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PgmNr 201: Identifying diagnoses beyond the exome: Lessons from challenging cases with compelling clinical phenotypes.

A. O'Donnell-Luria 1,2,3; M.H. Wojcik 1,2; K.R. Chao 1,3; J.K. Goodrich 1,3; L.S. Pais 1,3; E. England 1,3; E.G. Seaby 1,3; A.B. Byrne 1,4,5; B.B. Cummings 1,3; R.L. Collins 1,3; M. Lek 6; L. Gallacher 7,8; T.Y. Tan 7,8; K.M. Bujakowska 9; E.A. Pierce 9; P.B. Agrawal 1,2; C.A. Walsh 1,2; J.M. Verboon 1,10,11; V.G. Sankaran 1,10,11; C. Barnett 12; H. Scott 4,13; W.K. Chung 14; E.A. Estrella 2; C.C. Bruels 15; P.B. Kang 15,16; S. Pajusalu 6,17; K. Ounap 17; A.K. Lovgren 1,3; H.L. Rehm 1,3; D.G. MacArthur 1,3

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1) Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA; 2) Division of Genetics and Genomics, Boston Children’s Hospital, Boston, MA; 3) Analytic and Translational Genomics Unit (ATGU), Massachusetts General Hospital, Boston, MA; 4) Department of Genetics and Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia; 5) School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia; 6) Department of Genetics, Yale School of Medicine, New Haven, CT; 7) Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Melbourne, Australia; 8) Department of Paediatrics, University of Melbourne, Melbourne, Australia; 9) Ocular Genomics Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA; 10) Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA; 11) Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; 12) Paediatric and Reproductive Genetics Unit, Women's and Children's Hospital/SA Pathology, Adelaide, Australia; 13) Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, Australia; 14) Division of Molecular Genetics, Department of Pediatrics, Department of Medicine, Columbia University Irving Medical Center, New York, NY; 15) Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL; 16) Genetics Institute and Myology Institute, University of Florida, Gainesville, FL; 17) Department of Clinical Genetics, Institute of Clinical Medicine, University of Tartu and Tartu University Hospital, Tartu, Estonia

Next-generation sequencing has revolutionized clinical genetics, yet our ability to detect pathogenic variants remains incomplete. In many cases, clinicians have strong phenotypic or biochemical data implicating a specific genetic diagnosis but molecular testing, including gene sequencing and deletion/duplication testing, is unrevealing. Should we trust our clinical judgment? Is this testing “complete”? What should we do next?

Through the Broad Institute Center for Mendelian Genomics, we have sequenced and analyzed >5,000 rare disease families including many for which there was a strong clinical suspicion but initial negative testing. Here we review a large series of these cases for which we ultimately identified a diagnosis with exome, genome, and/or transcriptome sequencing. We characterize the spectrum of diagnoses found across ~30 cases and the reasons that the molecular diagnosis was originally missed. Conditions include many phenotypically well-characterized disorders such as Duchenne and Ullrich muscular dystrophy, Axenfeld Rieger, Meckel Gruber, and Marfan syndromes.

Variants in high GC content regions were initially missed by clinical exome (FOXC1). Small structural variants (including a partial exon deletion of FBN1) were missed on original testing. Copy neutral inversions (DMD) were entirely missed by Sanger sequencing, exome, array, and MLPA, and ultimately detected by capturing the breakpoints by genome sequencing. Noncoding variants that result in altered splicing including exon skipping or the creation of pseudoexons were detected by RNA-seq (COL6A1). In one case, an epimutation resulted in gene silencing (MMACHC). We find the clinical suspicion was correct in most cases, but the underlying causal variant(s) were cryptic to all standard genetic testing methodologies. A small number of cases were due to a phenocopy with a similar condition found to be the eventual diagnosis.

Overall, this cohort provides a view of the full allelic spectrum of pathogenic variants for a set of very well-characterized Mendelian phenotypes and disease genes, demonstrating several classes of pathogenic variation missed by standard genetic testing methodologies. These examples demonstrate the importance of careful clinical phenotyping in guiding the analysis of genetic results, particularly by prioritizing loci for deeper study. Finally, we describe our best practices used to identify elusive pathogenic variants to maximize diagnostic yield.