BACKGROUND: The apolipoprotein E () gene is a key genetic determinant of Alzheimer's disease (AD) risk, with the ε4 allele significantly increasing susceptibility. While the pathogenic effects of the ε4 allele are well established, the functional impact of distinct haplotype configurations within the broader ε3 and ε4 backgrounds remains poorly understood. This study investigates the role of intragenic sub haplotypes in modulating expression and their potential influence on AD progression. METHODS: We utilized Oxford Nanopore Technology (ONT) long-read sequencing to phase variants within a 4-kilobase comprising the locus in a cohort of 1,265 individuals with known 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 genotypes. Additionally, their influence on dementia progression in mild cognitive impairment (MCI) subjects was analyzed using adjusted Cox proportional hazards models. RESULTS: Our analysis identified 48 Single Nucleotide Variants (SNVs) within a 4-kilobase region containing the gene, including nine novel variants. Phasing of variants within the locus revealed 59 unique haplotypes in the Spanish population, which were grouped into five major haplogroups-ε2, ε3A, ε3B, ε4A, and ε4B-including two common haplogroups for each of the ε3 and ε4 isoforms. The ε4A haplogroup was associated with a significant decrease in APOE ε4 protein levels in CSF (p = 0.004), suggesting a regulatory mechanism that may mitigate the toxic gain-of-function effect typically attributed to the ε4 allele. Conversely, the ε3B haplogroup was linked to increased APOE ε3 protein levels in ε3/ε4 carriers (p = 0.025), potentially serving a compensatory role.These effects were independent of overall genotype and remained significant after adjusting for covariates. Both haplogroups (ε4A and ε3B) demonstrated protective effects in the progression from MCI to dementia, underscoring their potential relevance in Alzheimer's disease. CONCLUSIONS: This study provides new insights into the intragenic allelic variability of the gene, demonstrating that intragenic haplogroups within the ε3 and ε4 backgrounds can modulate isoform expression in ways that might modulate AD. Our findings highlight the importance of considering haplotype-specific effects when interpreting the functional impact of and in designing targeted therapeutic strategies. Further research is needed to explore the broader regulatory network of the locus and its interaction with neighboring loci in the 19q13 region.