Use of Endophenotypes in the Search for Alzheimer's Disease (AD) Risk Genes
The goal of this study is to combine data for gene expression quantitative trait loci (eQTL), with genetic data for cerebrospinal fluid (CSF) biomarkers and case control GWAS data to identify the functional alleles that influence risk for late onset AD. Using this approach we have identified novel genes that influence levels of tau protein in the cerebrospinal fluid and risk for AD. We have demonstrated that APOE genotype influences tau levels in the CSF independently of their effects on abeta levels suggesting that APOE has pleiotropic effects on AD risk. Combining eQTL and CSF biomarker data we seeking to identify the the AD susceptibility genes within each GWAS identified locus.
Cruchaga C, Kauwe JS, Harari O, Jin SC, Cai Y, Karch CM, Benitez BA, Jeng AT, Skorupa T, Carrell D, Bertelsen S, Bailey M, McKean D, Shulman JM, De Jager PL, Chibnik L, Bennett DA, Arnold SE, Harold D, Sims R, Gerrish A, Williams J, Van Deerlin VM, Lee VM, Shaw LM, Trojanowski JQ, Haines JL, Mayeux R, Pericak-Vance MA, Farrer LA, Schellenberg GD, Peskind ER, Galasko D, Fagan AM, Holtzman DM, Morris JC; GERAD Consortium; Alzheimer's Disease Neuroimaging Initiative (ADNI); Alzheimer Disease Genetic Consortium (ADGC), Goate AM. GWAS of Cerebrospinal Fluid Tau Levels Identifies Risk Variants for Alzheimer's Disease. Neuron. 2013; 78(2):256-68. PMCID: PMC3664945; PMID: 23562540
Modifier Genes that influence age at onset or protect against development of AD
Although most cases of Alzheimer's disease (AD) have an age at onset above age 80yrs the range in age at onset is huge with the earliest ages at onset occurring in individuals as young as the third decade of life while other individuals may live beyond one hundred years and remain cognitively normal. Even within specific risk groups, such as presenilin (PSEN) mutation carriers or apolipoportein E4 (APOE4) carriers, the range in age at onset can vary by several decades. For example individuals from an extended Colombian kindred who develop AD all carry the PSEN1E280A variant but the range in age at onset in this family has been reported to span three decades (35-62yrs. Similarly, individuals who carry an APOE4 allele may develop AD as early as 50 yrs while other individuals with this risk factor may be cognitively normal and older than 80yrs of age We hypothesize that in human populations there are both risk and protective alleles that influence the age at onset of AD. We will use GWAS data, exome chip data and whole genome/exome sequence data to identify common, low frequency and rare variants that influence age at onset of AD. Discovery analyses will focus on subjects of European ancestry but follow up analyses will also examine these variants/genes in minority populations, including family members from the PSEN1E280A Colombian kindred. Follow up of replicated genes/variants will include genetic studies to test whether these novel variants influence cerebrospinal fluid levels of beta-amlyoid or tau, implicating specific pathogenic mechanisms in AD. This project will use data generated by AD consortia including ADSP, ADGC, ADNI, CHARGE and GERAD. All data from this project will be deposited in NIAGADS and dbGAP to be used by the research community.
Benitez BA, Jin SC, Guerreiro R, Graham R, Lord J, Harold D, Sims R, Lambert JC, Gibbs JR, Bras J, Sassi C, Harari O, Bertelsen S, Lupton MK, Powell J, Bellenguez C, Brown K, Medway C, Haddick PC, van der Brug MP, Bhangale T, Ortmann W, Behrens T, Mayeux R, Pericak-Vance MA, Farrer LA, Schellenberg GD, Haines JL, Turton J, Braae A, Barber I, Fagan AM, Holtzman DM, Morris JC; The 3C Study Group, the EADI consortium, the Alzheimer's Disease Genetic Consortium (ADGC), Alzheimer's Disease Neuroimaging Initiative (ADNI), the GERAD Consortium, Williams J, Kauwe JS, Amouyel P, Morgan K, Singleton A, Hardy J, Goate AM, Cruchaga C. Missense variant in TREML2 protects against Alzheimer's disease. Neurobiol Aging 2014; 35(6):1510.e19-26. PMCID: PMC3750021; PMID: 24439484
Identification of Novel Alzheimer's disease genes using next generation sequencing
Over the last twenty years it has been apparent that genetic studies of Alzheimer's disease (AD) provide a powerful means to identifying the underlying disease mechanisms. Initial studies in early onset familial forms of the disease demonstrated that production of the protein (beta-amyloid) found in senile plaques is central to the disease process. Overproduction of beta-amyloid causes AD in Down Syndrome and in familial AD. More recently, genetic studies in late onset AD suggest that defects in clearance of beta amyloid may underlie the more common form of the disease.
