NEW YORK (GenomeWeb) – Using a new in-phase assay, researchers led by Duke University's Allen Roses believe they have captured key differences in APOE and TOMM40 haplotypes between African-American, Caucasian, and West African populations. The varying APOE and TOMM40 haplotypes in these populations may explain why African-Americans, despite harboring lower frequencies of the APOE4 risk allele compared to Caucasians, have similar rates of Alzheimer's disease.
The investigators have hypothesized that in African-Americans, many of the long alleles of TOMM40 are attached as an APOE3-Long haplotype that is rarely observed in Caucasians, in whom virtually all of the TOMM40 long alleles are in APOE4-Long haplotypes. "This would help explain why the APOE4 association with Alzheimer's has been variable in African-American populations over the past 20 years," Roses, Jefferson-Pilot professor of neurobiology and neurology at Duke University, told PGx Reporter.
Roses and his colleagues presented their preliminary analysis of samples from four ethnic groups in a poster at the Alzheimer’s Association International Conference this week in Copenhagen, Denmark. Their findings, if confirmed and elaborated upon in prospective analysis, may move the field closer to developing an algorithm that is customized across different ethnicities for determining the age at which a person is at risk for developing Alzheimer's-related cognitive decline. Furthermore, their work may also help inform the design of prevention and treatment trials for African-Americans and people of mixed ethnic groups.
Roses' startup Zinfandel Pharmaceuticals is already collaborating with Takeda Pharmaceutical on the Phase III TOMMORROW trial, in which the drugmakers are hoping to prove the efficacy and safety of low-dose pioglitazone, dubbed AD-4833, in delaying the onset of mild cognitive decline due to Alzheimer's in individuals at high genetic risk for the illness. AD-4833 in TOMMORROW has the same active ingredient as the marketed diabetes drug Actos, but has a different dosing schedule (0.8SR mg QD). In that same trial, researchers are hoping to validate an algorithm for predicting the age of onset for Alzheimer's-related mild cognitive decline in the next five years.
The age of onset risk prediction algorithm – which factors in people's age, and APOE and TOMM40 genotypes – was developed by Roses' team by analyzing samples from Caucasian cohorts. According to Roses, the addition of TOMM40 genotypes to APOE SNPs makes for a much more powerful algorithm for predicting late-onset Alzheimer's than just with APOE genotypes. APOE3 is the most common form of the gene, APOE4 is well known in the literature to heighten a person's risk for Alzheimer's, while the rarer APOE2 is thought to be protective against the disease. Testing to gauge whether a person carries one or two APOE4 risk alleles is informative for earlier Alzheimer's risk for only 30 percent of Caucasians.
However, as described in Clinical Pharmacology & Therapeutics by Roses and colleagues in a 2013 paper, the addition of TOMM40 – which happens to be in linkage disequilibrium with APOE – better elucidates the roles APOE2 and APOE3 in Alzheimer's risk, creating a risk prediction system that is informative for 97 percent of the Caucasian population. In that paper, Roses' team reported that among Caucasians the APOE-TOMM40 haplotypes could be differentiated by age of onset distributions, making the more common APOE3/3 and 2/3 genotypes informative with regard to the genetic risk for Alzheimer's.
This latest study expands on that earlier work and aims to study the role of APOE and TOMM40 in African-Americans. Researchers have long been stumped by why African-Americans develop Alzheimer's at similar rates to Caucasians in the US, but studies have not replicated APOE4 allele frequencies in African-Americans as consistently as in Caucasians. The work by Roses' team suggests again that this may be explained by an algorithm that factors in APOE-TOMM40 haplotypes.
The findings
In the study presented at the AAIC meeting, researchers used samples stored in the Coriell Cell Repository on Caucasians, African-Americans, the Maasai people in Kenya, and the Yoruban population in Nigeria.
Using various techniques to genetically analyze the samples, Roses and his group reported that a large proportion of the African-American population carries a long TOMM40'523 polymorphism connected to APOE3 that has only been seen in West Africans to date. Greater than 98 percent of Caucasians have long TOMM40'523 polymorphisms, but connected to APOE4 – and these haplotypes are also seen in African-Americans.
Additionally, the investigators identified in the study a short version of TOMM40'523 linked to APOE4 in West Africans and African-Americans. The short TOMM40-APOE4 haplotype, has been seen in West African populations, but observed very infrequently in Caucasians.
"African-American TOMM40 '523/APOE haplotype frequencies differ from both Caucasians and West Africans," the study authors concluded in the poster presented at the meeting in Copenhagen. "The haplotype relationship between APOE4 and TOMM40'523 in an African-American cohort identified a distinct difference in the frequency of APOE4 alleles in phase with a short TOMM40'523 allele, a combination rarely observed in Caucasian cohorts."
In particular, the researchers assert that the high proportion of African-Americans with the short TOMM40-APOE4 haplotype may reflect the impact of American history with slavery. "The large proportion of APOE4 alleles attached to the Short (T 15) allele has not been observed in any ... populations [other than West Africans] up to now and may be explained by genetic admixture, presumably occurring during intermixing of West Africans and Caucasians in the period of slavery in the US," they said in the poster. "The presence of short TOMM40 '523 alleles connected to APOE4 in approximately equal proportions to those connected to APOE3 in African-Americans may be explained by admixture in recent history, where Caucasians contributed mostly Long (T 28-29) APOE4-TOMM40 '523 chromosomes and West Africans contributed the Long (T 30-32) chromosomes."
