Search for the elusive haplotype of the APOE polymorphism associated with Alzheimer’s disease

The common APOE2/E3/E4 polymorphism is determined by two-site haplotypes: C112R and R158C. Due to strong linkage disequilibrium between the two sites, three of the four expected haplotypes/alleles ( E2, E3, E4 ) have been observed. Compared to the most common haplotype of E3 (C112 – R158), the E4 (R112 – R158) and E2 (C112 – C158) haplotypes are determined by a single-point mutation at codons 112 and 158, respectively. The fourth haplotype ( E5 ) having mutations at both sites (R112–C158) has been reported only as an incidental finding in three kindreds. To our knowledge, no systematic search has been done to determine its distribution in the general population. The objective of this study was to search for the elusive haplotype in 355 APOE 2/4 subjects derived from 14,819 genotyped subjects. A DNA fragment of 177bp from APOE 2/4 subjects was subcloned into competent bacterial cells to construct the phased haplotype clones followed by Sanger sequencing. We also used Whole-genome sequencing and RFLP assay to search for the fourth haplotype. All three strategies confirmed that the E4 and E2 alleles are present on opposite chromosomes, with no example having both alleles on the same chromosome, suggesting E5 might have minimum effect, if any, on disease risk.