Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-ß precursor protein (APP) and extracellular Aß42 and Aß40 (the 42- and 40-residue isoforms of the amyloid-ß peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aß42 and Aß40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Heritability and genetic variance of dementia with Lewy bodies.

Recent large-scale genetic studies have allowed for the first glimpse of the effects of common genetic variability in dementia with Lewy bodies (DLB), identifying risk variants with appreciable effect sizes. However, it is currently well established that a substantial portion of the genetic heritable component of complex traits is not captured by genome-wide significant SNPs. To overcome this issue, we have estimated the proportion of phenotypic variance explained by genetic variability (SNP heritability) in DLB using a method that is unbiased by allele frequency or linkage disequilibrium properties of the underlying variants. This shows that the heritability of DLB is nearly twice as high as previous estimates based on common variants only (31% vs 59.9%). We also determine the amount of phenotypic variance in DLB that can be explained by recent polygenic risk scores from either Parkinson's disease (PD) or Alzheimer's disease (AD), and show that, despite being highly significant, they explain a low amount of variance. Additionally, to identify pleiotropic events that might improve our understanding of the disease, we performed genetic correlation analyses of DLB with over 200 diseases and biomedically relevant traits. Our data shows that DLB has a positive correlation with education phenotypes, which is opposite to what occurs in AD. Overall, our data suggests that novel genetic risk factors for DLB should be identified by larger GWAS and these are likely to be independent from known AD and PD risk variants.

Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies.

Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson's and Alzheimer's diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.

Interaction of insulin and PPAR-α genes in Alzheimer's disease: the Epistasis Project.

Altered glucose metabolism has been described in Alzheimer's disease (AD). We re-investigated the interaction of the insulin (INS) and the peroxisome proliferator-activated receptor alpha (PPARA) genes in AD risk in the Epistasis Project, including 1,757 AD cases and 6,294 controls. Allele frequencies of both SNPs (PPARA L162V, INS intron 0 A/T) differed between Northern Europeans and Northern Spanish. The PPARA 162LL genotype increased AD risk in Northern Europeans (p = 0.04), but not in Northern Spanish (p = 0.2). There was no association of the INS intron 0 TT genotype with AD. We observed an interaction on AD risk between PPARA 162LL and INS intron 0 TT genotypes in Northern Europeans (Synergy factor 2.5, p = 0.016), but not in Northern Spanish. We suggest that dysregulation of glucose metabolism contributes to the development of AD and might be due in part to genetic variations in INS and PPARA and their interaction especially in Northern Europeans.

No evidence that extended tracts of homozygosity are associated with Alzheimer's disease.

We sought to investigate the contribution of extended runs of homozygosity in a genome-wide association dataset of 1,955 Alzheimer's disease cases and 955 elderly screened controls genotyped for 529,205 autosomal single nucleotide polymorphisms. Tracts of homozygosity may mark regions inherited from a common ancestor and could reflect disease loci if observed more frequently in cases than controls. We found no excess of homozygous tracts in Alzheimer's disease cases compared to controls and no individual run of homozygosity showed association to Alzheimer's disease.

The dopamine ß-hydroxylase -1021C/T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project.

BACKGROUND: The loss of noradrenergic neurones of the locus coeruleus is a major feature of Alzheimer's disease (AD). Dopamine ß-hydroxylase (DBH) catalyses the conversion of dopamine to noradrenaline. Interactions have been reported between the low-activity -1021T allele (rs1611115) of DBH and polymorphisms of the pro-inflammatory cytokine genes, IL1A and IL6, contributing to the risk of AD. We therefore examined the associations with AD of the DBH -1021T allele and of the above interactions in the Epistasis Project, with 1757 cases of AD and 6294 elderly controls. METHODS: We genotyped eight single nucleotide polymorphisms (SNPs) in the three genes, DBH, IL1A and IL6. We used logistic regression models and synergy factor analysis to examine potential interactions and associations with AD. RESULTS: We found that the presence of the -1021T allele was associated with AD: odds ratio = 1.2 (95% confidence interval: 1.06-1.4, p = 0.005). This association was nearly restricted to men < 75 years old: odds ratio = 2.2 (1.4-3.3, 0.0004). We also found an interaction between the presence of DBH -1021T and the -889TT genotype (rs1800587) of IL1A: synergy factor = 1.9 (1.2-3.1, 0.005). All these results were consistent between North Europe and North Spain. CONCLUSIONS: Extensive, previous evidence (reviewed here) indicates an important role for noradrenaline in the control of inflammation in the brain. Thus, the -1021T allele with presumed low activity may be associated with misregulation of inflammation, which could contribute to the onset of AD. We suggest that such misregulation is the predominant mechanism of the association we report here.

