From Genotype to Phased Haplotype: Multiplex Digital PCR-Based Haplotyping at the APOE Locus in Alzheimer’s Disease

Backgrounds: Alzheimer’s disease (AD) risk reflects multi-locus variant effects. Despite advances in genotyping techniques, current molecular haplotyping approaches involve intricate operational frameworks and incur high implementation costs, and most studies therefore rely on unphased genotypes. We developed a straightforward approach to reconstruct locus-wide haplotype structure within the APOE locus and assess for potential AD-risk haplotypes. Methods We analyzed 49 postmortem brain samples (40 AD and 9 Controls) heterozygous for APOE ε3/ε4. We developed a Multiplex Digital PCR assay to resolve allelic phase configurations of AD-associated SNPs across three closely linked loci within the APOE genomic region: TOMM40 (rs2075650), APOE (rs429358) and APOC1 (rs12721046). Gaussian mixture modeling was used to reconstruct sample-level phased haplotype structure. Results We achieved high-confidence reconstruction of three-SNP haplotypes spanning a 25.6 kilobase (kb) region. The diplotype frequencies did not differ significantly in AD and Control groups (G = 11.36, p = 0.25). A suggestive trend was observed between the AD and Control groups for TOMM40 rs2075650-A and APOC1 rs12721046-G haplotype (G = 6.50, p = 0.09). Conclusions Although underpowered and not statistically significant, this proof-of-concept shows that multiplex digital PCR can support simple, sample-level haplotype phasing at the APOE locus. This strategy provides a robust platform for mechanistic investigation and translational application by integrating phased haplotype configurations with three-dimensional chromatin architecture–associated regulatory dynamics, thereby informing locus-specific therapeutic targeting.