . g . humanhap550v1 used to genotype the 1958 birth cohort ( sanger ) cohort compared with the humanhap550v3 array used to genotype the 1958 birth cohort ( t1dgc )), only snps common to both versions were considered as present on that array . as such , snps included in our analysis fell into 4 different categories ; 1 ) 266 , 714 snps common to all 3 arrays and genotyped in all individuals ; 2 ) 202 , 516 snps common to the 610 and 550 arrays , but not present or without genotypes in individuals typed on the 300 array ; 3 ) 7 , 744 snps common to the 610 and 300 arrays , but not present or without genotypes in individuals typed on the 550 array ; 4 ) 105 , 614 snps with genotypes only in the 610 data ( see table s6 ). we assessed the effects of different missing data rate and hardy - weinberg filters , aiming to remove poorly performing snps without excluding markers that may show genuine association with ad . for each of the 4 snp categories , markers were excluded if they had a minor allele frequency ( maf )& lt ; 0 . 01 or a hardy - weinberg p ≦ 1 × 10 − 5 , in either cases or controls . snps with a maf ≧ 0 . 05 were excluded if they had a genotype missing rate of & gt ; 0 . 03 in either cases or controls ; for snps with a maf between 0 . 01 and 0 . 05 , a more stringent genotype missing rate threshold of 0 . 01 was employed . as a result of this basic snp qc 43 , 542 snps were excluded . ten principal components ( pcs ) were extracted using eigenstrat , as previously described . to determine if the pcs could assuage any population structure within our sample , we performed logistic regression tests of association with ad , sequentially including between 0 and 10 of the top pcs as covariates . the impact of including the pcs was evaluated by calculating the genomic control inflation factor , λ 21 . we found that including the first 4 pcs as covariates had the maximum impact on λ ( see table s7 ). to minimise inter - chip and inter - cohort differences that could result in an inflation of type i error rate , minor allele frequencies were compared between controls in the different groups using logistic regression analysis , incorporating the top 4 pcs as covariates as previously described . comparisons were only performed between individuals from the same geographical region ( i . e . british isles , germany or usa ) and included : 1 ) 1958 birth cohort ( sanger ) versus 1958 birth cohort ( t1dgc ); 2 ) 1958 birth cohort ( combined sanger and t1dgc ) versus 610 uk controls ; 3 ) 1958 birth cohort ( combined sanger and t1dgc ) versus als uk controls ; 4 ) 610 uk controls versus als uk controls ; 5 ) hnr cohort versus kora cohort ; 6 ) 610 german controls versus combined hnr and kora cohort ; 7 ) mayo controls versus als us controls ; 8 ) coriell cohort versus combined mayo and als us controls ; 9 ) 610 us controls versus combined mayo and als us controls ; 10 ) 610 us controls versus coriell cohort . moreover , as a result of comparisons 2 , 4 , 6 , 7 , 9 and 10 , elderly screened controls were compared with unscreened / population controls . for each of the 4 categories of snps , a quantile - quantile ( q - q ) plot was produced for each cohort control comparison , and the significance threshold employed to exclude snps was based on where the observed χ 2 statistics departed from the null expectation ( see table s8 and fig4 ). a further 9 , 828 snps were excluded as a result of these comparisons . thus , a total of 529 , 218 snps were analysed for association with ad in this study . snps were tested for association with ad using logistic regression , assuming an additive model . covariates were included in the logistic regression analysis to allow for geographical region and chip , i . e . to distinguish between 1 ) individuals from the british isles , 2 ) individuals from germany , 3 ) individuals from the us typed on the 610 or 550 chip , 4 ) individuals from the us typed on the 300 chip . it was not possible to include a covariate for each chip as only controls were genotyped on the 550 chip . similarly , it was not possible to include a covariate for each of the 8 groups , as only two included both cases and controls ( 610 and mayo groups ). the first 4 pcs extracted from eigenstrat were also included as covariates , as previously described . following analysis , 130 cluster plots were visually inspected for snps with a p - value ≦ 1 × 10 − 4 . thirteen snps showing poorly formed clusters were excluded . thus our analysis was based on 529 , 205 snps , and a conservative genome - wide significance threshold of 0 . 