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

Abstract The APOE gene is a key genetic determinant of Alzheimer’s disease (AD) risk. While the pathogenic effects of the ε4 allele are well established, the contribution of distinct haplotypic backgrounds within ε3 and ε4 remains poorly understood. Here, we used long-read Oxford Nanopore sequencing to identify and phase single nucleotide variants (SNVs) across a 3.9 kb region spanning APOE locus (3’ to 5’ UTR) in a large memory-clinic based cohort (N=1,266). We identified 48 SNVs, including nine novel variants, and reconstructed haplotypes through direct phasing. Our analysis revealed a structured haplotypic architecture, with two major configurations for ε3 (ε3a and ε3b) and ε4 (ε4a and ε4b), defined by the promoter variant rs405509 in phase with the canonical isoform-defining variants rs429358 and rs7412. The observed homoplasy in the rs405509, likely arising from a recombination or gene conversion event, complicates phasing by standard genetic approaches, highlighting the value of long-read sequencing for accurate characterization of APOE haplotypic diversity. Importantly, the ε4a haplogenotype was associated with reduced risk of progression from mild cognitive impairment to AD dementia compared to ε4b (APOE-stratified meta-analysis HR[95%CI]=0.564[0.400–0.794], p=0.001), while ε3b showed a borderline protective effect towards AD biomarker positivity in ε3ε3 subjects (OR[95%CI]=0.76[0.57–1.00], p=0.053). Notably, haplogenotypes had no detectable effect on CSF APOE levels, indicating that haplotype-dependent effects are not mediated by differences in total APOE abundance. This study provides new insights into the intragenic allelic variability of the APOE gene, highlighting 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. Competing Interest Statement The authors have declared no competing interest. Funding Statement Authors acknowledge the support of the Agency for Innovation and Entrepreneurship (VLAIO) grant N° PR067/21 and Janssen for the HARPONE project and the ADAPTED project the EU/EFPIA Innovative Medicines Initiative Joint Undertaking Grant N° 115975. Also, the Spanish Ministry of Science and Innovation, Proyectos de Generación de Conocimiento grants PID2021-122473OA-I00, PID2021-123462OB-I00 and PID2019-106625RB-I00. ISCIII, Acción Estratégica en Salud integrated in the Spanish National R+D+I Plan and financed by ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER ‘Una manera de hacer Europa’) grants PI17/01474, PI19/00335, PI22/01403 and PI22/00258. The support of CIBERNED (ISCIII) under the grants CB06/05/2004 and CB18/05/00010. The support from PREADAPT project, Joint Program for Neurodegenerative Diseases (JPND) grant N° AC19/00097, and from DESCARTES project, German Research Foundation (DFG). The support of Fundación bancaria 'La Caixa', Fundación ADEY, Fundación Echevarne and Grifols SA (GR@ACE project). ACF received support from the Instituto de Salud Carlos III (ISCIII) under the grant Sara Borrell (CD22/00125). PGG was supported by CIBERNED employment plan (CNV-304-PRF-866). IdR is supported by the ISCIII under the grant FI20/00215. AR is also supported by STAR Award. University of Texas System. Tx, United States, The South Texas ADRC. National Institute of Aging. National Institutes of Heath. USA. (P30AG066546), the Keith M. Orme and Pat Vigeon Orme Endowed Chair in Alzheimer's and Neurodegenerative Diseases (2024-2025) and Patricia Ruth Frederick Distinguished Chair for Precision Therapeutics in Alzheimer's and Neurodegenerative Diseases (2025-2028). Part of this study was also funded by the German Federal Ministry of Education and Research (BMBF) within the EU program JPND (Grant number: PreADAPT project 01ED2007A) and by BMBF (Grant numbers: DESCARTES project 01EK2102B and 01EK2102A). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Informed consents for lumbar puncture, genetic analyses, and the anonymized use of clinical records for research were approved by the Ethics Committee of the Hospital Clinic i Provincial de Barcelona (Barcelona, Spain) in accordance with Spanish biomedical laws (Law 14/2007, of July 3, on biomedical research; Royal Decree 1716/2011, of November 18). The study also adhered to the recommendations of the Declaration of Helsinki. The Harpone project, encompassing all the research described in this manuscript, has also been approved by the Ethics Committee of the Hospital de Bellvitge (Barcelona, Spain) (Acta 04/21). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes This revised version of the manuscript incorporates an updated analytical framework, including the joint analysis, genotype-stratified meta-analysis, and interaction models based on APOE haplotypes. We also introduce a new analysis including APOE Lumipulse measurements to validate the effect of APOE haplogroups on CSF APOE levels. All statistical analyses were conducted on R v4.1.1. In addition, the manuscript clarity and readability has been improved, and a more appropriate title has been selected. Data Availability All data produced in the present study are available upon reasonable request to the authors