Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.
Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study (GWAS), as well as an analysis of known genetic risk loci for AD dementia, using data from 4,914 brain autopsies. Genome-wide significance was observed for 7 genes and pathologic features of AD and related diseases. Twelve of the 22 genetic risk loci for clinically-defined AD dementia were confirmed in our pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for hallmark pathologic features of AD were strongly positive and linear. Our study discovered new genetic associations with specific pathologic features and aligned known genetic risk for AD dementia with specific pathologic changes in a large brain autopsy study of AD and related dementias.
Rare mutations in AβPP, PSEN1, and PSEN2 cause uncommon early onset forms of Alzheimer’s disease (AD), and common variants in MAPT are associated with risk of other neurodegenerative disorders. We sought to establish whether common genetic variation in these genes confer risk to the common form of AD which occurs later in life (>65 years). We therefore tested single-nucleotide polymorphisms at these loci for association with late-onset AD (LOAD) in a large case-control sample consisting of 3,940 cases and 13,373 controls. Single-marker analysis did not identify any variants that reached genome-wide significance, a result which is supported by other recent genome-wide association studies. However, we did observe a significant association at the MAPT locus using a gene-wide approach (p = 0.009). We also observed suggestive association between AD and the marker rs9468, which defines the H1 haplotype, an extended haplotype that spans the MAPT gene and has previously been implicated in other neurodegenerative disorders including Parkinson’s disease, progressive supranuclear palsy, and corticobasal degeneration. In summary common variants at AβPP, PSEN1, and PSEN2 and MAPT are unlikely to make strong contributions to susceptibility for LOAD. However, the gene-wide effect observed at MAPT indicates a possible contribution to disease risk which requires further study.
Alzheimer’s disease; amyloid-β protein precursor; genetics; human; MAPT protein; PSEN1 protein; PSEN2 protein
MAPT encodes for tau, the predominant component of neurofibrillary tangles that are neuropathological hallmarks of Alzheimer’s disease (AD). Genetic association of MAPT variants with late-onset AD (LOAD) risk has been inconsistent, although insufficient power and incomplete assessment of MAPT haplotypes may account for this.
We examined the association of MAPT haplotypes with LOAD risk in more than 20,000 subjects (n-cases = 9,814, n-controls = 11,550) from Mayo Clinic (n-cases = 2,052, n-controls = 3,406) and the Alzheimer’s Disease Genetics Consortium (ADGC, n-cases = 7,762, n-controls = 8,144). We also assessed associations with brain MAPT gene expression levels measured in the cerebellum (n = 197) and temporal cortex (n = 202) of LOAD subjects. Six single nucleotide polymorphisms (SNPs) which tag MAPT haplotypes with frequencies greater than 1% were evaluated.
H2-haplotype tagging rs8070723-G allele associated with reduced risk of LOAD (odds ratio, OR = 0.90, 95% confidence interval, CI = 0.85-0.95, p = 5.2E-05) with consistent results in the Mayo (OR = 0.81, p = 7.0E-04) and ADGC (OR = 0.89, p = 1.26E-04) cohorts. rs3785883-A allele was also nominally significantly associated with LOAD risk (OR = 1.06, 95% CI = 1.01-1.13, p = 0.034). Haplotype analysis revealed significant global association with LOAD risk in the combined cohort (p = 0.033), with significant association of the H2 haplotype with reduced risk of LOAD as expected (p = 1.53E-04) and suggestive association with additional haplotypes. MAPT SNPs and haplotypes also associated with brain MAPT levels in the cerebellum and temporal cortex of AD subjects with the strongest associations observed for the H2 haplotype and reduced brain MAPT levels (β = -0.16 to -0.20, p = 1.0E-03 to 3.0E-03).
These results confirm the previously reported MAPT H2 associations with LOAD risk in two large series, that this haplotype has the strongest effect on brain MAPT expression amongst those tested and identify additional haplotypes with suggestive associations, which require replication in independent series. These biologically congruent results provide compelling evidence to screen the MAPT region for regulatory variants which confer LOAD risk by influencing its brain gene expression.
