Inherited bleeding and platelet disorders (BPD) are highly heterogeneous and their diagnosis involves a combination of clinical investigations, laboratory tests and genetic screening. This review will outline some of the challenges that geneticists and experts in clinical hemostasis face when implementing high-throughput sequencing (HTS) for patient care. We will provide an overview of the strengths and limitations of the different HTS techniques that can be used to diagnose BPD. A HTS test is cost efficient and expected to increase the diagnostic rate with a possibility to detect unexpected diagnoses and decrease the turnaround time to diagnose patients. On the other hand, technical shortcomings, variant interpretation difficulties and ethical issues related to HTS for BPD will also be documented. Delivering a genetic diagnosis to patients is highly desirable to improve clinical management and allow family counseling, but making incorrect assumptions about variants and providing insufficient information to patients before initiating the test could be harmful. Data-sharing and improved HTS guidelines are essential to limit these major drawbacks of HTS.

Essentials• Gene panel tests are used for diagnostics of inherited bleeding and thrombotic disorders.• International data sharing is essential for variant curation and gene discovery studies.• Next-generation sequencing (NGS) contributes to gene discovery, but data interpretation remains difficult.• Data from the ISTH 2021 congress illustrates the added value of NGS methods. | INTRODUC TI ONPathways involved in hemostasis are very complex and involve interplays between platelets, coagulation, and fibrinolysis. 1 Their normal function is required to prevent excessive blood loss upon vessel injury (bleeding) or, on the other hand, the formation of blood clots (thrombosis). The critical balance between these pathways is regulated by diverse proteins, and genetic variants in genes that encode for these proteins are known to cause inherited forms of bleeding or thrombosis. This review focuses on insights related to this genetic regulation of bleeding and thrombosis.

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Pathogenic missense variants in SLFN14, which encodes an RNA endoribonuclease protein that regulates rRNA degradation, are known to cause inherited thrombocytopenia with impaired platelet aggregation and ATP secretion. Despite rather mild laboratory defects, these patients display an obvious bleeding phenotype. The function of SLFN14 in megakaryocyte (MK) and platelet biology is unknown. This study aims to model the disease in the immortalized megakaryocyte cell line imMKCL and characterize the platelet transcriptome in patients with a SLFN14 K219N variant. MK derived from heterozygous and homozygous SLFN14 K219N imMKCL and stem cells of blood from patients mainly presented with a defect in proplatelet formation and mitochondrial organization. SLFN14 defective platelets and mature MK showed signs of rRNA degradation while this was absent in undifferentiated imMKCL cells and granulocytes. Total platelet RNA was sequenced for two patients and 19 healthy controls. Differential gene expression analysis yielded a total of 2999 and 2888 significantly (|log2FC|>1, FDR<0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched for any biological pathway while upregulated genes were enriched for pathways involved in (mitochondrial) translation and transcription with a significant upregulation of 134 ribosomal protein genes (RPG). Upregulation of (M)RPG through increased mTORC1 signaling in SLFN14 K219N MK seems to be a compensatory response to rRNA degradation. Indeed, mTORC1 inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTORC1 coordinated ribosomal biogenesis as the disease mechanism for SLFN14-related thrombocytopenia.

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