APOE Haplotype Phasing Using ONT Long-Read Sequencing Reveals Two Common ϵ3 and ϵ4 intragenic haplotypes in the Spanish Population

BackgroundThe apolipoprotein E (APOE) gene is a key genetic determinant of Alzheimers disease (AD) risk, with the {varepsilon}4 allele significantly increasing susceptibility. While the pathogenic effects of the {varepsilon}4 allele are well established, the functional impact of distinct haplotype configurations within the broader {varepsilon}3 and {varepsilon}4 backgrounds remains poorly understood. This study investigates the role of intragenic sub haplotypes in modulating APOE expression and their potential influence on AD progression. MethodsWe utilized Oxford Nanopore Technology (ONT) long-read sequencing to phase variants within a 4-kilobase comprising the APOE locus in a cohort of 1,265 individuals with known APOE genotypes. We evaluated the impact of the identified intragenic haplotypes on APOE protein levels in cerebrospinal fluid (CSF) using the Olink platform, adjusting for demographic and molecular covariates. Statistical modeling was employed to assess the independent effects of these haplotypes alongside traditional APOE genotypes. Additionally, their influence on dementia progression in mild cognitive impairment (MCI) subjects was analyzed using adjusted Cox proportional hazards models. ResultsOur analysis identified 48 Single Nucleotide Variants (SNVs) within a 4-kilobase region containing the APOE gene, including nine novel variants. Phasing of variants within the APOE locus revealed 59 unique haplotypes in the Spanish population, which were grouped into five major haplogroups--{varepsilon}2, {varepsilon}3A, {varepsilon}3B, {varepsilon}4A, and {varepsilon}4B--including two common haplogroups for each of the {varepsilon}3 and {varepsilon}4 isoforms. The {varepsilon}4A haplogroup was associated with a significant decrease in APOE {varepsilon}4 protein levels in CSF (p = 0.004), suggesting a regulatory mechanism that may mitigate the toxic gain-of-function effect typically attributed to the {varepsilon}4 allele. Conversely, the {varepsilon}3B haplogroup was linked to increased APOE {varepsilon}3 protein levels in {varepsilon}3/{varepsilon}4 carriers (p = 0.025), potentially serving a compensatory role. These effects were independent of overall APOE genotype and remained significant after adjusting for covariates. Both haplogroups ({varepsilon}4A and {varepsilon}3B) demonstrated protective effects in the progression from MCI to dementia, underscoring their potential relevance in Alzheimers disease. ConclusionsThis study provides new insights into the intragenic allelic variability of the APOE gene, demonstrating that intragenic APOE haplogroups within the {varepsilon}3 and {varepsilon}4 backgrounds can modulate APOE isoform expression in ways that might modulate AD. Our findings highlight the importance of considering haplotype-specific effects when interpreting the functional impact of APOE and in designing targeted therapeutic strategies. Further research is needed to explore the broader regulatory network of the APOE locus and its interaction with neighboring loci in the 19q13 region.