The most recent late-onset Alzheimer’s disease (LOAD) genome-wide association study revealed genome-wide significant association of two new loci: rs744373 near BIN1 (p=1.6×10−11) and rs597668 near EXOC3L2/BLOC1S3/MARK4 (p=6.5×10−9). We have genotyped these variants in a large (3,287 LOAD, 4,396 controls), independent dataset comprising eleven case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and also tested for association using logistic regression adjusted by age-at-diagnosis, sex and APOE ε4 status. Meta-analysis results showed no evidence of series heterogeneity and logistic regression analysis successfully replicated the association of BIN1 (rs744373) with LOAD with an odds ratio (OR=1.17, p=1.1×10−4) comparable to that previously reported (OR=1.15). The variant near EXOC3L2 (rs597668) showed only suggestive association with LOAD (p=0.09) after correcting for the presence of the APOE ε4 allele. Addition of our follow-up data to the results previously reported increased the strength of evidence for association with BIN1 (11,825 LOAD, 32,570 controls, rs744373 Fisher combined p=3.8×10−20). We also tested for epistatic interaction between these variants and APOEε4 as well as with the previously replicated LOAD GWAS genes (CLU: rs11136000, CR1; rs3818361, and PICALM: rs3851179). No significant interactions between these genes were detected. In summary, we provide additional evidence for the variant near BIN1 (rs744373) as a LOAD risk modifier, but our results indicate that the effect of EXOC3L2 independent of APOE ε4 should be studied further.
Alzheimer Disease; Late Onset; Heterogeneity; Meta-Analysis; Case-Control Studies
Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls.
In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10−6) and 14 (IGHV1-67 p = 7.9×10−8) which indexed novel susceptibility loci.
The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.
Eleven susceptibility loci for late-onset Alzheimer’s disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer’s disease cases and 37,154 controls. In stage 2,11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer’s disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10−8) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer’s disease.
Nicastrin (NCSTN) is a component of the γ-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn−/−) cells and clonal NCSTN-BAC+/Ncstn−/− cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued γ-secretase activity and amyloid beta (Aβ) production in NCSTN-BAC+/Ncstn−/− lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease.
Nicastrin; Haplotype variation; Functional genomics; Alzheimer's disease; γ-Secretase complex
Recent genome-wide association studies (GWAS) of late-onset Alzheimer’s disease (LOAD) have identified single nucleotide polymorphisms (SNPs) which show significant association at the well-known APOE locus and at nineteen additional loci. Among the functional, disease-associated variants at these loci, missense variants are particularly important because they can be readily investigated in model systems to search for novel therapeutic targets. It is now possible to perform a low-cost search for these “actionable” variants by genotyping the missense variants at known LOAD loci already cataloged on the Exome Variant Server (EVS). In this proof-of-principle study designed to explore the efficacy of this approach, we analyzed three rare EVS variants in APOE, p.L28P, p.R145C and p.V236E, in our case control series of 9114 subjects. p.R145C proved to be too rare to analyze effectively. The minor allele of p.L28P, which was in complete linkage disequilibrium (D’ = 1) with the far more common APOE ϵ4 allele, showed no association with LOAD (P = 0.75) independent of the APOE ϵ4 allele. p.V236E was significantly associated with a marked reduction in risk of LOAD (P = 7.5×10−05; OR = 0.10, 0.03 to 0.45). The minor allele of p.V236E, which was in complete linkage disequilibrium (D’ = 1) with the common APOE ϵ3 allele, identifies a novel LOAD-associated haplotype (APOE ϵ3b) which is associated with decreased risk of LOAD independent of the more abundant APOE ϵ2, ϵ3 and ϵ4 haplotypes. Follow-up studies will be important to confirm the significance of this association and to better define its odds ratio. The ApoE p.V236E substitution is the first disease-associated change located in the lipid-binding, C-terminal domain of the protein. Thus our study (i) identifies a novel APOE missense variant which may profitably be studied to better understand how ApoE function may be modified to reduce risk of LOAD and (ii) indicates that analysis of protein-altering variants cataloged on the EVS can be a cost-effective way to identify actionable functional variants at recently discovered LOAD loci.