The beauty of genetic approaches to understanding disease is that they only rely on the underlying hypothesis that "genes play a role" in determining risk for disease. As a result they are not limited by our existing knowledge and have the potential to identify unexpected connections and thus uncover novel therapeutic targets.
The goal of this project is to use next generation sequencing approaches in late onset AD families to uncover novel risk genes. During the last twelve months we have used this approach in small-scale studies to identify two novel AD risk genes: TREM2 and PLD3. In this study we plan to obtain whole exome sequencing in 900 individuals from approximately 300 families to identify new genes that increase or decrease risk for AD. The basic strategy will be to look for rare or novel DNA variants that are present in affected family members but absent from unaffected family members and then to look across families to identify the genes or variants that show this pattern in multiple families.
Once we have evidence of novel AD genes from our families we will resequence these genes in an independent dataset to look for additional variants affecting risk. When we used this strategy for PLD3 we found that 8% of AD cases carried a rare DNA variant in this gene that increased their risk for disease two-fold. For genes with compelling evidence as risk factors for AD we will begin functional studies to uncover the mechanisms underlying disease risk. We will develop induced pluripotent stem cells from individuals carrying these novel variants in order to study the impact of the variants on cellular metabolism in "cells genetically predisposed to AD" and in "disease relevant cell types". We will perform theses experiments and others in collaboration with other members of the consortium. This integrated genetic and functional approach within a consortium of collaborating investigators has high potential to rapidly translate novel disease genes and pathways into druggable targets for AD treatment.
Cruchaga C, Karch CM, Jin SC, Benitez BA, Cai Y, Guerreiro R, Harari O, Norton J, Budde J, Bertelsen S, Jeng AT, Cooper B, Skorupa T, Carrell D, Levitch D, Hsu S, Choi J, Ryten M; UK Brain Expression Consortium (UKBEC), Hardy J, Ryten M, Trabzuni D, Weale ME, Ramasamy A, Smith C, Sassi C, Bras J, Gibbs JR, Hernandez DG, Lupton MK, Powell J, Forabosco P, Ridge PG, Corcoran CD, Tschanz JT, Norton MC, Munger RG, Schmutz C, Leary M, Demirci FY, Bamne MN, Wang X, Lopez OL, Ganguli M, Medway C, Turton J, Lord J, Braae A, Barber I, Brown K; The Alzheimer's Research UK (ARUK) Consortium, Passmore P, Craig D, Johnston J, McGuinness B, Todd S, Heun R, Kölsch H, Kehoe PG, Hooper NM, Vardy ER, Mann DM, Pickering-Brown S, Brown K, Kalsheker N, Lowe J, Morgan K, David Smith A, Wilcock G, Warden D, Holmes C, Pastor P, Lorenzo-Betancor O, Brkanac Z, Scott E, Topol E, Morgan K, Rogaeva E, Singleton AB, Hardy J, Kamboh MI, St George-Hyslop P, Cairns N, Morris JC, Kauwe JS, Goate AM. Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease. Nature 2014; 505(7484): 550-4. PMCID PMC:4050701; PMID: 24336208
Dominantly Inherited Alzheimer Network
During the last two decades many genes have been shown to cause autosomal dominant forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. Several of the most promising new therapeutics are based on the Aß hypothesis, a hypothesis strongly supported by the causative mechanisms of disease mutations in autosomal dominant families. As these putative therapeutics are tested in clinical trials there is a growing need to use the FAD kindreds both to understand the natural history of the earliest clinical and preclinical phases of the disease and to test the efficacy of the therapeutics in a setting, where if the Aß hypothesis is correct, they should have a dramatic effect on prognosis. During the last funding cycle we have developed a network of centers and have begun to characterize a large series of FAD kindreds with known disease-causing mutations. The goal of the next funding period will be to continue longitudinal follow up of these kindreds to identify the earliest detectable changes associated with development of disease and to characterize the temporal series of events that occurs up to and including the diagnosis of symptomatic AD. The goal of the Genetics Core of the DIAN initiative is to provide genetic information and useful biological and genomic materials to the research community for the study of AD. We have already collected samples from 373 individuals and extracted DNA. We will initiate collection of an additional 50 new individuals during the next funding period. Since these individuals are part of FAD kindreds with known causative mutations we will screen each sample for the known mutation in that family and genotype all families for known disease modifying alleles such as APOE e4. Blood from each new participant will be sent to NCRAD for banking in the cell repository. Currently NCRAD has immortalized cell lines on 359 individuals from 134 DIAN families. In this renewal application we will initiate collection of a blood sample at each assessment for longitudinal gene expression profiling. We will also undertake a pilot DNA methylation study using longitudinal DNA samples from a subset of the DIAN cohort. Under separate funding DIAN has also collected skin biopsies from 52 individuals representing 19 different mutations in APP (1), PSEN1(15) and PSEN2 (3).
