Ribosome dysfunction underlies SLFN14-related thrombocytopenia.

Pathogenic missense variants in SLFN14, which encode an RNA endoribonuclease protein that regulates ribosomal RNA (rRNA) degradation, are known to cause inherited thrombocytopenia (TP) with impaired platelet aggregation and adenosine triphosphate secretion. Despite mild laboratory defects, the patients displayed an obvious bleeding phenotype. However, the function of SLFN14 in megakaryocyte (MK) and platelet biology remains unknown. This study aimed to model the disease in an immortalized MK cell line (imMKCL) and to characterize the platelet transcriptome in patients with the 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; however, this was absent in undifferentiated imMKCL cells and granulocytes. Total platelet RNA was sequenced in 2 patients and 19 healthy controls. Differential gene expression analysis yielded 2999 and 2888 significantly (|log2 fold change| >1, false discovery rate <0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched in any biological pathway, whereas upregulated genes were enriched in pathways involved in (mitochondrial) translation and transcription, with a significant upregulation of 134 ribosomal protein genes (RPGs). The upregulation of mitochondrial RPGs through increased mammalian target of rapamycin complex 1 (mTORC1) signaling in SLFN14 K219N MK seems to be a compensatory response to rRNA degradation. 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 is the disease mechanism for SLFN14-related TP.

Hemostatic phenotypes and genetic disorders.

This review is focused on genetic regulators of bleeding and thrombosis with a focus on next-generation sequencing (NGS) technologies for diagnosis and research of patients with inherited disorders. The molecular diagnosis of hemostatic phenotypes relies on the detection of genetic variants in the 99 curated disease-causing genes implicated for bleeding, platelet, and thrombotic disorders through the use of multigene panel tests. In this review, we will provide an overview of the advantages and disadvantages of using such multigene panel tests for diagnostics. During the past decade, NGS technologies have also been used for the gene discovery of 32 novel genes involved in inherited hemostatic phenotypes. We will provide a brief overview of these genes and discuss what information (eg, linkage, consanguinity, multiple index cases with similar phenotypes, mouse models, and more) was used to support the gene discovery process. Next, we provide examples on how RNA sequencing is useful to explore disease mechanisms of novel and often unexpected genes. This review will summarize the important findings concerning NGS technologies for diagnostics and gene discovery that were presented at the ISTH 2021 conference. Finally, future perspectives in our field mainly deal with finding the needle in the haystack for some still unexplained patients and the need for exploring the noncoding gene space and rapid disease validation models.

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

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. An 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.