Exome sequencing in 116 patients with inherited thrombocytopenia that remained of unknown origin after systematic phenotype-driven diagnostic workup

Abstract

Inherited thrombocytopenias (IT) are genetic diseases characterized by low platelet count, sometimes associated with congenital defects or a predisposition to develop additional conditions. Next-generation sequencing has substantially improved our knowledge of IT, with more than 40 genes identified so far, but obtaining a molecular diagnosis remains a challenge especially for patients with non-syndromic forms, having no clinical or functional phenotypes that raise suspicion about specific genes. We performed exome sequencing (ES) in a cohort of 116 IT patients (89 families), still undiagnosed after a previously validated phenotype-driven diagnostic algorithm including a targeted analysis of suspected genes. ES achieved a diagnostic yield of 36%, with a gain of 16% over the diagnostic algorithm. This can be explained by genetic heterogeneity and unspecific genotype-phenotype relationships that make the simultaneous analysis of all the genes, enabled by ES, the most reasonable strategy. Furthermore, ES disentangled situations that had been puzzling because of atypical inheritance, sex-related effects or false negative laboratory results. Finally, ES-based copy number variant analysis disclosed an unexpectedly high prevalence of RUNX1 deletions, predisposing to hematologic malignancies. Our findings demonstrate that ES, including copy number variant analysis, can substantially contribute to the diagnosis of IT and can solve diagnostic problems that would otherwise remain a challenge.

Figure 1.

Pedigrees of families with inherited thrombocytopenia. (AC) Families with pathogenic variants in ABCG8 (A), WAS (B) and GATA1 (C). Double lines indicate consanguineous unions. Platelet count (x10⁹/L) and genotype are reported when available. wt: wt allele; mut: mutated allele.

Figure 2.

RUNX1 deletions. (A) RUNX1 deletions identified by exome sequencing were confirmed by chromosomal microarray analysis. Profiles from probands of Families 44, 66 and 72 (top to bottom panels) are shown. Highlighting indicates deleted regions. The top panel was produced by the UCSC Genome Browser (https://genome.ucsc.edu/) and shows the genomic positions and all genes included in the region. Segregation of deletions was evaluated by real-time polymerase chain reaction in Family.44 (B), Family.66 (C) and Family.72 (D). Genotype is reported when available.