Late-onset Alzheimer disease (LOAD) is a common disorder with a substantial genetic component. We postulate that many disease susceptibility variants act by altering gene expression levels.
We measured messenger RNA (mRNA) expression levels of 12 LOAD candidate genes in the cerebella of 200 subjects with LOAD. Using the genotypes from our LOAD genome-wide association study for the cis-single nucleotide polymorphisms (SNPs) (n = 619) of these 12 LOAD candidate genes, we tested for associations with expression levels as endophenotypes. The strongest expression cis-SNP was tested for AD association in 7 independent case-control series (2,280 AD and 2,396 controls).
We identified 3 SNPs that associated significantly with IDE (insulin degrading enzyme) expression levels. A single copy of the minor allele for each significant SNP was associated with ∼twofold higher IDE expression levels. The most significant SNP, rs7910977, is 4.2 kb beyond the 3′ end of IDE. The association observed with this SNP was significant even at the genome-wide level (p = 2.7 × 10−8). Furthermore, the minor allele of rs7910977 associated significantly (p = 0.0046) with reduced LOAD risk (OR = 0.81 with a 95% CI of 0.70-0.94), as expected biologically from its association with elevated IDE expression.
These results provide strong evidence that IDE is a late-onset Alzheimer disease (LOAD) gene with variants that modify risk of LOAD by influencing IDE expression. They also suggest that the use of expression levels as endophenotypes in genome-wide association studies may provide a powerful approach for the identification of disease susceptibility alleles.
= Alzheimer disease;
= confidence interval;
= genome-wide association study;
= late-onset Alzheimer disease;
= messenger RNA;
= odds ratio;
= single nucleotide polymorphism.
Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP.
Methods and results
Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser1177 in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH4) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH4 and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH4 bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91phox and SODs, thereby reducing production of superoxide anion in the aortas.
Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.
Amyloid-β precursor protein; Endothelial function; Superoxide anion; Tetrahydrobiopterin; Atherosclerosis
Neuronal network hyperexcitability underlies the pathogenesis of seizures and is a component of some degenerative neurological disorders such as Alzheimer’s disease (AD). Recently, the microtubule binding protein tau has been implicated in the regulation of network synchronization. Genetic removal of Mapt, the gene encoding tau, in AD models overexpressing amyloid-beta (Aβ) decreases hyperexcitability and normalizes the excitation/inhibition imbalance. Whether this effect of tau removal is specific to Aβ mouse models remains to be determined. Here we examined tau as an excitability modifier in the non-AD nervous system using genetic deletion of tau in mouse and Drosophila models of hyperexcitability. Kcna1−/− mice lack Kv1.1 delayed rectifier currents and exhibit severe spontaneous seizures, early lethality, and megencephaly. Young Kcna1−/− mice retained wild-type levels of Aβ, tau, and tau phospho-Thr231. Decreasing tau in Kcna1−/− mice reduced hyperexcitability and alleviated seizure-related comorbidities. Tau reduction decreased Kcna1−/− video-EEG recorded seizure frequency and duration as well as normalized Kcna1−/− hippocampal network hyperexcitability in vitro. Additionally, tau reduction increased Kcna1−/− survival and prevented megencephaly and hippocampal hypertrophy, as determined by MRI. Bang-sensitive Drosophila mutants display paralysis and seizures in response to mechanical stimulation, providing a complementary excitability assay for epistatic interactions. We found that tau reduction significantly decreased seizure sensitivity in two independent bang-sensitive mutant models, kcc and eas. Our results indicate that tau plays a general role in regulating intrinsic neuronal network hyperexcitability independently of Aβ overexpression and suggest that reducing tau function could be a viable target for therapeutic intervention in seizure disorders and antiepileptogenesis.
