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

Abstract

BackgroundInherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet counts and often disproportionate bleeding with over 30 genes currently implicated. Previously the UK‐GAPP study using whole exome sequencing (WES) identified a pathogenic variant in 19 of 47 (40%) patients of which 71% had variants in genes known to cause IT.AimsTo employ a targeted next‐generation sequencing platform to improve efficiency of diagnostic testing and reduce overall costs.MethodsWe have developed an IT‐specific gene panel as a pre‐screen for patients prior to WES using the Agilent SureSelectQXT transposon‐based enrichment system.ResultsThirty‐one patients were analyzed using the panel‐based sequencing, of which; 10% (3/31) were identified with a classified pathogenic variant, 16% (5/31) were identified with a likely pathogenic variant, 51% (16/31) were identified with variants of unknown significance, and 23% (7/31) were identified with either no variant or a benign variant.Discussion and ConclusionAlthough requiring further clarification of the impact of the genetic variations, the application of an IT‐specific next generation sequencing panel is an viable method of pre‐screening patients for variants in known IT‐causing genes prior to WES. With an added benefit of distinguishing IT from idiopathic thrombocytopenic purpura (ITP) and the potential to identify variants in genes known to have a predisposition to hematological malignancies, it could become a critical step in improving patient clinical management.

Inherited thrombocytopenias: history, advances and perspectives

“… 123 As the number of causal genes for all forms of inherited platelet disorders increased, BRIDGE-BPD and GAPP both put together platforms to test patients for potentially pathogenic variants against previously identified target genes concentrating on exomes, untranslated regions and selected intronic regions. 124 – 127 Indeed, GAPP designed a gene panel specific for inherited thrombocytopenias. 127 In contrast, the ThromboGenomics Consortium (Department of Haematology, University of Cambridge, UK) included a limited number of genes causal for other blood and thrombotic disorders; the panel was regularly updated and a large cohort of 2,396 patients screened.…”

“… 124 – 127 Indeed, GAPP designed a gene panel specific for inherited thrombocytopenias. 127 In contrast, the ThromboGenomics Consortium (Department of Haematology, University of Cambridge, UK) included a limited number of genes causal for other blood and thrombotic disorders; the panel was regularly updated and a large cohort of 2,396 patients screened. 125 , 126 The use of NGS and high-throughput procedures for diagnosing platelet disorders including thrombocytopenias has quickly expanded worldwide as is illustrated by reports from Italy, Japan, Spain, France, Holland and Scandinavia as well as North America.…”

“… 149 Despite current technological advances, a high proportion of new cases of inherited thrombocytopenia remain without diagnosis; this proportion is often estimated to be around 50% but can be as high as 70% or more depending on the extent to which patients with easily identified disorders have been prescreened. 127 , 128 , 150 , 151 Assigning a significance to the variants is key, because in the absence of confirmation with follow-up biological studies, including the use of mouse, zebrafish or Drosophila models or site-directed mutagenesis in megakaryocyte-related cells, the alternative is to use sophisticated bioinformatics; new variants are being variously graded as highly significant and likely pathogenic to those of unknown significance. 126 , 131 An early meta-analysis of genome-wide association studies identified 68 common single-nucleotide variants that influenced platelet count or platelet volume; genes already linked to inherited thrombocytopenia, such as THPO , GPIBA , TUBB1 and the pro-survival gene, BAK , were included, but the majority involved genes with no known link to megakaryopoiesis.…”

Genetic classification and confirmation of inherited platelet disorders: current status in Korea

“…To enable more accurate and definitive diagnostics, advanced genetic approaches, including nextgene ration sequencing, are required for patients with suspected IPD. 79,80) Although the NGS panel for IPDs has not yet been commercialized and popularized in Korea, interest in IPDs is increasing. As described in Table 2, Korean Pediatric Hematology Oncology Group recently attempted the genetic confirmation of IPDs using targeted exome sequencing as a multicenter study.…”

Diagnosis of Inherited Platelet Disorders on a Blood Smear

“…The spread of next-generation sequencing (NGS) greatly contributed to the unraveling of the genetic basis of several forms of IPD, and thus immediately became a significant diagnostic tool in the field. Using a small amount of blood with minor risk of pre-analytical artifacts, it can provide patients with a highly accurate diagnosis [4][5][6][7][66][67][68][69][70][71][72]. As it becomes cheaper to perform, it is not inconceivable that NGS could become the standard above other diagnostic techniques.…”