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

PURPOSE: To describe examples of missed pathogenic variants on whole exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing. METHODS: Guided by phenotypic information, three children with negative WES underwent targeted single gene testing. RESULTS: Individual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and an 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 non-coding 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 MRI 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, likely missed due to failure of alignment. CONCLUSIONS: These cases illustrate potential pitfalls of WES/NGS testing, and the importance of phenotype-guided molecular testing in yielding diagnoses.