05 / 529205 = 9 . 4 × 10 − 8 was employed . q - q plots of the test results are shown in fig5 . the overall genomic control inflation factor , λ , was calculated to be 1 . 037 . results are shown for snps with a p - value ≦ 1 × 10 − 4 in table s9 . a breakdown of minor allele frequencies in cases and controls is shown for genome - wide significant snps in table s10 . we genotyped snps in cases and controls from 5 european cohorts ( described in table s2 and s3 ). genotyping was performed at cardiff using the massarray and iplexgold systems ( sequenom , san diego , calif .) according to manufacturer &# 39 ; s recommendations . all assays were validated prior to use , based on optimisation in 30 reference centre d ′ etude du polymorphisme humain ( ceph ) parent - offspring trios . sample plates contained cases , controls and blank samples . quality control measures included independent double genotyping blind to sample identity and blind to the other rater , and where available comparison of our ceph genotypes to those in the hapmap ( www . hapmap . org ). in addition , 231 individuals included in the gwas were also genotyped on the sequenom platform . we calculated the average concordance rate for the 7 snps typed on both platforms to be 99 %. all genotyped snps had genotype call frequency rates & gt ; 90 % in the follow - up sample , and no snps had hwe p - value ≦ 0 . 05 in cases or controls . snps were tested for association with ad using logistic regression , assuming an additive model . covariates were included in the logistic regression analysis to allow for each cohort , i . e . 1 ) belgium , 2 ) mrc , 3 ) art , 4 ) bonn , 5 ) greek . we included genotype data from stages 1 and 2 in a meta - analysis for snps at the clu and picalm loci . in addition , we employed genotype data from the tgen study , a publicly available ad gwas dataset . this sample is comprised of 861 ad cases and 550 controls genotyped on the affymetrix 500k chip . if a snp of interest was not genotyped in our gwas or the tgen dataset , an attempt was made to impute genotypes in plink , using the 60 hapmap ceu founders as a reference panel . only imputed snps with an information content metric value greater than 0 . 8 were included in analysis ( see plink website ). snps were tested for association with ad using logistic regression , assuming an additive model . covariates were included in the logistic regression analysis to allow for geographical region and chip as in stage 1 and for cohort as in stage 2 . covariates included for the tgen sample distinguished between samples from the netherlands brain bank and samples from the usa . results of the meta - analysis are shown in table 1 and table s11 . we included genotype data from stages 1 and the full stage 2 sample in a meta - analysis for snps at the abca7 and ms4a loci . in addition , we employed genotype data from the tgen ( translational genomics research institute ) 22 and adni ( alzheimer &# 39 ; s disease neuroimaging initiative ) 23 studies , both publicly available ad gwas datasets , analysed in house for association to ad using a logistic regression assuming an additive model and including country of origin covariates for the tgen study 10 . we first performed an inverse variance weighted fixed effects meta - analysis of the gerad1 , adni and tgen datasets . the p - values from this meta - analysis were then combined with the publicly available p - values from the eadi1 ( european alzheimer &# 39 ; s disease initiative stage 1 ) 24 study using fisher &# 39 ; s combined probability test ( note that odds ratios and variances for all snps genotyped in the eadi1 study were not publicly available , thus precluding an inverse variance weighted meta - analysis of all 4 gwas ). the combined analysis tested 496763 autosomal snps . these snps passed qc in our stage 1 sample and each of the adni and eadi1 gwa studies ; 52391 of these snps were also genotyped and passed qc in the tgen gwas ( which unlike the other studies employed the affymetrix 500k array ). in the combined analysis , 61 snps were associated with ad at p ≦ 1 × 10 − 5 ( see table s14 ). for the five snps that showed evidence for association in our stage 2 sample ( p & lt ; 0 . 