SNPs in catalytic and regulatory subunits of calcineurin are associated with CSF tau levels and mRNA levels

Poster Presentations P4 African Americans versus Caucasian elderly individuals, and that presence of any T allele at GRN rs5848 may be associated with increased risk of cognitive decline and dementia. This polymorphism may be involved in processes which underlie previously reported racial differences in dementia rates. Analyses in larger samples, and longitudinal follow-up is required to confirm these observations. P4-133

GENOME-WIDE ASSOCIATION STUDY OF AGE AT ONSET OF ALZHEIMER’S DISEASE

Denise Harold1, Paul Hollingworth1, Marian Hamshere1, Peter Holmans1, Richard Abraham1, Rebecca Sims1, Amy Gerrish1, Simon Lovestone2, Carol Brayne3, Michael Gill4, Brian Lawlor4, Peter Passmore5, Markus Nothen6, Wolfgang Maier6, Gill Livingston7, Nicola Bass7, Alison Goate8, Steven Younkin9, Ammar Al-Chalabi10, Rhian Gwilliam11, Kevin Morgan12, Panos Deloukas11, Michael O’Donovan1, Michael Owen1, Julie Williams1, 1Cardiff University, Cardiff, United Kingdom; 2Institute of Psychiatry, London, United Kingdom; 3Cambridge University, Cambridge, United Kingdom; 4Trinity College Dublin, Dublin, Ireland; 5Queen’s University Belfast, Belfast, United Kingdom; 6University of Bonn, Bonn, Germany; 7University College London, London, United Kingdom; 8Washington University School of Medicine, St. Louis, MO, USA; 9Mayo Clinic, Jacksonville, FL, USA; 10Kings College London, London, United Kingdom; 11 Sanger Institute, Cambridge, United Kingdom; 12University of Nottingham, Nottingham, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a common debilitating disorder with a prevalence that rises steeply with age from below 1% at 65 years to as high as 40% after the age of 90. Previous studies have suggested that genetic variation contributes not only to AD susceptibility, but also to variation in the age at onset (AAO) of disease symptoms. Objective: To identify susceptibility variants influencing age at onset of Alzheimer’s disease. Methods: We conducted a genome-wide association study (GWAS) of age at onset of AD using the Illumina human610-quad beadchip. Age at onset was defined as the age at which the individual, family or caregiver first noted cognitive problems sufficient to interfere with independent daily activities. After stringent quality control, our GWAS sample was comprised of 3240 AD cases. All cases met criteria for either probable (NINCDS-AARDA, DSM-IV) or definite (CERAD) AD. AAO was employed as the dependent variable in a linear regression analysis. Geographical information and significant components from a multidimensional scaling analysis were included as covariates to control for population stratification. We have also performed a GWAS of AD (4047 cases, 2486 elderly controls and 6377 population controls); the results of this study will also be presented at ICAD. Results: The SNP most significantly associated with age at onset was on chromosome 19, and was tightly linked to the APOE locus. This is unsurprising as APOE e4 alleles have previously been associated with reduced age at onset of AD. In addition, a number of novel variants were identified that influence AAO. Conclusions: We have conducted a powerful genome-wide association study, identifying novel variants that influence age at onset of AD. P4-134

LONGEVITY GENOTYPE IS ASSOCIATED WITH A REDUCED RATE OF MEMORY DECLINE AND REDUCED INCIDENCE OF DEMENTIA: RESULTS FROM THE EINSTEIN AGING STUDY

Richard B. Lipton1, Amy E. Sanders1, Laurie Ozelius2, Mindy Katz1, Carol A. Derby1, Gil Atzmon1, Cuiling Wang1, Nir Barzilai1, 1Albert Einstein College of Medicine, Bronx, NY, USA; 2Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: We estimated rates of memory decline and relative risks of dementia in carriers and non-carriers of a minor (V) allele at codon 405 of the CETP gene (isoleucine to valine, I405V; NCBI dbSNP rs5882) in a population-based longitudinal aging study. CETP allelic variants have been associated with human exceptional longevity. We have previously reported that V405 homozygosity is associated with preserved global cognition in Ashkenazi Jewish (AJ) centenarians and that the V-allele is overrepresented in non-demented AJ subjects in the Einstein Aging Study (EAS). Case-control studies of CETP polymorphisms and dementia suscep-