Replication by the Epistasis Project of the interaction between the genes for IL-6 and IL-10 in the risk of Alzheimer's disease.

BACKGROUND: Chronic inflammation is a characteristic of Alzheimer's disease (AD). An interaction associated with the risk of AD has been reported between polymorphisms in the regulatory regions of the genes for the pro-inflammatory cytokine, interleukin-6 (IL-6, gene: IL6), and the anti-inflammatory cytokine, interleukin-10 (IL-10, gene: IL10). METHODS: We examined this interaction in the Epistasis Project, a collaboration of 7 AD research groups, contributing DNA samples from 1,757 cases of AD and 6,295 controls. RESULTS: We replicated the interaction. For IL6 rs2069837 AA x IL10 rs1800871 CC, the synergy factor (SF) was 1.63 (95% confidence interval: 1.10-2.41, p = 0.01), controlling for centre, age, gender and apolipoprotein E epsilon4 (APOEepsilon4) genotype. Our results are consistent between North Europe (SF = 1.7, p = 0.03) and North Spain (SF = 2.0, p = 0.09). Further replication may require a meta-analysis. However, association due to linkage disequilibrium with other polymorphisms in the regulatory regions of these genes cannot be excluded. CONCLUSION: We suggest that dysregulation of both IL-6 and IL-10 in some elderly people, due in part to genetic variations in the two genes, contributes to the development of AD. Thus, inflammation facilitates the onset of sporadic AD.

A comprehensive screening of copy number variability in dementia with Lewy bodies.

The role of genetic variability in dementia with Lewy bodies (DLB) is now indisputable; however, data regarding copy number variation (CNV) in this disease has been lacking. Here, we used whole-genome genotyping of 1454 DLB cases and 1525 controls to assess copy number variability. We used 2 algorithms to confidently detect CNVs, performed a case-control association analysis, screened for candidate CNVs previously associated with DLB-related diseases, and performed a candidate gene approach to fully explore the data. We identified 5 CNV regions with a significant genome-wide association to DLB; 2 of these were only present in cases and absent from publicly available databases: one of the regions overlapped LAPTM4B, a known lysosomal protein, whereas the other overlapped the NME1 locus and SPAG9. We also identified DLB cases presenting rare CNVs in genes previously associated with DLB or related neurodegenerative diseases, such as SNCA, APP, and MAPT. To our knowledge, this is the first study reporting genome-wide CNVs in a large DLB cohort. These results provide preliminary evidence for the contribution of CNVs in DLB risk.

Mendelian adult-onset leukodystrophy genes in Alzheimer's disease: critical influence of CSF1R and NOTCH3.

Mendelian adult-onset leukodystrophies are a spectrum of rare inherited progressive neurodegenerative disorders affecting the white matter of the central nervous system. Among these, cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy, cerebroretinal vasculopathy, metachromatic leukodystrophy, hereditary diffuse leukoencephalopathy with spheroids, and vanishing white matter disease present with rapidly progressive dementia as dominant feature and are caused by mutations in NOTCH3, HTRA1, TREX1, ARSA, CSF1R, EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5, respectively. Given the rare incidence of these disorders and the lack of unequivocally diagnostic features, leukodystrophies are frequently misdiagnosed with common sporadic dementing diseases such as Alzheimer's disease (AD), raising the question of whether these overlapping phenotypes may be explained by shared genetic risk factors. To investigate this intriguing hypothesis, we have combined gene expression analysis (1) in 6 different AD mouse strains (APPPS1, HOTASTPM, HETASTPM, TPM, TAS10, and TAU) at 5 different developmental stages (embryo [E15], 2, 4, 8, and 18 months), (2) in APPPS1 primary cortical neurons under stress conditions (oxygen-glucose deprivation) and single-variant-based and single-gene-based (c-alpha test and sequence kernel association test (SKAT)) genetic screening in a cohort composed of 332 Caucasian late-onset AD patients and 676 Caucasian elderly controls. Csf1r was significantly overexpressed (log2FC > 1, adj. p-value < 0.05) in the cortex and hippocampus of aged HOTASTPM mice with extensive Aß dense-core plaque pathology. We identified 3 likely pathogenic mutations in CSF1R TK domain (p.L868R, p.Q691H, and p.H703Y) in our discovery and validation cohort, composed of 465 AD and mild cognitive impairment (MCI) Caucasian patients from the United Kingdom. Moreover, NOTCH3 was a significant hit in the c-alpha test (adj p-value = 0.01). Adult-onset Mendelian leukodystrophy genes are not common factors implicated in AD. Nevertheless, our study suggests a potential pathogenic link between NOTCH3, CSF1R, and sporadic late-onset AD, which warrants further investigation.