05 ), summary data ( including odds ratios and variances ) were obtained from the charge ( cohorts for heart and aging research in genomic epidemiology ) 25 and eadi1 studies , such that all data ( i . e ., our stage 1 & amp ; 2 , eadi1 , adni , tgen and charge ) were combined in an inverse - variance weighted fixed effects meta - analysis ( total sample of up to 11607 cases and 31871 controls ). cochran &# 39 ; s q - test was performed and i 2 calculated to assess heterogeneity . although the same direction of effect was observed in all studies analyzed for the cr1 snp rs3818361 , there was some evidence of heterogeneity ( cochran &# 39 ; s q test p = 0 . 02 , i 2 = 65 %). a random effects meta - analysis of this snp was also performed for comparison ( see table s15 ). we also tested the gws snps for relationships with age at onset ( aao ). to this end , age at onset ( in years ) was employed as the dependent variable in a linear regression analysis and an additive model was assumed . aao data was available for 2 , 856 ad cases . covariates were included in the logistic regression analysis to allow for geographical region and chip , i . e . to distinguish between 1 ) cases from the british isles , 2 ) cases from germany , 3 ) cases from the us typed on the 610 chip , 4 ) cases from the us typed on the 300 chip . results are shown in table s12 . in addition , we stratified our sample based on presence / absence of at least 1 apoe ε4 allele . we had apoe genotype data for 6045 individuals ; our ε4 - positive sample consisted of 2 , 203 ad cases and 632 controls ; our ε4 - negative sample consisted of 1 , 446 cases and 1 , 764 controls . we performed genome - wide tests for association with ad in each sub - sample , but no snp achieved genome - wide significance ( data not shown ). we assessed our results to determine if we observed more significant snps at stage 1 than would be expected by chance . we first removed snps within 500 kb either side of risk snps , i . e . rs429358 ( the apoe ε4 snp ), rs11136000 ( clu ) and rs3851179 ( picalm ). we thus excluded 170 “ apoe ” snps , 290 “ clu ” snps and 257 “ picalm ” snps . of the 528 , 448 remaining snps we estimated 397 , 224 . 7 “ independent ” tests using the algorithm we described in ( 15 ). of 16 snps significant at a significance level α = 10 − 5 ( excluding apoe , clu and picalm snps ) we estimated 12 . 6 “ independent ” tests . we calculated mean ( n * α = 397224 . 7 * 10 − 5 ≈ 4 . 0 ) and variance ( n * α *( 1 − α )= 3 . 97 ) of the expected number of significant tests at α = 10 − 5 level using the binomial distribution . thus the probability of observing 12 . 6 significant tests is p = 7 . 5 × 10 − 6 . preliminary analysis of novel gws loci : to further explore relationships within the two associated gene regions ( clu and picalm ) we selected additional snps from each , which either showed some evidence for association at stage 1 ( p ≦ 0 . 001 ) or were putative functional snps in linkage disequilibrium ( d ′& gt ; 0 . 3 ) with the novel gws hits ( see table s11 ). predicted functional snps were identified using pupasuite ( http :// bioinfo . cipf . es / pupasuite / www / index . jsp ) 26 . these were tested for association with ad in an independent sample and also checked for association in the tgen gwas . four additional snps were selected from the clu locus , one which showed some evidence at stage 1 ( rs7012010 ; p = 8 × 10 − 4 ) and three which had putative functional significance but were not typed in stage 1 ( rs3087554 , rs9331888 and rs7982 , see table s11 ). rs7012010 showed no evidence for association in the original stage 2 sample , some evidence in the tgen dataset ( p = 0 . 