O2-02-07

S34

Oral O2-03: APP Processing and Interaction

with five different genes in this region. Conclusions: Investigation of an even more dense SNP coverage will be required for the two regions on chromosome 12 (⬃167 Kb at 6.5-6.6Mb and ⬃220 Kb at 9.0-9.2 Mb) that cumulatively include at least 20 different genes. O2-02-07

GENETIC VARIANTS ON CHROMOSOME 9 ARE ASSOCIATED WITH LATE-ONSET ALZHEIMER’S DISEASE, VARIATION IN ALLELE-SPECIFIC GENE EXPRESSION, AND DIFFERENTIAL APOPTOTIC RESPONSE

Yonghong Li1, Andrew Grupe1, Charles Rowland1, Petra Nowotny2, John S.K. Kauwe2, Scott Smemo2, Anthony Hinrichs2, Kristina Tacey1, Shirley Kwok1, Joseph Catanese1, John Sninsky1, Thomas J. White1, Paul Hollingworth3, Sandra L. Harris4, Arnold Levine5, Fabienne Wavrant-De Vrieze6, John Hardy6, Michael O’Donovan3, Simon Lovestone7, John Morris2, Leon J. Thal8, Michael Owen3, Julie Williams3, Alison Goate2, 1Celera Diagnostics, Alameda, CA, USA; 2Washington University, St. Louis, MO, USA; 3Cardiff University, Cardiff, United Kingdom; 4Robert Wood Johnson Medical School, New Brunswick, NJ, USA; 5Institute for Advanced Study, Princeton, NJ, USA; 6 National Institute on Aging, Bethesda, MD, USA; 7Institute of Psychiatry, London, United Kingdom; 8University of California, San Diego, San Diego, CA, USA. Contact e-mail: [email protected] Background: Whole genome linkage scans of late-onset Alzheimer’s disease (LOAD) have implicated candidate positional loci on chromosome 9, but the specific gene(s) has not been definitively identified. Objective: To identify chromosome 9 gene(s) that is associated with risk of developing LOAD. Methods: Genotyping single nucleotide polymorphisms (SNPs) using 3 LOAD case control sample sets. Expression and functional analyses of the candidate gene/mutation. Results: We report the association of two SNPs in a strong biological and positional candidate gene with LOAD after scanning SNPs on chromosome 9. SNP-1 was significantly associated with LOAD in our discovery case-control sample set (P⬍0.05, 2 sided) and replicated in each of the two validation case-control sample sets (P⬍0.05, 1 sided). A meta-analysis of the combined sample sets, totaling 914 cases and 1065 controls, showed an allelic P value of 0.0006 and an odds ratio of 0.79 (95%CI: 0.69 to 0.90). Minor allele homozygotes had a consistent lower risk than major allele homozygotes and the odds ratio in the combined sample increased to 0.55 (95%CI: 0.40 to 0.74), while the risk for heterozygous subjects was not significantly different from that of major allele homozygotes. The gene is located in the center of a previously identified linkage peak on chromosome 9. A second SNP, SNP-2, which is in high linkage disequilibrium with SNP-1 (r2⫽0.68), was also significantly associated with LOAD (meta P⫽0.0017). In addition, transcripts of the gene showed differential allelic expression, and both SNPs were significantly associated with the allele-specific expression (P values range from 0.015 to ⬍0.0001). Furthermore, SNP-1 genotype was significantly correlated to the response of human cell lines to pro-apoptotic stimuli (P⬍0.05). Conclusion: Variants of the chromosome 9 gene contribute to the etiology of LOAD. O2-02-08