P471

tibility have reported inconsistent findings. Methods: Analyses are based on data from the EAS, a longitudinal study of cognition, aging and dementia. We recruited a systematic sample of initially nondemented adults over the age of 70 and followed them annually with clinical evaluations and neuropsychological tests. Using linear mixed models, we tested the influence of V405 homozygosity on performance on Free Recall from the Free and Cued Selective Reminding Test, an established neuropsychological test of episodic memory. Dementia was diagnosed in consensus conferences according to DSM-IV criteria. We examined time-to-dementia in individuals with and without a V-allele using Cox proportional hazard models adjusted for demographic variables (including race), medical comorbidities and baseline cognitive function. Results: Of 422 individuals (61% female, 68% Caucasian, mean age 78.2 years, mean education 13.9 years) followed for a mean of 3.8 years, 26 developed incident dementia. Allele frequency of V405 was 43.7%; 91 participants (21.5%) were homozygous and 188 (44.5%) were heterozygous. V405 homozygotes had a reduced rate of linear decline in Free Recall in comparison with those homozygous for the I allele (linear rate difference ¼ 0.24, p¼0.034). In adjusted models, V405 was associated with reduced dementia risk (HR 0.29 [95% confidence interval 0.11 - 0.75; p¼0.01]). Conclusions: Presence of any V-allele at CETP codon 405 was associated with a reduced rate of memory decline and with reduced incidence of dementia in a broadly representative sample, not just AJs. Since CETP is associated with exceptional longevity, in case-control studies the protective effect associated with V405 may be attenuated by prolonged survival in cases having the favorable polymorphism. P4-135

COMBINED SNP AND COPY NUMBER VARIATION HAPLOTYPES AT THE MAPT LOCUS

Norbert Sule, Yanchun Li, Kinga Szigeti, Baylor College of Medicine, Houston, TX, USA. Contact e-mail: [email protected] Background: MAPT haplotypes have been associated with various neurodegenerative diseases, including progressive supranuclear palsy and Alzheimer disease. The strength of the association is dependent on haplotype sampling and can be improved by cladistic analysis. MAPT is located on chromosome 17q21.31 within a haplotype block with strong linkage disequilibrium in Caucasians. The LD is caused by a 900kb inversion polymorphism containing MAPT, suppressing recombination in the region. The inversion polymorphism identifies the H2 haplotype from the H1 haplotype clade. The H2 lineage is rare in Africans and almost absent in Asians, but occurs at a frequency of 20% in Europeans. There are at least five distinct CNV haplotypes in LD with MAPT. Methods: HapMap genotypes and genomotypes were inferred from the publicly available Affymetrix 6.0 dataset. The AD subjects, controls and brain DNA were genotyped on the same platform. We used the Birdseed algorithm for genotype calls and a Hidden-Markov-Model to infer CNV status. We phased the SNP and subsequently the combined SNP and CNV haplotypes by FastPhase. Expression profiling of the brain samples was performed on Illumina humanRefSeq-6 array. Standard immunohistochemical methods were used to evaluate tau pathology. Amyloid was visualized by Congo red. Results: We used the HapMap trios to evaluate the accuracy of the phasing algorithm (FastPhase) and found segregation of the SNP/CNV haplotypes in 29 trios. Subsequently we studied the distribution of the SNP haplotypes, CNV genomotypes and the combined SNP and CNV haplotypes in a pilot cohort of AD and normal controls. Brain expression and tau pathology was evaluated in 10 post mortem human samples in the context of the CNV present. Conclusions: Using combined SNP and CNV haplotypes may enhance the association signal between the MAPT locus and AD. P4-136

SNPS IN CATALYTIC AND REGULATORY SUBUNITS OF CALCINEURIN ARE ASSOCIATED WITH CSF TAU LEVELS AND MRNA LEVELS

Carlos Cruchaga, John S. Kauwe, Kevin Mayo, Sarah Bertelsen, Petra Nowotny, Aarti R. Shah, Anne M. Fagan, David M. Holtzman, John C. Morris, Alison M. Goate, Washington University School of Medicine, St.Louis, MO, USA. Contact e-mail: [email protected]