033 ) but did not reach gws levels of significance in the meta - analysis ( p = 1 × 10 − 4 ). two of the remaining snps showed no evidence for association , whereas the third rs7982 , a synonymous snp in strong ld ( r 2 = 1 in hapmap ceu individuals ; r 2 = 0 . 95 in extension sample ) with the gws snp in clusterin , did show association in the original stage 2 sample , with a similar magnitude of effect ( meta p = 8 × 10 − 10 ; stage 1 genotypes imputed ). we selected 8 additional snps for follow - up from the picalm locus : half showed significant evidence for association in stage 1 and half were chosen for their putative function ( see table s11 ). three of the first four snps showed evidence for association in both the original stage 2 and tgen samples with the fourth showing association in the tgen dataset alone . producing combined p - values of : rs677909 , p = 3 × 10 − 8 ; rs541458 , p = 5 × 10 − 9 ; rs543293 , p = 1 × 10 − 8 ; and rs7941541 p = 3 × 10 − 9 , none of which exceeded that of the original gws snp . all of these snps were in high ld with the original gws snp . the only potentially functional snps which showed good evidence for association were rs561655 which is within a putative transcription factor binding site and rs592297 which is a synonomous snp in exon 5 of the gene which may influence a putative exon splicing enhancer . however , neither of these snps showed the strength of evidence for association observed for rs3851179 , the gws snp , producing meta p = 1 × 10 − 7 and p = 2 × 10 − 7 , respectively . our results provide compelling evidence that the genes described herein are true susceptibility genes for ad . however , a further striking implication of our findings is the support for additional disease mechanisms that go beyond aβ accumulation . three , and possibly four of the recently identified ad susceptibility loci ( clu , cr1 , abca7 & amp ; the ms4a gene cluster ) have known functions in the immune system , specifically the classical complement pathway . furthermore , both picalm and bin1 are involved in clathrin - mediated endocytosis and apoe , clu and abca7 in lipid processing . a p values were obtained by performing an inverse - variance weighted meta - analysis of the stage 1 , adni and tgen , and combining the resultant p - values with those from the eadi1 study using fisher &# 39 ; s combined probability test . c rs11894266 failed to optimize , rs13010581 genotyped as proxy ( r2 = 1 , d ′ = 1 ), d p - values based on inverse variance weighted meta - analysis of stage 1 & amp ; 2 , adni , tgen and eadi1 for snps rs3764650 , rs670139 , rs744373 , rs610932 & amp ; rs3818361were 2 . 8 × 10 − 7 , 2 . 9 × 10 − 6 , 3 . 6 × 10 − 9 , 3 . 8 × 10 − 7 and 2 . 5 × 10 − 12 , respectively . † mean age at death for autopsy confirmed samples only ( n = 246 ). age at onset data is not available for these participants . ‡ age at onset only available for a proportion of the sample sample size and descriptive statistics for the original stage 2 sample . ‡ age at onset only available for a proportion of the sample † age at interview not available for 438 ad cases and 104 controls . age at death is provided for these subjects where available . ‡ age at onset data only available for less than 75 % of the sample note that 311 individuals were included in both the coriell and als control cohort . a population controls were not excluded if there was a discrepancy between reported gender and genotype - determined gender . are based on analysis of snps common to the iilumina 610 - quad , significance thresholds ( χ 2 values ) employed to exclude snps showing snps showing association with ad ( p ≦ 1 × 10 − 4 ) in the gwas . * this snp was genotyped in a subsample of 3333 cases and 6460 controls . snps selected for follow - up genotyping . p - values in the gwas , the extension sample , a previous ad gwas ( tgen ), and the combined sample ( meta ) are also shown . all p - values are two - tailed . test of association between genome - wide significant snps and age at onset of alzheimer &# 39 ; s disease . stage 1 p - values and details of snp selection for stage 2 genotyping and stage 3 meta - anlaysis ( r 2 = 1 ) was genotyped in gerad2 ( p = 0 . 