14 May 2010. By some measures, ApoE found its first companions in 2009 when scientists reported three additional genes robustly linked to late-onset Alzheimer disease. Now, in this week’s JAMA, a genomewide association study (GWAS) of more than 35,000 people—the largest to date for AD—has revealed two more risk genes and confirmed two of the 2009 loci. None confer nearly as much risk as ApoE, but all have reached genomewide significance in multiple independent GWASs—something the vast majority of genes emerging from smaller GWASs fail to do. One of the novel genes in the current analysis is near BIN1, which encodes a protein involved in clathrin-mediated trafficking. The second lies close to two genes (BLOC1S3 and MARK4) in pathways linked to AD pathology. The previously identified CLU and PICALM also came up as major hits, yet did not improve AD risk prediction when added to a model that included age, gender, and ApoE status. Nevertheless, these and other small-effect genes may help guide researchers to biological pathways that contribute to AD pathogenesis.
For AD genetics research, and GWASs in particular, 2009 was a banner year. At the International Conference on Alzheimer’s Disease in Vienna (see ARF related conference story) and a few months later in two Nature Genetics papers (Lambert et al., 2009; Harold et al., 2009), scientists announced what looked to be the first three AD risk genes besides ApoE to reach genomewide significance in several independent GWASs. Those genes were CLU (clusterin/apolipoprotein J), PICALM (phosphatidylinositol-binding clathrin assembly protein), and CR1 (complement receptor 1).
In the current study, an international research team led by Monique Breteler of Erasmus University, Rotterdam, The Netherlands, and Sudha Seshadri of Boston University, Massachusetts, started by analyzing GWAS data from six
cohorts in the U.S. and Europe. Two cohorts came from larger case-control studies (Translational Genomics Research Institute public release database and the Mayo AD GWAS); the others came from CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology). More than 2,000 of the nearly 17,000 participants had AD. Because the cohorts had their DNA sequenced on different platforms and may have had other slight differences due to geography, the investigators figured it may be inappropriate to simply pool all datasets right from the start, Seshadri told ARF.
Rather than doing a more conventional GWAS—which pulls top hits from a large pool of samples and replicates them in independent cohorts—Seshadri and colleagues used a three-stage sequential approach. In the first stage, they identified 2,708 single nucleotide polymorphisms (SNPs) with moderate AD association in the six initial cohorts. All but 38 (on 10 loci) were left in the dust after the second round, which subjected the first-stage SNPs to stricter criteria in an expanded sample set containing the original six plus an additional European cohort. The strongest hits proceeded to the final stage—replication in a meta-analysis of all second-round samples plus a different non-overlapping cohort—bringing the total number analyzed to 8,371 AD cases among some 35,000 participants. Besides ApoE, just four SNPs emerged from the final gauntlet with genomewide significance—PICALM, CLU, and two novel loci on chromosomes 2 (near BIN1) and 19 (near BLOC1S3 and MARK4). These four associations were confirmed in an independent Spanish sample set.
It is interesting that the winners’ circle lacked CR1, the third gene identified in the 2009 GWAS. However, 13 SNPs within CR1 fell just below the predefined cutoff, and did reach genomewide significance in a post-hoc analysis. “We believe it’s real,” Seshadri said.
Using data on incident AD from the two largest CHARGE cohorts that had the information available, the researchers determined that PICALM and CLU did little to improve existing models for predicting AD risk. This is not surprising. In existing models, age and gender already explain a large part of the risk, suggested coauthor Philippe Amouyel of Institute Pasteur, Lille, France, who led one of the 2009 GWASs (Lambert et al., 2009). “Thus, adding one or two susceptibility genes or any other risk factor will not, in a general population sample, largely improve this prediction,” he wrote in an e-mail to ARF.
However, those genes may still prove useful in a clinical setting. Amouyel made this point by citing an example from heart disease. Genetic variability in HMG (3-hydroxy-3-methyl-glutaryl)-CoA reductase explains less than 10 percent of the variance of low-density-lipoprotein blood cholesterol levels. Yet this enzyme is currently the major target of statins, one of the most widely used drugs for staving off heart disease.
Similarly, targeting AD risk genes with small effects could help keep disease at bay. If interventions could wipe out the disease-related effects of the handful of genes identified thus far, “you could remove 20 to 30 percent of all AD cases,” said coauthor Julie Williams of Cardiff University, U.K. For common diseases like AD, “we all have some risk factors,” she said. “But it’s the accumulation of risk factors that is important. The accumulation takes us to a point that tips us into a disease state. By simply removing some effects of these risk factors, you can actually take people out of the risk zone.” It might also postpone age of onset. “Disease that starts at age 75 may not develop until age 90, for example,” she said.
Aside from clinical relevance, one of the most exciting things coming out of GWASs may be the identification of potential biological pathways that influence AD pathology. “A pattern is developing in the genes we identify,” Williams said. “These are not random genes. They are telling us a story.” One story from the current paper relates to BIN1, which was just underneath the threshold for genomewide significance in her 2009 GWAS (Harold et al., 2009). “Now it is a definite hit,” she said. “Both PICALM and BIN1 affect the same process in the brain—clathrin-mediated endocytosis and trafficking. You’ve got two genes that are telling us something new about components or factors that trigger disease.”
Beyond the potential biological insight, the study represents somewhat of a methodological milestone. “I think it's encouraging that researchers within the field are willing to pool data and are more enthusiastic to do that,” Williams said. In her mind, the gold standard would be a huge GWAS where multiple datasets are treated uniformly with the same quality control measures and pooled in a structured way, “so we can really extract all the information we can, and follow up what comes out of that in independent samples,” she said, noting this has succeeded for other complex diseases such as diabetes and hypertension. “That would be a really powerful way to get at more genes undoubtedly out there that are related to AD.”
At present, she is heading up a GWAS that will involve around 50,000 people. “I’m optimistic that we will find several more genes within the next few months,” Williams told ARF. Seshadri predicts GWASs could net 20 to 25 new loci over the next five years.
Seshadri expects future GWASs to have deeper computation and more complex analytical approaches—sequencing areas of interest, for instance, or looking at methylation and gene-environment interactions. “Maybe a gene only has an effect if you’re obese in midlife,” Seshadri said.
In the meantime, the current data reinforce the idea that “at the individual level, it makes far more sense to focus on things we know affect risk prediction and development of disease,” Seshadri said. “I would focus more on midlife vascular risk factors than on trying to determine whether you have the minor or major allele of the CLU locus.”
Potential impact of environmental interactions was also highlighted by Nancy Pedersen of Karolinska Institutet, Stockholm, Sweden, in her JAMA commentary on the present GWAS. “Very large sample sizes, on the order of those in GWAS consortia such as those Seshadri et al. report, are necessary for detecting significant gene-environment interactions,” she wrote. “Possibly more could be gained by focusing efforts in these consortia on incorporating information on environmental risk and protective factors in further collaborative efforts than in further pursuit of gene identification or replication.”—Esther Landhuis.
Seshadri S, Fitzpatrick AL, Ikram MA, DeStefano AL, Gudnason V, Boada M, Bis JC, Smith AV, Carassquillo MM, Lambert JC, et al. for the CHARGE, GERAD1 and EADI1 Consortia. Genome-wide Analysis of Genetic Loci Associated With Alzheimer Disease. JAMA. 12 May 2010. Abstract
Pedersen NL. Reaching the Limits of Genome-Wide Significance in Alzheimer Disease. JAMA. 12 May 2010. Abstract