Use of Targeted High-Throughput Sequencing for Genetic Classification of Patients with Bleeding Diathesis and Suspected Platelet Disorder

Abstract

Inherited platelet disorders (IPD) form a rare and heterogeneous disease entity that is present in about 8% of patients with non-acquired bleeding diathesis. Identification of the defective cellular pathway is an important criterion for stratifying the patient's individual risk profile and for choosing personalized therapeutic options. While costs of high-throughput sequencing technologies have rapidly declined over the last decade, molecular genetic diagnosis of bleeding and platelet disorders is getting more and more suitable within the diagnostic algorithms. In this study, we developed, verified, and evaluated a targeted, panel-based next-generation sequencing approach comprising 59 genes associated with IPD for a cohort of 38 patients with a history of recurrent bleeding episodes and functionally suspected, but so far genetically undefined IPD. DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS, RBM8A, FERMT3, P2YR12, and MYH9 served as controls during the validation process. In 40% of 35 patients analyzed, we were able to finally detect 15 variants, eight of which were novel, in 11 genes, ACTN1, AP3B1, GFI1B, HPS1, HPS4, HPS6, MPL, MYH9, TBXA2R, TPM4, and TUBB1, and classified them according to current guidelines. Apart from seven variants of uncertain significance in 11% of patients, nine variants were classified as likely pathogenic or pathogenic providing a molecular diagnosis for 26% of patients. This report also emphasizes on potentials and pitfalls of this tool and prospectively proposes its rational implementation within the diagnostic algorithms of IPD.

Curated disease‐causing genes for bleeding, thrombotic, and platelet disorders: Communication from the SSC of the ISTH

“…However, over the past decade, high‐throughput sequencing has become the primary means of identifying disease‐causing genetic variants . Different diagnostic gene panel tests for BTPD have been developed using targeted or exome sequencing . Interestingly, when comparing the gene content of these different genetic panel tests, significant differences were observed.…”

Strengths and limitations of high‐throughput sequencing for the diagnosis of inherited bleeding and platelet disorders

“…Different HTS studies for diagnostics of BPD have been published but for this review, we have only focussed on the studies that have used the guidelines for variant classification as formulated by the American College of Medical Genetics (ACMG) in 2015 [10]. Figure 2 provides an overview of the diagnostic rates obtained in these studies that however differ in terms of used HTS methods (TS or WES), studied pathologies (platelet disorders, coagulation disorders and bleeding of unknown etiology) and the number of genes and patients included [11][12][13][14][15][16][17][18][19][20][21]. In addition, these studies have used different inclusion criteria ranging from the selection of only patients with a known or suspected etiology (studies with typically high diagnostic rates) to patients with bleeding of unknown etiology and normal laboratory parameters (studies with very low diagnostic rates).…”

“…Other HTS studies have also reported variants in more than one gene but without further specifications if these had a clinical impact for the patient or family [11,[14][15][16][17][18]20]. Such examples of mostly digenic inheritance have recently also been documented in case reports.…”

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.…”