Investigating the genetic architecture of dementia with Lewy bodies: a two-stage genome-wide association study.

BACKGROUND: Dementia with Lewy bodies is the second most common form of dementia in elderly people but has been overshadowed in the research field, partly because of similarities between dementia with Lewy bodies, Parkinson's disease, and Alzheimer's disease. So far, to our knowledge, no large-scale genetic study of dementia with Lewy bodies has been done. To better understand the genetic basis of dementia with Lewy bodies, we have done a genome-wide association study with the aim of identifying genetic risk factors for this disorder. METHODS: In this two-stage genome-wide association study, we collected samples from white participants of European ancestry who had been diagnosed with dementia with Lewy bodies according to established clinical or pathological criteria. In the discovery stage (with the case cohort recruited from 22 centres in ten countries and the controls derived from two publicly available database of Genotypes and Phenotypes studies [phs000404.v1.p1 and phs000982.v1.p1] in the USA), we performed genotyping and exploited the recently established Haplotype Reference Consortium panel as the basis for imputation. Pathological samples were ascertained following autopsy in each individual brain bank, whereas clinical samples were collected after participant examination. There was no specific timeframe for collection of samples. We did association analyses in all participants with dementia with Lewy bodies, and also only in participants with pathological diagnosis. In the replication stage, we performed genotyping of significant and suggestive results from the discovery stage. Lastly, we did a meta-analysis of both stages under a fixed-effects model and used logistic regression to test for association in each stage. FINDINGS: This study included 1743 patients with dementia with Lewy bodies (1324 with pathological diagnosis) and 4454 controls (1216 patients with dementia with Lewy bodies vs 3791 controls in the discovery stage; 527 vs 663 in the replication stage). Results confirm previously reported associations: APOE (rs429358; odds ratio [OR] 2·40, 95% CI 2·14-2·70; p=1·05 × 10-48), SNCA (rs7681440; OR 0·73, 0·66-0·81; p=6·39 × 10-10), an GBA (rs35749011; OR 2·55, 1·88-3·46; p=1·78 × 10-9). They also provide some evidence for a novel candidate locus, namely CNTN1 (rs7314908; OR 1·51, 1·27-1·79; p=2·32 × 10-6); further replication will be important. Additionally, we estimate the heritable component of dementia with Lewy bodies to be about 36%. INTERPRETATION: Despite the small sample size for a genome-wide association study, and acknowledging the potential biases from ascertaining samples from multiple locations, we present the most comprehensive and well powered genetic study in dementia with Lewy bodies so far. These data show that common genetic variability has a role in the disease. FUNDING: The Alzheimer's Society and the Lewy Body Society.

Mannan binding lectin and viral hepatitis.

Mannan binding lectin (MBL) is a pattern recognition molecule of the innate immune system that binds to sugars on the surface of invading micro-organisms. Target binding, complement activation and other functions of MBL are dependent on the presence of multiple carbohydrate recognition domains. Several polymorphisms in the promoter and structural regions of MBL2 adversely affect the plasma concentration and oligomeric state of MBL. The possession of mutant alleles has been linked to disease outcome for a variety of bacterial and viral infections. Viral hepatitis is caused by unrelated viruses referred to as hepatitis virus A-E. The disease usually has both acute and chronic phases, the latter leading to cirrhosis and hepatocellular carcinoma. Hepatitis viruses B and C (HBV and HCV, respectively) are a significant cause of morbidity worldwide. HBV encodes envelope glycoproteins termed large, middle, and small that may exist in glycosylated or unglycosylated forms on the virion. An interaction between HBV glycoproteins and MBL has been demonstrated in vitro. Significant associations between MBL levels, determined by MBL2 haplotypes, and HBV persistence and disease progression have been described. HCV encodes two highly glycosylated envelope proteins, E1 and E2, which are potential targets for interaction with MBL. Mutant MBL2 haplotypes have been linked to disease progression and response to therapy in HCV infection. Here we summarise the effect of MBL2 polymorphisms on MBL function and how this may relate to disease outcome in HBV and HCV infection.

Analysis of C9orf72 repeat expansions in a large international cohort of dementia with Lewy bodies.

C9orf72 repeat expansions are a common cause of amyotrophic lateral sclerosis and frontotemporal dementia. To date, no large-scale study of dementia with Lewy bodies (DLB) has been undertaken to assess the role of C9orf72 repeat expansions in the disease. Here, we investigated the prevalence of C9orf72 repeat expansions in a large cohort of DLB cases and identified no pathogenic repeat expansions in neuropathologically or clinically defined cases, showing that C9orf72 repeat expansions are not causally associated with DLB.

