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A comprehensive iterative approach is highly effective in diagnosing individuals who are exome negative



Sixty to seventy-five percent of individuals with rare and undiagnosed phenotypes remain undiagnosed after exome sequencing (ES). With standard ES reanalysis resolving 10–15% of the ES negatives, further approaches are necessary to maximize diagnoses in these individuals.


In 38 ES negative patients an individualized genomic–phenotypic approach was employed utilizing (1) phenotyping; (2) reanalyses of FASTQ files, with innovative bioinformatics; (3) targeted molecular testing; (4) genome sequencing (GS); and (5) conferring of clinical diagnoses when pathognomonic clinical findings occurred.


Certain and highly likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping, and targeted testing identifying variants that were undetected or not prioritized on prior ES. GS diagnosed 3/18 individuals with structural variants not amenable to ES. Additionally, tentative diagnoses were made in 3 (8%), and in 5 individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%).


Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without GS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.

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This work has received support by the National Institutes of Health (NIH) Common Fund through the Office of Strategic Coordination/Office of the NIH Director (U01HG007672, to Shashi V and Goldstein DB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Correspondence to Vandana Shashi MD, MBBS.

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David Goldstein is a founder of and holds equity in Pairnomix and Praxis, serves as a consultant to AstraZeneca, and has research supported by Janssen, Gilead, Biogen, AstraZeneca, and UCB. The remaining authors declare no conflicts of interest.

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  • Exome sequencing
  • Genome sequencing
  • Undiagnosed diseases
  • Rare diseases
  • Phenotyping

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Fig. 1