Prognostic importance of direct assignment of parent-of-origin via long-read genome and epigenome sequencing in retinoblastoma

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Abstract

BACKGROUND. Current clinical sequencing methods cannot effectively detect DNA methylation and allele-specific variation to provide parent-of-origin information from the proband alone. Parent-of-origin effects can lead to differential disease and the inability to assign this in de novo cases limits prognostication in the majority of affected individuals with retinoblastoma, a hereditary cancer with suspected parent-of-origin effects. METHODS. To directly assign parent-of-origin in retinoblastoma patients, genomic DNA was extracted from blood samples for sequencing using a programmable, targeted single-molecule long-read DNA genomic and epigenomic approach. This allowed germline variant calling and simultaneous haplotype-resolved CpG methylation in subjects with familial (n=7) and de novo (n=9) retinoblastoma. RESULTS. Targeted long-read sequencing allowed phasing genomic variation with a differentially methylated region in intron 2 of the RB1 gene to confirm parent-of-origin in known familial samples. Leveraging this approach allowed us to directly assign parent-of-origin rapidly in simple and complex de novo cases from the proband alone. The ability to assign parent-of-origin in all cases of retinoblastoma showed that harboring disease-causing variants on the paternally inherited allele, whether arising familial or de novo, is associated with more advanced cancer staging at presentation and significantly greater risk of chemotherapy failure (P=0.002). CONCLUSION. This study demonstrates the diagnostic potential of multi-omic long-read profiling to unveil the parent-of-origin effect in hereditary cancer. The approach in this work will be instrumental in assigning parent-of-origin to other genetic diseases using local and distant imprinting signals in the genome. FUNDING. National Eye Institute, NIH (K08EY033789); Gerber Foundation; Research to Prevent Blindness

Authors

Andrew W. Stacey, Kenji Nakamichi, Jennifer Huey, Jeffrey Stevens, Natalie Waligorski, Erin E. Crotty, Russell N. Van Gelder, Debarshi Mustafi