Influence of Coding Variability in APP-Aß Metabolism Genes in Sporadic Alzheimer's Disease.

The cerebral deposition of Aß42, a neurotoxic proteolytic derivate of amyloid precursor protein (APP), is a central event in Alzheimer's disease (AD)(Amyloid hypothesis). Given the key role of APP-Aß metabolism in AD pathogenesis, we selected 29 genes involved in APP processing, Aß degradation and clearance. We then used exome and genome sequencing to investigate the single independent (single-variant association test) and cumulative (gene-based association test) effect of coding variants in these genes as potential susceptibility factors for AD, in a cohort composed of 332 sporadic and mainly late-onset AD cases and 676 elderly controls from North America and the UK. Our study shows that common coding variability in these genes does not play a major role for the disease development. In the single-variant association analysis, the main hits, none of which statistically significant after multiple testing correction (1.9e-4

ABCA7 p.G215S as potential protective factor for Alzheimer's disease.

Genome-wide association studies (GWASs) have been effective approaches to dissect common genetic variability underlying complex diseases in a systematic and unbiased way. Recently, GWASs have led to the discovery of over 20 susceptibility loci for Alzheimer's disease (AD). Despite the evidence showing the contribution of these loci to AD pathogenesis, their genetic architecture has not been extensively investigated, leaving the possibility that low frequency and rare coding variants may also occur and contribute to the risk of disease. We have used exome and genome sequencing data to analyze the single independent and joint effect of rare and low-frequency protein coding variants in 9 AD GWAS loci with the strongest effect sizes after APOE (BIN1, CLU, CR1, PICALM, MS4A6A, ABCA7, EPHA1, CD33, and CD2AP) in a cohort of 332 sporadic AD cases and 676 elderly controls of British and North-American ancestry. We identified coding variability in ABCA7 as contributing to AD risk. This locus harbors a low-frequency coding variant (p.G215S, rs72973581, minor allele frequency = 4.3%) conferring a modest but statistically significant protection against AD (p-value = 0.024, odds ratio = 0.57, 95% confidence interval = 0.41-0.80). Notably, our results are not driven by an enrichment of loss of function variants in ABCA7, recently reported as main pathogenic factor underlying AD risk at this locus. In summary, our study confirms the role of ABCA7 in AD and provides new insights that should address functional studies.

Genome-wide analysis of genetic correlation in dementia with Lewy bodies, Parkinson's and Alzheimer's diseases.

The similarities between dementia with Lewy bodies (DLB) and both Parkinson's disease (PD) and Alzheimer's disease (AD) are many and range from clinical presentation, to neuropathological characteristics, to more recently identified, genetic determinants of risk. Because of these overlapping features, diagnosing DLB is challenging and has clinical implications since some therapeutic agents that are applicable in other diseases have adverse effects in DLB. Having shown that DLB shares some genetic risk with PD and AD, we have now quantified the amount of sharing through the application of genetic correlation estimates, and show that, from a purely genetic perspective, and excluding the strong association at the APOE locus, DLB is equally correlated to AD and PD.

Investigating the role of rare coding variability in Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP) in late-onset Alzheimer's disease.

The overlapping clinical and neuropathologic features between late-onset apparently sporadic Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and other neurodegenerative dementias (frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, and Creutzfeldt-Jakob disease) raise the question of whether shared genetic risk factors may explain the similar phenotype among these disparate disorders. To investigate this intriguing hypothesis, we analyzed rare coding variability in 6 Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP), in 141 LOAD patients and 179 elderly controls, neuropathologically proven, from the UK. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least 1 rare variant in the genes studied. We report a novel variant in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), absent in controls and both likely pathogenic. Our findings support previous studies, suggesting that (1) rare coding variability in PSEN1 and PSEN2 may influence the susceptibility for LOAD and (2) GRN, MAPT, and PRNP are not major contributors to LOAD. Thus, genetic screening is pivotal for the clinical differential diagnosis of these neurodegenerative dementias.

Exome sequencing identifies 2 novel presenilin 1 mutations (p.L166V and p.S230R) in British early-onset Alzheimer's disease.

