Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders

Abstract

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.

Figure 1.

Classification of the 92 IPD patients sequenced with a novel HTS platform. Ninety-two unrelated patients with a suspicion of IPD were enrolled in the project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders”. Patients fell into 2 main groups: on the left, a validation group comprising 10 IPD patients harboring known pathogenic variants identified by Sanger sequencing (Online Supplementary Table S1), and on the right, a study group of 82 IPD patients with unknown molecular pathology. DNA from all patients was sequenced with an HTS platform targeting 72 genes (Table 1), as described in the Methods. The identified genetic variants were prioritized and assessed for pathogenicity, as stated in the Methods. IPD: inherited platelet disorder; HTS: high-throughput sequencing.