A rare variant in the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) gene has been reported to be a genetic risk factor for Alzheimer’s disease by two independent groups (Odds ratio between 2.9-4.5). Given the key role of TREM2 in the effective phagocytosis of apoptotic neuronal cells by microglia, we hypothesized that dysfunction of TREM2 may play a more generalized role in neurodegeneration. With this in mind we set out to assess the genetic association of the Alzheimer’s disease-related risk variant in TREM2 (rs75932628, p.R47H) with other related neurodegenerative disorders.
The study included 609 patients with frontotemporal dementia, 765 with amyotrophic lateral sclerosis, 1493 with Parkinson’s disease, 772 with progressive supranuclear palsy, 448 with ischemic stroke and 1957 controls subjects free of neurodegenerative disease. A significant association was observed for the TREM2 p.R47H substitution in susceptibility to frontotemporal dementia (OR = 5.06; p-value = 0.001) and Parkinson’s disease (OR = 2.67; p-value = 0.026), while no evidence of association with risk of amyotrophic lateral sclerosis, progressive supranuclear palsy or ischemic stroke was observed.
Our results suggest that the TREM2 p.R47H substitution is a risk factor for frontotemporal dementia and Parkinson’s disease in addition to Alzheimer’s disease. These findings suggest a more general role for TREM2 dysfunction in neurodegeneration, which could be related to its role in the immune response.
TREM2; Frontotemporal dementia; Parkinson disease; Genetic association
Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer’s disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5′UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02–0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.
The risk of Alzheimer’s disease (AD) is strongly determined by genetic factors and recent genome-wide association studies (GWAS) have identified several genes for the disease risk. In addition to the disease risk, age-at-onset (AAO) of AD has also strong genetic component with an estimated heritability of 42%. Identification of AAO genes may help to understand the biological mechanisms that regulate the onset of the disease. Here we report the first GWAS focused on identifying genes for the AAO of AD. We performed a genome-wide meta analysis on 3 samples comprising a total of 2,222 AD cases. A total of ~2.5 million directly genotyped or imputed SNPs were analyzed in relation to AAO of AD. As expected, the most significant associations were observed in the APOE region on chromosome 19 where several SNPs surpassed the conservative genome-wide significant threshold (P<5E-08). The most significant SNP outside the APOE region was located in the DCHS2 gene on chromosome 4q31.3 (rs1466662; P=4.95E-07). There were 19 additional significant SNPs in this region at P<1E-04 and the DCHS2 gene is expressed in the cerebral cortex and thus is a potential candidate for affecting AAO in AD. These findings need to be confirmed in additional well-powered samples.
Genome-wide association study; age-at-onset; Alzheimer’s disease; single-nucleotide polymorphisms; meta analysis
Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR, and SPINK1 variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated at PRSS1-PRSS2 (1×10-12) and x-linked CLDN2 (p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). The PRSS1 variant affects susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07.
GRB-associated binding protein 2 (GAB2) represents a compelling genome-wide association signal for late-onset Alzheimer’s disease (LOAD) with reported odds ratios (ORs) ranging from 0.75–0.85. We tested eight GAB2 variants in four North American Caucasian case-control series (2,316 LOAD, 2,538 controls) for association with LOAD. Meta-analyses revealed ORs ranging from (0.61–1.20) with no significant association (all p>0.32). Four variants were hetergeneous across the populations (all p<0.02) due to a potentially inflated effect size (OR = 0.61–0.66) only observed in the smallest series (702 LOAD, 209 controls). Despite the lack of association in our series, the previously reported protective association for GAB2 remained after meta-analyses of our data with all available previously published series (11,952-22,253 samples; OR = 0.82–0.88; all p<0.04). Using a freely available database of lymphoblastoid cell lines we found that protective GAB2 variants were associated with increased GAB2 expression (p = 9.5×10−7−9.3×10−6). We next measured GAB2 mRNA levels in 249 brains and found that decreased neurofibrillary tangle (r = −0.34, p = 0.0006) and senile plaque counts (r = −0.32, p = 0.001) were both good predictors of increased GAB2 mRNA levels albeit that sex (r = −0.28, p = 0.005) may have been a contributing factor. In summary, we hypothesise that GAB2 variants that are protective against LOAD in some populations may act functionally to increase GAB2 mRNA levels (in lymphoblastoid cells) and that increased GAB2 mRNA levels are associated with significantly decreased LOAD pathology. These findings support the hypothesis that Gab2 may protect neurons against LOAD but due to significant population heterogeneity, it is still unclear whether this protection is detectable at the genetic level.
