Whole exome sequencing identifies a mutation in thrombomodulin as the genetic cause of a suspected platelet disorder in a family with normal platelet function.

Here, we describe a mother and son with a lifelong bleeding tendency and posttraumatic bleeding who were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study with a suspected platelet function disorder. However, despite a clinically significant bleeding score, both had normal platelet counts and normal platelet function. The patients' blood was analyzed by light transmission aggregometry and genotyping by whole exome sequencing, as outlined by the GAPP study. Approximately 25 000 genetic variants were found for each patient as a result of sequencing and were filtered using a specialized bioinformatics pipeline. A heterozygous variant displaying autosomal dominant inheritance (c.1611 C>A) was found in the gene THBD which encodes the glycoprotein thrombomodulin. This sequence change results in a stop codon (p.Cys537Stop) and truncation of the protein and has been previously described in two other families with bleeding events which suggests it may be a recurrent mutation. In summary, this study shows that patients with a suspected platelet disorder but who present with a normal pattern of platelet aggregation should be investigated for defects in nonplatelet genes.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Recurrent activating STAT5B N642H mutation in myeloid neoplasms with eosinophilia.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.

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

BACKGROUND: Inherited 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. AIMS: To employ a targeted next-generation sequencing platform to improve efficiency of diagnostic testing and reduce overall costs. METHODS: We have developed an IT-specific gene panel as a pre-screen for patients prior to WES using the Agilent SureSelectQXT transposon-based enrichment system. RESULTS: Thirty-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 CONCLUSION: Although 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.