682 ) snp was not selected for stage 2 as a proxy snp rs3818361 ( r 2 = 0 . 956 ) was genotyped through gerad2 ( p = 0 . 009 ) snp was not selected for stage 2 as a proxy snp rs3818361 ( r 2 = 1 ) was genotyped through gerad2 ( p = 0 . 009 ) snp was not selected for stage 2 as a proxy snp rs610932 ( r 2 = 0 . 88 ) was genotyped through gerad2 ( p = 0 . 028 ) snp was not selected for stage 2 as it is in ld with rs610932 ( r 2 = 0 . 75 ) which was genotyped through gerad2 ( p = 0 . 028 ) snp was not selected for stage 2 as a proxy snp rs670139 ( r 2 = 1 ) was genotyped through gerad2 ( p = 0 . 0009 ) snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the apoe locus snp was not selected for stage 2 as it is located at the clu locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus snp was not selected for stage 2 as it is located at the picalm locus nb : the position of the snp of interest within the flanking sequence is depicted by the square brackets . the nucleotide change at this snp is displayed within the square brackets . 1 . abraham , r . a genome - wide association study for late - onset alzheimer &# 39 ; s disease using dna pooling . bmc med . genomics 1 , 44 ( 2008 ). 2 . beecham , g . genome - wide association study implicates a chromosome 12 risk locus for late - onset alzheimer disease . am . j . hum . genet . 84 , 35 - 43 ( 2009 ). 3 . bertram , l . genome - wide association analysis reveals putative alzheimer &# 39 ; s disease susceptibility loci in addition to apoe . am . j . hum . genet . 83 , 623 - 632 ( 2008 ). 4 . carrasquillo , m . genetic variation in pcdh11x is associated with susceptibility to late - onset alzheimer &# 39 ; s disease . nat . genet . 41 , 192 - 198 ( 2009 ). 5 . coon , k . a high - density whole - genome association study reveals that apoe is the major susceptibility gene for sporadic late - onset alzheimer &# 39 ; s disease . j . clin . psychiatry 68 , 613 - 618 ( 2007 ). 6 . grupe , a . evidence for novel susceptibility genes for late - onset alzheimer &# 39 ; s disease from a genome - wide association study of putative functional variants . hum . mol . genet . 16 , 865 - 873 ( 2007 ). 7 . li , h . candidate single - nucleotide polymorphisms from a genomewide association study of alzheimer disease . arch . neurol . 65 , 45 - 53 ( 2008 ). 8 . reiman , e . gab2 alleles modify alzheimer &# 39 ; s risk in apoe epsilon4 carriers . neuron 54 , 713 - 720 ( 2007 ). 9 . harold , d . et al . genome - wide association study identifies variants at clu and picalm associated with alzheimer &# 39 ; s disease . nat genet 41 , 1088 - 1093 ( 2009 ). 10 . hollingworth , p . et al . evidence that abca7 and ms4a are novel susceptibility loci for alzheimer &# 39 ; s disease and further support for bin1 and cr1 . nature genetics submitted , 11 . wichmann , h ., gieger , c . & amp ; illig , t . kora - gen - resource for population genetics , controls and a broad spectrum of disease phenotypes . gesundheitswesen 67 suppl 1 , s26 - 30 ( 2005 ). 12 . birnbaum , s . et al . key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24 . nat . genet 41 , 473 - 477 ( 2009 ). 13 . hillmer , a . m . et al . susceptibility variants for male - pattern baldness on chromosome 20p11 . nat . genet 40 , 1279 - 1281 ( 2008 ). 14 . brouwers , n . et al . genetic variability in progranulin contributes to risk for clinically diagnosed alzheimer disease . neurology 71 , 656 - 664 ( 2008 ). 15 . teo , y . y . et al . a genotype calling algorithm for the illumina beadarray platform . bioinformatics 23 , 2741 - 2746 ( 2007 ). 16 . clayton , d . g . et al . population structure , differential bias and genomic control in a large - scale , case - control association study . nat genet 37 , 1243 - 1246 ( 2005 ). 17 . moskvina , v ., craddock , n ., holmans , p ., owen , m . j . & amp ; 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