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Poster Presentations P4

Background: Cerebrospinal fluid (CSF) levels of tau protein are increased in Alzheimer’s disease (AD), and correlate with the presence of tangles in the brain suggesting they may be a useful biomarker for AD. Quantitative traits such as CSF tau levels offer more power than case-control tests in identifying genes influencing risk for AD. Objective: To identify SNPs in genes related to tau metabolism that are associated with CSF tau and phosphorylated tau181 (ptau181) levels that could be new risk factors for AD. Methods: We genotyped 384 SNPs in 35 genes related to tau posttranslational modification. CSF tau and ptau181 levels were available for 374 individuals from the Washington University ADRC and for 400 individuals from ADNI. ANCOVA was used to test for association between genotypes and CSF tau and ptau181 levels. False discovery rate was used for multiple test correction. PPP3R1 and PPP3CA mRNA levels in parietal lobe from 42 non-demented and 78 demented individuals were quantified by real-time PCR. Results: 15 SNPs in 11 genes were associated with either tau or ptau181 in the ADRC CSF series. Upon follow-up of the significant SNPs in the ADNI series only SNPs located in the regulatory (PPP3R1; rs1868402) and in the catalytic (PPP3CA; rs17030739) subunits of the phosphatase PP2B (calcineurin) replicated. In a combined analysis of the two series, rs1868402 (p¼6.28x10-05) and rs17030739 (p¼2.05x10-04) were the most significant SNPs. Gene expression analyses showed that PPP3R1 mRNA levels are higher in AD cases compared to controls (p¼0.0001) and that in controls the minor allele of rs1868402 is associated with lower PPP3R1 expression (p¼0.009). PPP3R1 and PPP3CA mRNA levels are highly correlated in the WU ADRC brains (Spearman correlation factor¼0.71; p¼1.33310-20) and in the publicly available GEO database (Series GSE8919) of 193 neuropathologically normal human brain samples (p¼6.35310-21). These results suggest the presence of common regulatory elements in both genes. Conclusions: Our results suggest genetic variants in both the catalytic and the regulatory subunits of calcineurin could increase tau pathology and be associated with risk for AD or earlier age at onset. P4-137

AGE, APOE GENOTYPE, GENDER AND SERIAL POSITION EFFECTS IN HEALTHY ADULTS

Nunzio Pomara1, Linlin Yi2, Amanda Schmeltz2, John J. Sidtis1, 1Nathan Kline Institute/NYU School of Medicine, Orangeburg, NY, USA; 2Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected]. org Background: The serial position effect is a phenomenon in which immediate recall of items at the end (recency) and beginning of a word list (primacy) is better than for mid-list items. Persons with Alzheimer’s disease (AD) show disproportionate recency effects and a loss of primacy effects that are thought to reflect distinct memory systems disrupted by hippocampal lesions. Aging, female sex, and the e4-allele are established risk factors for AD. Methods: We examined if these factors produced changes in serial position in cognitively intact adults similar to those found in AD. All subjects (n¼383; 151 males, mean age 69.97 6 7.76 yrs; 232 females, 68.28 6 8.73 yrs) had MMSE scores between 28-30 and were administered the Auditory Verbal Learning Test (AVLT). The sub-group of subjects with APOE genotyping consisted of 229 subjects, 161 e4 negative (69.73 6 8.85 yrs) and 68 e4 positive (69.43 6 8.57 yrs). Results: An ANOVA examining the effects of position, e4, decade and sex revealed significant serial position effects in each age group. Age and sex effects tended to occur in the middle positions where recall tended to be lower. Age effects also occurred at the initial position. With respect to e4, the youngest e4 positive group had better performance across most serial positions, but this e4 effect reversed with increasing age such that the e4 positive group became worse than the e4 negative group with age. Conclusions: The presence of e4 does not produce AD-like changes in serial position in aging normal subjects on this paradigm. P4-138

EXAMINATION OF TOMM40, APOE, AND PCDH11X IN CARIBBEAN HISPANICS WITH LATE-ONSET ALZHEIMER’S DISEASE

Joseph H. Lee1, Rong Cheng1, Ekaterina Rogaeva2, Peter St. GeorgeHyslop2, Richard Mayeux1, 1Columbia University, New York, NY, USA;

