Evaluation of the diagnostic capabilities of nanopore long read sequencing and splice variant analysis in muscular dystrophies

Topic:

Pre-Clinical Research

Poster Number: 138

Author(s):

Christine Bruels, PhD, Paul & Sheila Wellstone Muscular Dystrophy Center & Dept of Neurology, Univ of Minnesota Med School, Hannah Little, Paul & Sheila Wellstone Muscular Dystrophy Center & Dept of Neurology, Univ of Minnesota Med School, Audrey Daugherty, University of Minnesota, Seth Stafki, Paul & Sheila Wellstone Muscular Dystrophy Center & Dept of Neurology, Univ of Minnesota Med School, Elicia Estrella, MS, LCGC, Department of Neurology, Boston Children’s Hospital, Emily McGaughy, Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Don Truong, Paul & Sheila Wellstone Muscular Dystrophy Center & Dept of Neurology, Univ of Minnesota Med School, Lynn Pais, Center for Mendelian Genomics, Program in Medical and Population Genetics, Broad Institute, Vijay Ganesh, Center for Mendelian Genomics, Program in Medical and Population Genetics, Broad Institute, Anne O'Donnell-Luria, MD, PhD, Center for Mendelian Genomics, Program in Medical and Population Genetics, Broad Institute, Heather Stalker, Division of Genetics, Department of Pediatrics, University of Florida College of Medicine, Yang Wang, PerkinElmer Genomics, Christin Collins, PerkinElmer Genomics, Andrea Behlmann, PerkinElmer Genomics, Richard Lemmers, Department of Human Genetics, Leiden University Medical Center, Silvère van der Maarel, Department of Human Genetics, Leiden University Medical Center, Regina Laine, MSN, PNP-BC, CNRN, Department of Neurology, Boston Children’s Hospital, Partha Ghosh, MD, Department of Neurology, Boston Children’s Hospital, Basil Darras, MD, Department of Neurology, Boston Children’s Hospital, Carla Zingariello, MD, Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Christina Pacak, Paul & Sheila Wellstone Muscular Dystrophy Center & Dept of Neurology, Univ of Minnesota Med School, Louis Kunkel, Division of Genetics and Genomics, Boston Children’s Hospital, Peter Kang, MD, University of Minnesota

Background: Many patients with muscular dystrophies remain genetically undiagnosed despite clinical diagnostic testing including second-generation short read sequencing (SRS) and standard variant analyses of that sequence data.

Objectives: We propose that a significant proportion of these undiagnosed individuals harbor either structural variants (SVs) or cryptic splice sites which are not easily detected by SRS and exome analysis alone. Nanopore long-read sequencing (LRS), a form of third-generation whole genome sequencing, can be used to identify a higher proportion of such SVs and cryptic splice sites in these unsolved families.

Results: We enrolled 11 individuals with muscular dystrophy who lacked complete genetic diagnoses after clinical genetic testing and one asymptomatic individual with a potential DMD duplication. We performed nanopore LRS on genomic DNA samples from these individuals. Nanopore LRS detected likely pathogenic SVs in 3 of these families, in the DMD and LAMA2 genes. Two of these SVs were previously undetected. In addition, we confirmed that the asymptomatic individual harbored a duplication in DMD that was in tandem rather than translocated as initially suspected, and we confirmed a likely pathogenic single nucleotide variant (SNV) in SMCHD1 in another family. Nanopore LRS also detected intronic SNVs in DMD that are predicted to alter splicing in 2 families. All SNVs that were previously detected on clinical genetic testing in these individuals were confirmed on nanopore LRS.

Conclusions: Overall, nanopore LRS identified or confirmed pathogenic variants in all families studied, and identified previously undetected pathogenic SVs and SNVs in 5 of the 11 symptomatic families. This study highlights the utility of nanopore LRS to identify and characterize a broad range of pathogenic variants in individuals with muscular dystrophy, from SNVs to SVs. This technology has the potential to provide a more streamlined means of obtaining complete molecular diagnoses for patients with muscular dystrophy and other Mendelian disorders.