Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

Duke Scholars

Author Sarah Rapisardo Long Pathology

Author Allyn McConkie-Rosell Pediatrics, Medical Genetics

Author Sujay Mansukhlal Kansagra Pediatrics, Neurology

Author Mays Antoine Dairi Ophthalmology, Neuro-Ophthalmology

Author Jane Sanders Bellet Dermatology

Author Martha Ann Keels Surgery

Author Joan Mary Jasien Pediatrics, Neurology

Author Peter George Kranz Radiology, Neuroradiology

Author Richard Joseph Noel Pediatrics, Gastroenterology, Hepatology and Nutrition

Author Shashi Kumar Nagaraj Pediatrics, Nephrology

Author Robert Lark Orthopaedic Surgery

Author Vandana Shashi Pediatrics, Medical Genetics