The limitation of genetic testing in diagnosing patients suspected for congenital platelet defects

To the Editor: Congenital platelet defects (CPD) are rare disorders of primary hemostasis caused by congenital defects in platelet production or function. Identification of CPDs is challenging due to the lack of awareness resulting in late or missing referrals, the lack of diagnostic criteria, absence or limitations of laboratory tests and poor standardization of the available tests. However, an accurate diagnosis is important for proper counseling andmanagement of patients and to avoid ineffective and potentially harmful treatments due tomisdiagnosis, like idiopathic thrombocytopenic purpura (ITP). DNA-based analysis has become increasingly important for diagnosing CPDs. Genetic analysis can be useful to confirm a suspected phenotypic diagnosis, and to identify patients with an increased risk for associated pathologies, such as myelofibrosis (NBEAL2), renal insufficiency (MYH9) and hematological malignancies (RUNX1). The International Society for Thrombosis and Hemostasis (ISTH) currently recommends to perform genetic analysis as a third-line investigation, that is, after extensive phenotyping and functional analyses have confirmed the presence of a platelet disorder. Recent studies on the efficacy of genetic testing in selected patients with platelet disorders have suggested that genetic analysis could be moved “upward” in the diagnostic approach in order to simplify and hasten the diagnosis of CPDs. However, it remains unclear whether genetic analysis should be performed as a first-line investigation, alongside initial functional analysis of platelet function in unselected patients in whom a congenital platelet disorder is suspected. In the Thrombocytopathy in the Netherlands (TiN) study, we assessed the diagnostic value of genetic analysis performed in parallel with routine laboratory tests in a prospective cohort of patients suspected of having a CPD. Three categories of patients were included in the study: (a) patients suspected of having a CPD based on previous abnormal platelet counts, light transmission aggregometry (LTA) results or platelet ADP content without a molecular diagnosis (n = 96) (b) patients suspected of having a CPD based on a predominantly mucocutaneous bleeding tendency compatible with a CPD, in whom other known causes of bleeding were excluded and in whom previous LTA results were normal (n = 39), and (c) patients suspected of having a CPD based on a predominantly mucocutaneous bleeding tendency compatible with a CPD, in whom other known causes of bleeding were excluded, newly referred for platelet function testing (n = 21). Laboratory tests were performed for platelet count, aggregation response to four agonists, nucleotide content, surface receptor expression with flow cytometry and whole-exome sequencing (WES) with a selected 76 gene panel (Table S1). A CPD was diagnosed when an abnormal platelet count or function was found on at least two separate occasions, of which one was in our diagnostic laboratory. A possible CPD was diagnosed when an abnormal platelet function was found once in our diagnostic laboratory, or when abnormal platelet function test results were inconsistent with previous findings. In line with the American College of Medical Genetics guidelines, a genetic variant was stated to be causal when a (likely) pathogenic variant (class 4 or 5, respectively) was identified in one or more of the selected genes that corresponded to the platelet phenotype. In patients with previously abnormal laboratory results, a CPD was confirmed in 61 of 96 (64%) patients, and a possible CPD was diagnosed in four of 96 (4%) patients. Eight of 96 (8%) patients received a molecular diagnosis, and in 11 of 96 (11%) patients a variant of unknown significance was identified (Table 1). In patients with previously normal LTA results and in newly referred patients, a possible CPD was diagnosed in 10 of 39 (26%) and six of 21 (29%) patients, respectively. No causal genetic variants were identified in these patients. We included several subgroups of patients suspected of having a CPD to properly assess when genetic analysis should be performed in the diagnostic procedure. Our study shows that the diagnostic yield of genetic analysis is limited in patients suspected for a CPD, since only 5% (8/156) of patients received a molecular diagnosis. This is in contrast to the diagnostic rate of 47.8% for platelet count defects, and 26.1% for platelet function defects reported in a recent study. There, 2396 patients with bleeding, thrombotic, and platelet disorders (BTPD) were screened with a panel of 96 BTPD-associated genes, in which the number of platelet associated genes was similar to our gene panel. However, their diagnostic rate included variants of unknown significance, resulting in an overestimation. Leaving out variants of unknown significance strongly reduced the diagnostic rate. The differences between their and our study are also related to patient-selection. Our study reflects the real-life population of patients suspected for a CPD referred to outpatient clinics of hemophilia treatment centers Their study included patients with a previously ascertained pathogenic variant, or patients with phenotypes strongly indicative of a particular disorder on the basis of laboratory abnormalities, with a high likelihood of having an inherited BTPD. In patients with either normal laboratory assays or assays not diagnostic of an established disorder, they reported a diagnostic rate of only 3.2%. Studies performed in the Iberian Peninsula, with a gene panel similar to ours, reported the identification of a molecular defect in 40% and 68% of patients with (suspected) CPDs. Their studies included large numbers of patients with Glanzmann thrombasthenia, © 2019 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.