2 University of Toronto, Toronto, ON, Canada. Contact e-mail: jhl2@ columbia.edu

Background: Controversies remain as to whether the TOMM40 gene independently contributes to Alzheimer disease (AD) or whether it is due to its close proximity to the APOE gene. We examined this question using cases and controls of Caribbean Hispanic ancestry, an admixture of Spaniards, Amerindians, and Africans. The population may offer an advantage in testing associations because we can use a variable linkage disequilibrium arising from the admixture to assess genetic contributions. We tested the association between TOMM40, APOE and AD. Subsequently, we evaluated the association between AD and a novel gene, PCDH11X, in this cohort. Methods: We studied 549 cases and 544 controls of Caribbean Hispanic ancestry. For TOMM40 and APOE, we evaluated 7 SNPs (median gap¼2.7kb). To measure the degree of admixture, we used ancestry identification markers to classify each individual. We then applied the same approach to evaluate rs5984894 and 27 other SNPs in PCDH11X (median gap¼7.8kb). We first conducted a single and multi-point association study using all subjects, and then repeated the analysis within one sex. Results: We observed the strongest positive association with rs2075650 in TOMM40 (p¼0.0003543), and the coding SNPs for APOE were significant (p¼6.6E-7) without adjusting for ethnic cluster. When restricted to the Caucasian cluster, rs2075650 and rs8106922 in TOMM40 showed a strong association (p¼0.000193). However, the same SNPs failed to show significant association in the African cluster. rs5984894 in PCDH11X was not associated with AD in females (p¼0.996), and the lack of association persisted after restricting to either ethnic cluster (p_Caucasians¼ 0.8275; p_Africans¼ 0.9824). We did not observe significant association for other SNPs. The results remained unchanged for the combined analysis. Conclusions: In this set of Caribbean Hispanics, the variant in TOMM40 appears to have a modest influence on AD in those with the Caucasian, but not in African, background. Given positive associations in homogenous Caucasian samples of published studies and the negative finding in the African cluster, the influence of TOMM40 will be modest at best and it may be partially due to a hitchhiking effect. This set of Caribbean Hispanics did not support any association with PCDH11X. P4-139

REDUCED NEPRILYSIN ACTIVITY IS ASSOCIATED WITH INCREASED FORMATION OF PYROGLUTAMYLATED AMYLOID-b PEPTIDES THROUGH UPREGULATION OF AMINOPEPTIDASES AND GLUTAMINYL CYCLASE IN BRAIN

Nobuhisa Iwata1, Satoshi Tsubuki1, Matthias Staufenbiel2, Takaomi C. Saido1, 1RIKEN Brain Science Institute, Wako, Saitama, Japan; 2 Novartis Institute of Biomedical Research Basel, CH-4002 Basel, Switzerland. Contact e-mail: [email protected] Background: Amyloid-b peptides (Abs) accumulated in Alzheimer’s disease brain consist mostly of amino-terminally truncated and modified Ab, AbN3pyroglutamate(N3pE), and are different from those secreted from neurons. AbN3pE is more resistant to proteolytic degradation (4-fold), and is more easily self-aggregated (250-fold) than amino-terminally intact Ab. Genetic deficiency of neprilysin (NEP), a potent physiological Ab-degrading enzyme, promotes accumulation of AbN3pE and increases thioflavin-positive amyloid plaques in the brains of APP tg mice. Thus, AbN3pE appears to act as a seed, promoting formation of amyloid plaques. Due to a reduction in NEP activity, Ab appears to be discharged to abnormal metabolic pathway(s), in which exopeptidases, such as aminopeptidase (AP) or dipeptidyl peptidase (DPP), and glutaminyl cyclase (QC) may be involved. In the present study, we investigated the involvement of these enzymes in the formation of AbN3pE. Methods: Ab-degrading activities of APA, APN and DPP IV immunopurified from mouse brains were measured using synthetic Ab as a substrate and an HPLC system. Protein amounts of QC and the above peptidases in the brains of 4- and 18-month-old APP tg and NEP-deficient APP tg mice were analyzed using Western blotting with their specific antibodies. Results: APA, APN and DPP IV showed comparable Ab-degrading activities to NEP, and the activity of DPP IV was about 10-fold higher than that of APA and