Expanded glutamine repeats of the ataxin-2 (ATXN2) protein cause spinocerebellar ataxia type 2 (SCA2), a rare neurodegenerative disorder. More recent studies have suggested that expanded ATXN2 repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) via an RNA-dependent interaction with TDP-43. Given the phenotypic diversity observed in SCA2 patients, we set out to determine the polymorphic nature of the ATXN2 repeat length across a spectrum of neurodegenerative disorders. In this study, we genotyped the ATXN2 repeat in 3919 neurodegenerative disease patients and 4877 healthy controls and performed logistic regression analysis to determine the association of repeat length with the risk of disease. We confirmed the presence of a significantly higher number of expanded ATXN2 repeat carriers in ALS patients compared with healthy controls (OR = 5.57; P= 0.001; repeat length >30 units). Furthermore, we observed significant association of expanded ATXN2 repeats with the development of progressive supranuclear palsy (OR = 5.83; P= 0.004; repeat length >30 units). Although expanded repeat carriers were also identified in frontotemporal lobar degeneration, Alzheimer's and Parkinson's disease patients, these were not significantly more frequent than in controls. Of note, our study identified a number of healthy control individuals who harbor expanded repeat alleles (31–33 units), which suggests caution should be taken when attributing specific disease phenotypes to these repeat lengths. In conclusion, our findings confirm the role of ATXN2 as an important risk factor for ALS and support the hypothesis that expanded ATXN2 repeats may predispose to other neurodegenerative diseases, including progressive supranuclear palsy.
Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n = 197, temporal cortex n = 202) and with other brain pathologies (non–AD, cerebellar n = 177, temporal cortex n = 197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ±100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non–ADs (q<0.05, p = 7.70×10−5–1.67×10−82). Of these, 2,089 were also significant in the temporal cortex (p = 1.85×10−5–1.70×10−141). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10−6). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9–3.3 fold enrichment (p<10−6) of significant cisSNPs with suggestive AD–risk association (p<10−3) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non–CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings have implications for the design and interpretation of eGWAS in general and the use of brain expression quantitative trait loci in the study of human disease genetics.
Genetic variants that regulate gene expression levels can also influence human disease risk. Discovery of genomic loci that alter brain gene expression levels (brain expression quantitative trait loci = eQTLs) can be instrumental in the identification of genetic risk underlying both central nervous system (CNS) and non–CNS diseases. To systematically assess the role of brain eQTLs in human disease and to evaluate the influence of brain region and pathology in eQTL mapping, we performed an expression genome-wide association study (eGWAS) in 773 brain samples from the cerebellum and temporal cortex of ∼200 autopsied subjects with Alzheimer's disease (AD) and ∼200 with other brain pathologies (non–AD). We identified ∼3,000 significant associations between cisSNPs near ∼700 genes and their cerebellar transcript levels, which replicate in ADs and non–ADs. More than 2,000 of these associations were reproducible in the temporal cortex. The top cisSNPs are enriched for both CNS and non–CNS disease-associated variants. We identified novel and confirmed previous cisSNP/transcript associations for many disease loci, suggesting gene expression regulation as their mechanism of action. These findings demonstrate the reproducibility of the eQTL approach across different brain regions and pathologies, and advocate the combined use of gene expression and disease GWAS for identification and functional characterization of human disease-associated variants.