Early-onset Alzheimer's disease (EOAD) represents 1%-2% of the Alzheimer's disease (AD) cases, and it is generally characterized by a positive family history and a rapidly progressive symptomatology. Rare coding and fully penetrant variants in amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are the only causative mutations reported for autosomal dominant AD. Thus, in this study we used exome sequencing data to rapidly screen rare coding variability in APP, PSEN1, and PSEN2, in a British cohort composed of 47 unrelated EOAD cases and 179 elderly controls, neuropathologically proven. We report 2 novel and likely pathogenic variants in PSEN1 (p.L166V and p.S230R). A comprehensive catalog of rare pathogenic variants in the AD Mendelian genes is pivotal for a premortem diagnosis of autosomal dominant EOAD and for the differential diagnosis with other early onset dementias such as frontotemporal dementia (FTD) and Creutzfeldt-Jakob disease (CJD).

The sex-specific associations of the aromatase gene with Alzheimer's disease and its interaction with IL10 in the Epistasis Project.

Epistasis between interleukin-10 (IL10) and aromatase gene polymorphisms has previously been reported to modify the risk of Alzheimer's disease (AD). However, although the main effects of aromatase variants suggest a sex-specific effect in AD, there has been insufficient power to detect sex-specific epistasis between these genes to date. Here we used the cohort of 1757 AD patients and 6294 controls in the Epistasis Project. We replicated the previously reported main effects of aromatase polymorphisms in AD risk in women, for example, adjusted odds ratio of disease for rs1065778 GG=1.22 (95% confidence interval: 1.01-1.48, P=0.03). We also confirmed a reported epistatic interaction between IL10 rs1800896 and aromatase (CYP19A1) rs1062033, again only in women: adjusted synergy factor=1.94 (1.16-3.25, 0.01). Aromatase, a rate-limiting enzyme in the synthesis of estrogens, is expressed in AD-relevant brain regions ,and is downregulated during the disease. IL-10 is an anti-inflammatory cytokine. Given that estrogens have neuroprotective and anti-inflammatory activities and regulate microglial cytokine production, epistasis is biologically plausible. Diminishing serum estrogen in postmenopausal women, coupled with suboptimal brain estrogen synthesis, may contribute to the inflammatory state, that is a pathological hallmark of AD.

Missense variant in TREML2 protects against Alzheimer's disease.

TREM and TREM-like receptors are a structurally similar protein family encoded by genes clustered on chromosome 6p21.11. Recent studies have identified a rare coding variant (p.R47H) in TREM2 that confers a high risk for Alzheimer's disease (AD). In addition, common single nucleotide polymorphisms in this genomic region are associated with cerebrospinal fluid biomarkers for AD and a common intergenic variant found near the TREML2 gene has been identified to be protective for AD. However, little is known about the functional variant underlying the latter association or its relationship with the p.R47H. Here, we report comprehensive analyses using whole-exome sequencing data, cerebrospinal fluid biomarker analyses, meta-analyses (16,254 cases and 20,052 controls) and cell-based functional studies to support the role of the TREML2 coding missense variant p.S144G (rs3747742) as a potential driver of the meta-analysis AD-associated genome-wide association studies signal. Additionally, we demonstrate that the protective role of TREML2 in AD is independent of the role of TREM2 gene as a risk factor for AD.

Discovery by the Epistasis Project of an epistatic interaction between the GSTM3 gene and the HHEX/IDE/KIF11 locus in the risk of Alzheimer's disease.

Despite recent discoveries in the genetics of sporadic Alzheimer's disease, there remains substantial "hidden heritability." It is thought that some of this missing heritability may be because of gene-gene, i.e., epistatic, interactions. We examined potential epistasis between 110 candidate polymorphisms in 1757 cases of Alzheimer's disease and 6294 control subjects of the Epistasis Project, divided between a discovery and a replication dataset. We found an epistatic interaction, between rs7483 in GSTM3 and rs1111875 in the HHEX/IDE/KIF11 gene cluster, with a closely similar, significant result in both datasets. The synergy factor (SF) in the combined dataset was 1.79, 95% confidence interval [CI], 1.35-2.36; p = 0.00004. Consistent interaction was also found in 7 out of the 8 additional subsets that we examined post hoc: i.e., it was shown in both North Europe and North Spain, in both men and women, in both those with and without the ε4 allele of apolipoprotein E, and in people older than 75 years (SF, 2.27; 95% CI, 1.60-3.20; p < 0.00001), but not in those younger than 75 years (SF, 1.06; 95% CI, 0.59-1.91; p = 0.84). The association with Alzheimer's disease was purely epistatic with neither polymorphism showing an independent effect: odds ratio, 1.0; p ≥ 0.7. Indeed, each factor was associated with protection in the absence of the other factor, but with risk in its presence. In conclusion, this epistatic interaction showed a high degree of consistency when stratifying by sex, the ε4 allele of apolipoprotein E genotype, and geographic region.