For the study, Polymorphic DNA Technologies gauged APOE and TOMM40 genotypes in samples using a "nested" PCR technique – where for each SNP the sample was amplified using "outside" oligonucleotide primers and then reamplified with a second set of "inside" primers – followed by Sanger sequencing. Professor Dmitry Goldgaber's lab at Stony Brook University used a novel PCR method with which researchers imputed the precise lengths of the TOMM40'523 polymorphism in samples.
Researchers also employed an assay, performed by Polymorphic, to gauge the haplotype phases between APOE alleles and different lengths of the TOMM40'523 polymorphisms. This in-phase assay enables researchers to sequence the entire lengths of the each chromosomal strand so that the various strands can be differentiated, and not just inferred from Caucasian data, Roses explained. "The method allows greater certainty for assigning the APOE-TOMM40'523 haplotypes," he said.
The assay being used in the TOMMORROW study measures the TOMM40 PolyT, but the area between TOMM40'523 and APOE is not sequenced. This works in the TOMMORROW study, since it is powered for the Caucasian population, in which the majority of people with APOE3/4 genotype have a long TOMM40-APOE4 haplotype. "In Caucasians the APOE3/4 genotype is consistent," Roses said. "There is no [age of onset] assignment difficulty when an individual has a heterozygous genotype like TOMM40'523 Short-T16, LongT30, and approximately 2 Kb away also has the APOE3/4 genotype."
However, the in-phase assay was necessary to observe that in African-Americans with an APOE3/3 genotype, there is a long TOMM40 allele attached to an APOE3 allele. "This appears inconsistent until the distinct APOE3-TOMM40 Long haplotype was observed in many West Africans and African-Americans," Roses noted. This observation provides "an explanation for the African-American variability [seen] based on APOE4 as the single marker over the past two decades, and highly supportive of the influence of the TOMM40-Long [allele] in the genetics of Alzheimer’s Disease."
If variations in APOE-TOMM40 haplotypes in different ethnic groups are confirmed in ongoing studies to predict age of Alzheimer's onset, then gauging TOMM40 polymorphisms alongside APOE genotypes will be particularly important in African-American and Asian populations, in which APOE4 frequencies are low. Furthermore, "these findings demonstrate the importance of determining an individual's genetic architecture and not inferring genetics from societal or national labels," Roses and his colleagues concluded in their poster.
Future directions
Roses and his colleagues urge that these results need to be confirmed in prospective age of onset evaluations to find out whether the varying APOE-TOMM40 haplotypes seen in African-Americans can accurately predict their risk of cognitive decline due to late-onset Alzheimer's disease. In the TOMMORROW study being conducted by Zinfandel and Takeda, researchers will enroll normal African-American subjects but their risk will be imputed based on the algorithm developed based on data from Caucasians. As such, the 5,800-subject TOMMORROW is sized for the Caucasian population, and will only generate exploratory findings on African-Americans and other subjects of non-Caucasian ethnicities.
"However, if the identical APOE-TOMM40 haplotype in any individual [of other ethnicities] appears to be similar to those haplotypes present in Caucasians, additional testing would come fast," Roses said. "The haplotypes are highly ethnically variable so a drug that works would then be tested. Other drug companies could also test new compounds, although these would be difficult to design without using a pioglitazone comparator and [without] licensing TOMM40'523." The US Patent and Trademark Office has granted Roses and his colleagues a defined usage patent on the Alzheimer's onset risk prediction algorithm, which would require other labs to garner a license before marketing a commercial test using the algorithm, but wouldn't restrict the use of the algorithm in research.
Roses' team has created mouse models with which they could study TOMM40 variations further, but they need additional research funding to pursue prospective analysis and to investigate the TOMM40-based risk algorithm as part of an Alzheimer's prevention drug trial.
The new phased haplotype assay available through Polymorphic and used in the latest analysis of African American populations is much more precise than the allele specific testing being employed in the TOMMORROW study, according to Roses, and could potentially be used to gauge APOE-TOMM40 haplotypes for all ethnic groups. "The TOMMORROW trial is [already] underway with specified validated assays, and it would be very disruptive to try to add each improvement to a multi-year trial," he noted "However, improvements in assays and in phase sequencing can be examined later as exploratory data."
Roses is conducting several collaborative exploratory studies regarding the role of APOE-TOMM40 haplotypes in Alzheimer's in various populations, including other prospective studies in Caucasians, African-Americans, Japanese, and other ethnic groups. However, performing the in-phase assay is expensive. So, for now, Roses is attempting to lower costs by first first gauging APOE and TOMM40 genotypes then retesting the APOE heterozygotes (APOE3/4) using the in-phase assay.
"Presumably, if next-generation sequencing concentrates on accurate sequencing of long [TOMM40] polyT lengths, a simpler technology for measuring in-phase haplotypes will be available," Roses reflected over e-mail. "However, by the time the data are available from the TOMMORROW study with respect to [drug] efficacy, new APOE-TOMM40 haplotypes in various ethnic populations will be defined and then tested for specific age of onset distribution in those populations to understand how best to apply these new 'non-Caucasian,' highly informative haplotypes accurately."