Alzheimer disease (AD) and frontotemporal dementia (FTD) are two frequent forms of primary neurodegenerative dementias with overlapping clinical symptoms. Pathogenic mutations of the amyloid precursor protein (APP) and presenilins 1 and 2 (PSEN1, PSEN2) genes have been linked to familial early-onset forms of AD; however, more recently mutations in the common FTD genes encoding the microtubule associated protein tau (MAPT), progranulin (GRN) and C9ORF72, have also been reported in clinically diagnosed AD patients. To access the contribution of mutations in a well-characterized series of patients, we systematically performed genetic analyses of these EOAD and FTD genes in a novel cohort of 227 unrelated probands clinically diagnosed as probable AD which were ascertained at Mayo Clinic Florida between 1997 and 2011. All patients showed first symptoms of dementia before 70 years. We identified 9 different pathogenic mutations in the EOAD genes in a total of 11 patients explaining 4.8% of the patient population. Two mutations were novel: PSEN1 p.Pro218Leu and PSEN2 p.Phe183Ser. Importantly, mutations were also identified in all FTD genes: one patient carried a MAPT p.R406W mutation, one patient carried the p.Arg198Glyfs19X loss-of-function mutation in GRN and two patients were found to carry expanded GGGGCC repeats in the non-coding region of C9ORF72. Together the FTD genes explained the disease in 1.8% of our probable AD population. The identification of mutations in all major FTD genes in this novel cohort of clinically diagnosed AD patients underlines the challenges associated with the differential diagnosis of AD and FTD resulting from overlapping symptomatology and has important implications for molecular diagnostic testing and genetic counseling of clinically diagnosed AD patients. Our findings suggest that in clinically diagnosed AD patients, genetic analyses should include not only the well-established EOAD genes APP, PSEN1 and PSEN2 but also genes that are usually associated with FTD. Finally, the overall low frequency of mutation carriers observed in our study (6.6%) suggests the involvement of other as yet unknown genetic factors associated with AD.
Alzheimer’s disease; frontotemporal dementia; amyloid precursor protein; presenilin 1; presenilin 2; progranulin; microtubule associated protein tau; C9ORF72; mutation; diagnosis.
Glutathione S-transferase omega-1 and 2 genes (GSTO1, GSTO2), residing within an Alzheimer and Parkinson disease (AD and PD) linkage region, have diverse functions including mitigation of oxidative stress and may underlie the pathophysiology of both diseases. GSTO polymorphisms were previously reported to associate with risk and age-at-onset of these diseases, although inconsistent follow-up study designs make interpretation of results difficult. We assessed two previously reported SNPs, GSTO1 rs4925 and GSTO2 rs156697, in AD (3,493 ADs vs. 4,617 controls) and PD (678 PDs vs. 712 controls) for association with disease risk (case-controls), age-at-diagnosis (cases) and brain gene expression levels (autopsied subjects).
We found that rs156697 minor allele associates with significantly increased risk (odds ratio = 1.14, p = 0.038) in the older ADs with age-at-diagnosis > 80 years. The minor allele of GSTO1 rs4925 associates with decreased risk in familial PD (odds ratio = 0.78, p = 0.034). There was no other association with disease risk or age-at-diagnosis. The minor alleles of both GSTO SNPs associate with lower brain levels of GSTO2 (p = 4.7 × 10-11-1.9 × 10-27), but not GSTO1. Pathway analysis of significant genes in our brain expression GWAS, identified significant enrichment for glutathione metabolism genes (p = 0.003).
These results suggest that GSTO locus variants may lower brain GSTO2 levels and consequently confer AD risk in older age. Other glutathione metabolism genes should be assessed for their effects on AD and other chronic, neurologic diseases.
GSTO genes; Disease risk; Gene expression; Association
KIBRA SNP rs17070145 was identified in a GWAS of memory performance, with some but not all follow-up studies confirming association of its T allele with enhanced memory. This allele was associated with reduced Alzheimer's disease (AD) risk in one study, which also found overexpression of KIBRA in memory-related brain regions of ADs. We genotyped rs17070145 and 14 additional SNPs in 2571 LOADs vs. 2842 controls, including African-Americans. We found significantly reduced risk for rs17070145 T allele in the older African-American subjects (p=0.007) and a suggestive effect in the older Caucasian series. Meta-analysis of this allele in >8000 subjects from our and published series showed a suggestive protective effect (p=0.07). Analysis of episodic memory in control subjects did not identify associations with rs17070145, though other SNPs showed significant associations in one series. KIBRA showed evidence of overexpression in the AD temporal cortex (p=0.06) but not cerebellum. These results suggest a modest role for KIBRA as a cognition and AD risk gene, and also highlight the multifactorial complexity of its genetic associations.