DUPLICATION OF THE APP LOCUS IN A DUTCH FAMILY WITH EARLY-ONSET ALZHEIMER DEMENTIA

Kristel Sleegers1, Nathalie Brouwers1, Ilse Gijselinck1, Jessie Theuns1, Dirk Goossens1, Jurgen Del-Favero1, Marc Cruts1, Cornelia M. van Duijn2, Christine Van Broeckhoven1, 1VIB8-Molecular Genetics, Antwerpen, Belgium; 2Epidemiology & Biostatistics, Erasmus MC, Rotterdam, The Netherlands. Contact e-mail: [email protected] Background and Objective: Evidence is accumulating that genetic variability in the expression of APP, encoding the amyloid beta precursor protein, can cause Alzheimer dementia (AD) through relative increase in

production of the pathogenic peptide Abeta42. Early evidence came from the observation of AD, Abeta deposits and congophilic amyloid angiopathy (CAA) in patients with trisomy 21, and a report of APP duplication in sporadic AD. Recently, Rovelet-Lecrux et al. described a duplication of the APP locus on chromosome 21 in families with autosomal dominant earlyonset AD (EOAD) and concurrent CAA. In EAOD patients we identified heterozygous APP promoter mutations that increased APP expression by nearly two-fold, mimicking APP trisomy 21. Methods: We investigated the contribution of copy number variants of APP to EAOD in a populationbased Dutch sample using real-time PCR. The sample included probands of 11 families with autosomal dominant EAOD. Results: We identified a duplication of APP in one proband, in whom mutations in the known AD genes were absent. Linkage analysis in this family had previously resulted in inconclusive linkage to the APP region (LOD score⬍1). The duplication was present in two affected siblings and absent from all healthy siblings ⬎50 years. Fluorescence in situ hybridization of interphase nuclei confirmed the presence of a duplication of APP in this family. Brain autopsy in two affected individuals was compatible with definite AD, but also revealed extensive CAA. Clinically, the disease presented with progressive memory loss at 55 years on average, extending to other areas of cognition and accompanied by seizures and roaming behavior. The mean duration of the disease was 6.5 years. None of the APP duplication carriers has had intracerebral hemorrhage. Semi-quantitative multiplex PCR studies are ongoing to delineate the length of the duplicated area. Conclusions: We are currently assessing the presence of duplications in an extended sample of familial and sporadic EAOD patients from the same region, but our preliminary data suggest that duplication of APP might explain 9% of autosomal dominant EAOD, which corroborates with the recently reported findings. Our results further confirm that duplications of APP give rise to AD dementia with concurrent CAA. MONDAY, JULY 17, 2006 ORAL O2-03 APP PROCESSING AND INTERACTION O2-03-01

NOVEL PROTEINS ACT AS GATEKEEPERS AND CONTROL ACCESS OF APP TO ALPHA- AND BETA-SECRETASE

Stefan F. Lichtenthaler, Susanne Scho¨bel, Stephanie Neumann, Sylvia Fischer, Christian Haass, University of Munich, Munich, Germany. Contact e-mail: [email protected] Background: Ectodomain shedding of APP by ␣- and ␤-secretase is a key regulatory step in the generation of the A␤ peptide. However, little is known about mechanisms that control access of APP to ␣- and ␤-secretase and, thus, A␤ generation. Objective: To identify proteins with such a function, we carried out a genome-wide expression-cloning screen. Methods and Results: We identified a novel membrane protein that strongly reduces APP cleavage by ␣- and ␤-secretase, suggesting that it limits access of APP to both proteases. Additionally, several proteins were obtained that strongly increased APP ␣-cleavage but only had a mild effect on APP ␤-cleavage. These proteins include the APP homolog APLP1, a member of the endophilin family of endocytic and signal transducing proteins, and SNX31, which is a novel member of the sorting nexin (SNX) family of intracellular trafficking proteins. Mechanistical analysis was carried out in HEK293 and COS cells using overexpression and RNAimediated knock-down experiments. This revealed that the three proteins control the rate of APP endocytosis and thus determine how much APP is available for ␣-secretase cleavage at the cell surface or for ␤-secretase cleavage in the endosomes. The proteins affected APP endocytosis through different mechanisms. APLP1 affected complex formation of APP with FE65 and the LDL-receptor related protein, which is required for efficient endocytosis of APP. In contrast, endophilin and SNX31 affected the dynamin-dependent endocytosis of APP, potentially by influencing the coupling of APP to the endocytic machinery. Despite their different mech-