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.

A comprehensive targeted next‐generation sequencing panel for genetic diagnosis of patients with suspected inherited thrombocytopenia

“…This detection rate is in keeping with other recent previous large‐scale targeted panel sequencing studies and the application of WES to patients with IT of unknown etiology 4 , 12 , 20 , 21 . One possible explanation for the inflated detection rate for panel based platforms is the relative increase in average read coverage when compared to WES analysis, especially at the point of variation.…”

“…Since the discovery of disease inheritance patterns in disorders such as Bernard Soulier Syndrome (BSS), genetic studies of thrombocytopenia have been a vital tool in determining megakaryocyte and platelet physiology 1. As a result of parallel whole exome and whole genome sequencing over the past 5‐10 years, we are discovering increasing numbers of novel genes and variants with a critical role in platelet production, physiology, and function 2, 3, 4, 5…”

High-throughput sequencing for rapid diagnosis of inherited platelet disorders: a case for a European consensus

“…In Europe, in parallel, a Birmingham-based UK group led by Professor Steve Watson looked specifically at UK patients with IPDs in the Genotyping and Phenotyping of Platelets (GAPP) study. 7 The data obtained from these groups and others world-wide 8,9 helped Bastida et al 4 to compose their gene platform. However, one disadvantage of selected gene platforms is that successful identification of disease-causing variants is limited to the genes tested, and this may explain the 22 cases where the causal gene defect was not found in the Spanish study.…”

“…In the current edition of Haematologica, Bastida et al 4 present a high-quality pilot study introducing highthroughput gene sequencing into the mainstream of genetic diagnostic practice for IPDs. Working in the context of a national collaboration, these authors designed a novel platform to identify gene variants in 82 patients, the majority of whom come from the Iberian Peninsula, with IPDs of previously undiagnosed molecular causes.…”

TBXA2R gene variants associated with bleeding

“…Beyond these rare population variants, which are associated with strong TPα receptor phenotypes, there are a number of reported common or low-frequency variants that are predicted to alter TXA 2 -mediated platelet responses but which have not been functionally analyzed in patients (18–20). For example, TBXA2 missense variants observed in population databases that predict Val80Glu and Ala160Thr substitution in the TPα receptor have been expressed in a novel megakaryocyte-based system in an attempt to recapitulate the impact on human platelet responses (18).…”