Alzheimer's disease; Association studies in genetics; Case control studies
We sought to identify new susceptibility loci for Alzheimer’s disease (AD) through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer’s Disease Genetic Consortium (ADGC). First, we undertook a combined analysis of four genome-wide association datasets (Stage 1) and identified 10 novel variants with P≤1×10−5. These were tested for association in an independent sample (Stage 2). Three SNPs at two loci replicated and showed evidence for association in a further sample (Stage 3). Meta-analyses of all data provide compelling evidence that ABCA7 (meta-P 4.5×10−17; including ADGC meta-P=5.0×10−21) and the MS4A gene cluster (rs610932, meta-P=1.8×10−14; including ADGC meta-P=1.2×10−16; rs670139, meta-P=1.4×10−9; including ADGC meta-P=1.1×10−10) are novel susceptibility loci for AD. Second, we observed independent evidence for association for three suggestive loci reported by the ADGC GWAS, which when combined shows genome-wide significance: CD2AP (GERAD+ P=8.0×10−4; including ADGC meta-P=8.6×10−9), CD33 (GERAD+ P=2.2×10−4; including ADGC meta-P=1.6×10−9) and EPHA1 (GERAD+ P=3.4×10−4; including ADGC meta-P=6.0×10−10). These findings support five novel susceptibility genes for AD.
The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study (GWAS) of late-onset Alzheimer disease (LOAD) using a 3 stage design consisting of a discovery stage (Stage 1) and two replication stages (Stages 2 and 3). Both joint and meta-analysis analysis approaches were used. We obtained genome-wide significant results at MS4A4A [rs4938933; Stages 1+2, meta-analysis (PM) = 1.7 × 10−9, joint analysis (PJ) = 1.7 × 10−9; Stages 1–3, PM = 8.2 × 10−12], CD2AP (rs9349407; Stages 1–3, PM = 8.6 × 10−9), EPHA1 (rs11767557; Stages 1–3 PM = 6.0 × 10−10), and CD33 (rs3865444; Stages 1–3, PM = 1.6 × 10−9). We confirmed that CR1 (rs6701713; PM = 4.6×10−10, PJ = 5.2×10−11), CLU (rs1532278; PM = 8.3 × 10−8, PJ = 1.9×10−8), BIN1 (rs7561528; PM = 4.0×10−14; PJ = 5.2×10−14), and PICALM (rs561655; PM = 7.0 × 10−11, PJ = 1.0×10−10) but not EXOC3L2 are LOAD risk loci1–3.
Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNP)s that are essentially unequivocally associated with peripheral cholesterol. Since the alleles of the APOE gene, which modulate peripheral cholesterol metabolism, and midlife plasma cholesterol are both associated with Alzheimer's disease (AD) risk, we have evaluated the hypothesis that SNPs associated with plasma cholesterol are also associated with AD.
Seventeen non-APOE SNPs reproducibly associated with cholesterol per GWAS were tested for association with AD in ~2,000 AD and ~4,000 non-AD subjects. As a group, these SNPs are associated with AD. Two SNPs in particular, rs3846662 and rs1532085, are associated with AD risk and age-of-onset. Additionally, rs3846662 was associated with HMGCR exon 13 splicing in human liver but not brain, possibly obscured by CNS cell-type heterogeneity. However, rs3846662 was associated with HMGCR exon 13 splicing in liver- and brain-derived cell lines.
Cholesterol-associated SNPs outside of APOE confer a global risk for AD. Rs3846662 and rs1532085 are associated with both AD risk and age-of-onset. Rs3846662 is associated with HMGCR exon 13 inclusion. Since rs3846662 affects AD risk and age-of-onset as well as statin responsiveness, this SNP may confound clinical trials evaluating the protective effects of statins on AD.
Alzheimer; genetics; cholesterol; GWAS; SNP; HMGCR; statin