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

Scientific Research Question: Inherited platelet disorders (IPD) comprise rare and heterogeneous disorders that are found in about eight percent of patients with non-acquired bleeding diathesis. Identification of the underlying defective cellular mechanism is an important criterion for stratifying the patient's individual risk profile and for choosing personalised therapeutic options. In this study, we aimed to identify molecular diagnoses in a cohort of patients with suspected, but so far genetically undefined IPD by using high-throughput sequencing techniques.

Methodology: We developed, verified, and evaluated a targeted, panel-based next generation sequencing (NGS) approach comprising 59 genes previously associated with IPD. Twenty-six children or adolescents and twelve adults were included into the study. Each patient had been evaluated in specialised centres in Germany and Austria due to a mild to severe bleeding diathesis and considered to have a familial, syndromal or functionally diagnosed platelet disorder with early onset in life. Of these, 18 patients (47%) suffered from isolated thrombocytopenia, 16 (42%) from a platelet function disorder, and four from a combined defect (11%). After initial classification using in silico filtering programmes and gene databases (phase#1), variants were classified in a second step with respect to clinical phenotype, segregation analysis, literature, in vitro data, and animal models (phase#2). DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS, RBM8A, FERMT3, P2YR12, and MYH9 genes served as controls during the validation process (Figure 1).

Findings: The sequencing procedure and subsequent data processing confirmed the known mutations in the five DNA samples including a complete heterozygous loss of the RBM8A gene. Three out of 38 data sets failed quality control and had thus to be excluded from further analysis. In 40% of the 35 analysed patients, we detected 17 variants (phase#1 variant assessment in the 12 genes ACTN1, AP3B1, FLNA, GFI1B, HPS1, HPS4, HPS6, MPL, MYH9, TBXA2R, TPM4, and TUBB1), of which nine were novel. In five cases, variants of uncertain significance (VUS, class 3) were categorized to be likely benign (class 2) or likely pathogenic (class 4) after further functional assays or segregational analyses (phase#2 variant assessment). Finally, nine variants were classified as likely pathogenic (class 4) or pathogenic (class 5) providing a molecular diagnosis for 26% of patients (Figure 1).

Conclusion: The authors consider that the presented NGS gene panel is capable of expanding molecular genetic testing to patients that have failed standard approaches to functionally diagnose or to genetically define IPD. The study demonstrates that, after careful variant assessment according to current guidelines, this technique substantially enriches diagnostic algorithms at definite steps and thus improves patient care.

Fig. 1 Flow chart of analysis procedure and variant assessment.

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