General research—OtherAuthors: Lilian Cordova1,3, Vahid Akbari1,2, Tiffany Leung4, Katherine Dixon1,5, Kieran O’Neill2, Alexandra Roston1,3, Eugene Cheung3, Chuyi Zheng3, Alshanee Sharma3, Yaoqing Shen2, Janine Senz6, Yanni Wang4,6, Daniel Chan4, Alexandra Fok3, Carol Cremin3, Jennifer Nuk3, Angela Bedard3, Melanie O’Loughlin1,3, Angela Inglis3, Allison Mindlin3, Salma Shickh3, Mary-Jill Asrat Mary-Jill Asrat3, Adam Kahnamelli3, Quan Hong7, Steve Bilobram2, Simon Chan2, Robin Coope2, Eric Chuah 2, Hyun-Wu Lee 2, Yongjun Zhao2, Miruna Bala2, Karen Mungall2, Andy Mungall2, Richard Moore2, Louis Lefebvre1, Brandon Bernard8, Dean Regier7,11, Alice Virani9, Fabio Feldman3, Janessa Laskin6, Marco Marra1,2, Kara Maxwell13, Stephen B. Gruber14, David Schaeffer10,12, Daniel Renouf8,12, Sophie Sun8,3, Stephen Yip10,15, Peter Lansdorp1,4, Steven J.M. Jones1,2, Kasmintan Schrader 1,3Institutions: 1Department of Medical Genetics, University of British Columbia, Vancouver, Canada, 2Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, Canada., 3Hereditary Cancer Program, BC Cancer, Vancouver, Canada., 4Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada., 5Department of Pediatric Pathology and Laboratory Medicine, 6Department of Molecular Oncology, BC Cancer, Vancouver, Canada, 7School of Population and Public Health, University of British Columbia, Vancouver, Canada, 8Medical Oncology, BC Cancer, Vancouver, Canada, 9Ethics and Spiritual Care, Provincial Health Services Authority, 10Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada, 11Canadian Centre for Applied Research in Cancer Control, Cancer Control Research, BC Cancer, Vancouver, Canada, 12Pancreas Centre BC, Vancouver, Canada, 13Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, USA; Abramson Cancer Center, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Corporal Michael Crescenz Veterans Affairs Medical Center, Philadelphia, PA, USA., 14City of Hope National Medical Center, Duarte, California, 15Cancer Genetics and Genomics Lab, Vancouver, Canada, 16*Equal contribution, **Equal contribution

Background and Aim: Assigning the parent-of-origin (PofO) of autosomal variants without parental DNA has long limited both research and clinical practice. Parent-of-Origin-Aware genomic analysis (POAga) overcomes this barrier by integrating chromosome-scale haplotyping with DNA-methylation profiles at imprinted loci, enabling single-sample PofO determination (Akbari, Hanlon et al. Cell Genomics 2023). Blinded testing in retrospective hereditary-cancer cohorts is validating POAga’s analytical accuracy, and prospective pilot clinical implementation is informing its utility for variant curation, risk management, and cascade testing in Tier 1 cancer syndromes. Collectively, these efforts are building a real-world catalogue of fully phased, PofO-resolved genomes from individuals carrying pathogenic variants in cancer-susceptibility genes.

Methods/Clinical Presentation/Preliminary Data: Under research-ethics approval, blood samples are being collected from carriers of pathogenic variants in hereditary-cancer genes representing a range of ages, sexes, ethnicities, cancer histories, and known or unknown parental segregation. Strand-seq and long-read whole-genome sequencing are being used to generate haplotypes, phase imprinted-locus methylation, and assign PofO with POAga.

Results/Discussion/Project Plan and Timeline: To date, 250 samples have yielded 255 independent pathogenic variants distributed across 19 genes: BRCA2 (n = 42), BRCA1 (39), MLH1 (27), MSH2 (29), MSH6 (24), SDHD (22), PMS2 (19), PALB2 (14), ATM (13), CDH1 (9), EPCAM (2), SDHAF2 (2), CHEK2 (3), MUTYH (2), TP53 (4), CDKN2A (1), POT1 (1), RAD51D (1), and SDHC (1). PofO was successfully assigned for 221 variants (86.7%). Concordance with known or reconstructed segregation was 98.6% (218/221). PofO remained unresolved in 34 variants (13.3%), primarily due to polymorphic imprinted methylation or extended runs of homozygosity that impeded phasing.

Conclusions/Requirements for Collaboration: These data support POAga’s ability to accurately assign PofO from routine blood samples in hereditary cancer patients. Continued case accrual and method refinement aim to resolve currently unassigned variants, further define real-world performance, and solidify POAga’s potential to transform genetic risk assessment, clinical management, and cascade testing.

Keywords: Parent-of-Origin-Aware genomic analysis, long-read sequencing, Strand-seq, methylation, imprinting, phasing, Parent-of-Origin-Aware genomic analysis, long-read sequencing, Strand-seq, methylation, imprinting, phasing