Bateman RJ, Xiong C, Benzinger TL, Fagan AM, Goate A, Fox NC, Marcus DS, Cairns NJ, Xie X, Blazey TM, Holtzman DM, Santacruz A, Buckles V, Oliver A, Moulder K, Aisen PS, Ghetti B, Klunk WE, McDade E, Martins RN, Masters CL, Mayeux R, Ringman JM, Rossor MN, Schofield PR, Sperling RA, Salloway S, Morris JC; Dominantly Inherited Alzheimer Network. Clinical and biomarker changes in dominantly inherited Alzheimer's disease. N Engl J Med. 2012 Aug 30;367(9):795-804. doi: 10.1056/NEJMoa1202753. Epub 2012 Jul 11. Erratum in: N Engl J Med. 2012 Aug 23;367(8):780. PMID: 22784036
Role of AD risk variants in phospholipase D3 on APP metabolism and amyloid pathology
Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) in the brain. The mechanism by which amyloid precursor protein (APP) is proteolyzed to generate Aβ is well understood; however, the role that other genes play in APP trafficking and Aβ production, clearance, and aggregation are poorly understood. APOE4 is a major risk factor for late onset AD that produces genotype-specific differences in Aβ clearance rates, illustrating the value of studying risk variants to understand AD pathogenesis. However, APOE4 only accounts for 50% of the genetic risk associated with late onset AD. We have recently identified several coding variants in the phospholipase D3 (PLD3) gene that double risk for late onset AD. Our preliminary data demonstrate that PLD3 is highly expressed in neurons and in regions of the brain that are most susceptible to AD pathology. Overexpression of PLD3 in cultured cells decreases extracellular Aβ levels while shRNA silencing of PLD3 increases extracellular Aβ levels. We also demonstrate that APP and PLD3 interact by co-immunoprecipitation in cultured cells and human brain tissue. Together, these findings demonstrate that PLD3 plays a role in APP metabolism that contributes to AD pathogenesis; however, the mechanisms underlying these observations remain unclear. The goal of this proposal is to begin to define the molecular mechanisms by which PLD3 influences AD risk. To accomplish this goal, we will clarify the role that PLD3 plays in APP processing in vitro and in vivo. First, we will define the effect of PLD3 on APP and Aβ trafficking in vitro in immortalized cell lines and human induced pluripotent stem cell (iPSC)-derived neurons from PLD3 risk variant carriers. Next, we will define the effect of PLD3 on APP cleavage in vitro in immortalized cell lines and human iPSC-derived neurons from PLD3 risk variant carriers. Finally, we will determine the effect of PLD3 on APP processing and Aβ generation in vivo by using viral vectors to drive overexpression or silencing of PLD3 in hippocampal neurons in an AD mouse model. These studies will increase our understanding of the precise mechanisms by which genetic risk variants in PLD3, regulate APP and Aβ metabolism, will provide novel insights into the underlying disease pathogenesis, and will allow for the identification of novel therapeutic targets for this devastating disease.
Cruchaga C, Karch CM, Jin SC, Benitez BA, Cai Y, Guerreiro R, Harari O, Norton J, Budde J, Bertelsen S, Jeng AT, Cooper B, Skorupa T, Carrell D, Levitch D, Hsu S, Choi J, Ryten M; UK Brain Expression Consortium (UKBEC), Hardy J, Ryten M, Trabzuni D, Weale ME, Ramasamy A, Smith C, Sassi C, Bras J, Gibbs JR, Hernandez DG, Lupton MK, Powell J, Forabosco P, Ridge PG, Corcoran CD, Tschanz JT, Norton MC, Munger RG, Schmutz C, Leary M, Demirci FY, Bamne MN, Wang X, Lopez OL, Ganguli M, Medway C, Turton J, Lord J, Braae A, Barber I, Brown K; The Alzheimer's Research UK (ARUK) Consortium, Passmore P, Craig D, Johnston J, McGuinness B, Todd S, Heun R, Kölsch H, Kehoe PG, Hooper NM, Vardy ER, Mann DM, Pickering-Brown S, Brown K, Kalsheker N, Lowe J, Morgan K, David Smith A, Wilcock G, Warden D, Holmes C, Pastor P, Lorenzo-Betancor O, Brkanac Z, Scott E, Topol E, Morgan K, Rogaeva E, Singleton AB, Hardy J, Kamboh MI, St George-Hyslop P, Cairns N, Morris JC, Kauwe JS, Goate AM. Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease. Nature 2014; 505(7484): 550-4. PMCID PMC:4050701