Plowing through phylogenetic analyses, researchers led by Allen Roses and Michael Lutz of Duke University in Durham, North Carolina, and Eric Reiman of Banner Alzheimer’s Institute in Phoenix, Arizona, came upon a poly-T variant within intron 6 of Tomm40 that greatly improved predictions for when ApoE3 carriers might develop AD. In particular, among autopsy-confirmed ApoE3/4 patients, those with two copies of the long Tomm40 variant (more than 20 poly-T repeats)—aka the “long/longs”—developed AD about eight years earlier than the “short/longs,” who had a copy of the short Tomm40 variant (20 or fewer poly-T repeats) along with the long version (Roses et al., 2009; see also ARF related news story). In this earlier study, the researchers analyzed several independent cohorts of patients whose LOAD onset ages were documented in medical records.

Because retrospective data can be unreliable, the scientists sought to reproduce those findings in prospective studies of people with known ApoE and Tomm40 status who are being followed with neuropsychological testing for future development of MCI or AD. On an ICAD poster, Richard Caselli of Mayo Clinic, Scottsdale, Arizona, and colleagues including Reiman and Roses, reported preliminary data from 30 participants in the first of several prospective studies in progress for five to 19 years. In short, the results came out as predicted: the “long/long” group developed incident MCI or AD about nine years earlier than the “short/longs” (onset age 73 versus 82). The cohort was too small to correct for ApoE genotype, Caselli noted, but the earlier age of onset in the long/longs did hold for both ApoE3/4 (n = 10) and ApoE3/3 (n = 11) subgroups.

The Tomm40 length variants also seem to track with other defining measures of AD—namely, brain atrophy and cognition. These preliminary studies involved participants of a longitudinal cohort study called WRAP (Wisconsin Registration for Alzheimer’s Prevention) that started in 2001 under the leadership of Mark Sager at the University of Wisconsin in Madison. Participants around a mean age of 54 enter the study asymptomatic and get cognitive testing every few years. Some also receive brain imaging through ancillary studies led by Sterling Johnson, also at the University of Wisconsin. Forty-six percent of the subjects are ApoE4-positive. Mining the data on 1,400 study participants, the researchers uncovered differences in white matter (measured by diffusion tensor imaging), brain activity (measured by functional magnetic resonance imaging of AD-relevant areas such as hippocampus), and certain measures of learning. Somewhat surprisingly, “the differences were based on whether or not their parents had AD,” Sterling said in his ICAD talk. “ApoE wasn’t really giving us all the explanatory power we needed. So we looked for other genetic and lifestyle factors that might predict [the parental history connection].”

Puzzling over these findings, which were reported last fall at the Clinical Trials on Alzheimer’s Disease meeting in Las Vegas (see ARF conference story), the researchers recalled the recent buzz over Tomm40 and wondered whether Tomm40 length variants might help tease out the differences they had seen related to family history. Johnson focused on E3 homozygotes because of their curious bimodal distribution on AD risk charts. Though E3 has historically been regarded as the risk-neutral ApoE variant, in reality, there is a subgroup of E3 carriers who seem just as prone to AD as people with the high-risk E4 allele. Johnson’s team analyzed 120 healthy E3/3 WRAP participants (mean age 57), assessing their Tomm40 status and measuring gray matter volume in the ventral posterior cingulate and precuneus (brain regions affected early in AD) by structural MRI. Comparing participants with two “short” Tomm40 alleles to those with two “long” Tomm40 alleles, the researchers found that the latter had lower gray matter volume in the analyzed brain areas.

Sager and colleagues analyzed more than 700 asymptomatic WRAP participants (mean age 54) with a family history of AD, and similarly compared short/short and long/long subgroups for cognitive differences. Consistent with their greater brain atrophy, the long/long subjects, regardless of ApoE genotype, did worse on several measures of the Auditory Verbal Learning Test.

If confirmed in larger samples, the findings may be “very important to explain why some E3/3s develop AD at earlier ages,” said Yadong Huang of Gladstone Institute of Neurological Disease at the University of California, San Francisco. Among E3 homozygotes, about a quarter have the long/long Tomm40 genotype that confers greater AD risk.

The new data may also hint at possible synergistic effects between ApoE and Tomm40 at mitochondria, which help maintain synapses and falter in early AD. Huang and colleagues have shown that proteolytic fragments of ApoE, which form more commonly from E4 than E3, interact with neuronal mitochondria, throwing off membrane potential and contributing to cytoskeletal structures that contain phosphorylated tau (Chang et al., 2005). Tomm40 is a mitochondrial membrane protein needed for shuttling proteins into the organelle. “If Tomm40 causes problems, then when the ApoE fragment comes in, that might make it even worse,” Huang speculated.

Whether and how the Tomm40 poly-T variants influence mitochondrial function to begin with remain unclear. Because they are intronic, the polymorphs do not affect Tomm40’s protein sequence and have yet to demonstrate effects on expression, leaving in question their biological effect in neurons, suggested John Hardy of University College London, U.K., in an e-mail to ARF. In his view, it seems more likely, for now, that LOAD risk variability derives from ApoE promoter polymorphisms that govern expression of ApoE (Lambert et al., 2002; Lambert et al., 1997). Hardy noted that this mechanism plays out in another disease, where missense variants in complement factor H have been shown to influence gene expression and predisposition to macular degeneration (Li et al., 2006).

Still, the ICAD data suggest the Tomm40 length variants “clearly have some effect—especially on E3/3s, where there are no confounding effects due to E4,” Huang told ARF. Whether those effects involve synergism with ApoE remains to be seen. On the one hand, studies with transgenic mice that express human E4 have shown that E4 alone can drive cognitive decline. That suggests to Huang that E4 messes with learning and memory independent of Tomm40, since mice are unlikely to have the same Tomm40 length variants that have been studied in people. In collaboration with Roses, Huang hopes to do the converse experiment—that is, put the human Tomm40 “long” allele into transgenic mice with or without ApoE4—to see whether Tomm40 effects require E4.

In the meantime, Roses has submitted an application to the U.S. Food and Drug Administration for a prevention trial in which the Tomm40 genotype would serve as a key criterion for selecting high-risk patients to test an investigational AD drug. The trial, called Opportunity for the Prevention of Alzheimer’s (OPAL), would enroll cognitively normal seniors from the ages of 60 to 87 and judge, based on age and Tomm40, whether they are at high or low risk for developing AD in the next five years. Most low-risk participants would go into the placebo arm while the high-risk group is randomized to receive drug or placebo. In this manner, the five-year study would, as Roses hopes, serve a dual purpose: validate Tomm40 as a genetic marker, and test the ability of an investigational AD drug to delay LOAD onset. The trial could start next year, Roses told ARF.—Esther Landhuis.