Characterization of the poly-T variant in the TOMM40 gene in diverse populations.
We previously discovered that a polymorphic, deoxythymidine-homopolymer (poly-T, rs10524523) in intron 6 of the TOMM40 gene is associated with age-of-onset of Alzheimers disease and with cognitive performance in elderly. Three allele groups were defined for rs10524523, hereafter 523, based on the number of T-residues: Short (S, T?19), Long (L, 20?T?29) and Very Long (VL, T?30). Homopolymers, particularly long homopolymers like 523, are difficult to genotype because slippage occurs during PCR-amplification. We initially genotyped this locus by PCR-amplification followed by Sanger-sequencing. However, we recognized the need to develop a higher-throughput genotyping method that is also accurate and reliable. Here we describe a new 523 genotyping assay that is simple and inexpensive to perform in a standard molecular genetics laboratory. The assay is based on the detection of differences in PCR-fragment length using capillary electrophoresis. We discuss technical problems, solutions, and the steps taken for validation. We employed the novel assay to investigate the 523 allele frequencies in different ethnicities. Whites and Hispanics have similar frequencies of S/L/VL alleles (0.45/0.11/0.44 and 0.43/0.09/0.48, respectively). In African-Americans, the frequency of the L-allele (0.10) is similar to Whites and Hispanics; however, the S-allele is more prevalent (0.65) and the VL-allele is concomitantly less frequent (0.25). The allele frequencies determined using the new methodology are compared to previous reports for Ghanaian, Japanese, Korean and Han Chinese cohorts. Finally, we studied the linkage pattern between TOMM40-523 and APOE alleles. In Whites and Hispanics, consistent with previous reports, the L is primarily linked to ?4, while the majority of the VL and S are linked to ?3. Interestingly, in African-Americans, Ghanaians and Japanese, there is an increased frequency of the 523S-APOE?4 haplotype. These data may be used as references for 523 allele and 523-APOE haplotype frequencies in diverse populations for the design of research studies and clinical trials.