The major spliceosome includes five small nuclear RNA (snRNAs), U1, U2, U4, U5 and U6, each of which is encoded by multiple genes. We recently showed that mutations in RNU4-2, the gene that encodes the U4-2 snRNA, cause one of the most prevalent monogenic neurodevelopmental disorders. Here, we report that recurrent germline mutations in RNU2-2 (previously known as pseudogene RNU2-2P), a 191-bp gene that encodes the U2-2 snRNA, are responsible for a related disorder. By genetic association, we identified recurrent de novo single-nucleotide mutations at nucleotide positions 4 and 35 of RNU2-2 in nine cases. We replicated this finding in 16 additional cases, bringing the total to 25. We estimate that RNU2-2 syndrome has a prevalence of ~20% that of RNU4-2 syndrome. The disorder is characterized by intellectual disability, autistic behavior, microcephaly, hypotonia, epilepsy and hyperventilation. All cases display a severe and complex seizure phenotype. We found that U2-2 and canonical U2-1 were similarly expressed in blood. Despite mutant U2-2 being expressed in patient blood samples, we found no evidence of missplicing. Our findings cement the role of major spliceosomal snRNAs in the etiologies of neurodevelopmental disorders.

Platelets are generated and released into the bloodstream from their precursor cells, megakaryocytes that reside in the bone marrow. Though platelets have no nucleus or DNA, they contain a full transcriptome that, during platelet formation, is transported from the megakaryocyte to the platelet. It has been described that transcripts in platelets can be translated into proteins that influence platelet response. The platelet transcriptome is highly dynamic and has been extensively studied using microarrays and, more recently, RNA sequencing (RNA-seq) in relation to diverse conditions (inflammation, obesity, cancer, pathogens and others). In this review, we focus on bulk and single-cell RNA-seq studies that have aimed to characterize the coding transcriptome of healthy megakaryocytes and platelets in humans. It has been noted that bulk RNA-seq has limitations when studying in vitro-generated megakaryocyte cultures that are highly heterogeneous, while single-cell RNA-seq has not yet been applied to platelets due to their very limited RNA content. Next, we illustrate how these methods can be applied in the field of inherited platelet disorders for gene discovery and for unraveling novel disease mechanisms using RNA from platelets and megakaryocytes and rare disease bioinformatics. Next, future perspectives are discussed on how this field of coding transcriptomics can be integrated with other next-generation technologies to decipher unexplained inherited platelet disorders in a multiomics approach.

Thrombomodulin (TM) expressed on endothelial cells regulates coagulation. Specific nonsense variants in the TM gene, THBD, result in high soluble TM levels causing rare bleeding disorders. In contrast, although THBD variants have been associated with venous thromboembolism, this association remains controversial. A multigene panel was used to diagnose 601 patients with inherited bleeding or thrombotic disorders. This resulted in the identification of 8 THBD variants for 6 patients with a thrombotic (C175S, A282P, L433P, P501L, G502R, and P508L) and 2 patients with a bleeding (P260A and T478I) phenotype. These were all classified as variants of uncertain significance, and we here aimed to assess their functional role in coagulation. For this purpose, soluble and cell membrane–bound recombinant TM were produced in Expi293F cells. L433P TM showed a marked decrease in the inhibition of thrombin generation and complete inhibition of protein C and thrombin activatable fibrinolysis inhibitor (TAFI) activation. Soluble C175S TM showed decreased inhibition of thrombin generation and protein C activation, whereas no effect was observed for cell membrane-bound recombinant TM. For the other TM variants, no effect on thrombin generation, protein C, or TAFI activation could be observed. Surface plasmon resonance analysis showed no thrombin-TM binding in the presence of L433P because this residue is located at their interaction site. In conclusion, our study shows the functional effects of L433P TM and potentially C175S TM, which are compatible with an increased thrombosis risk. THBD variants are rare but can be relevant to both bleeding and thrombosis. Functional assays for these variants are critical to understand their roles.

Platelets are anucleate cells produced in the bone marrow and derived from large progenitor cells called megakaryocytes (MKs). Platelets receive RNA transcripts from their progenitorial MKs during thrombopoiesis. However, the correspondence between platelet and MK transcriptomes is poorly understood, particularly in the context of germline mutations that cause platelet formation defects or thrombocytopenia. We have studied the effects of two such mutations on MK and platelet transcriptomes. We generated immortalized MK cell line (imMKCL)‐based models of Bernard–Soulier syndrome and IKZF5 ‐related thrombocytopenia. MKs derived from imMKCLs with either a homozygous deletion of GP9 ( GP9 ^−/− ) or a heterozygous Y121F variant in IKZF5 ( IKZF5 ^WT/Y121F ) exhibited reduced proplatelet formation (reductions of 96% and 57%, respectively). Platelets from patients with either GP9 ^−/− or IKZF5 ^WT/Y121F genotypes had broad transcriptomic dysregulation, suggesting that (pro)platelet formation defects due to mutations in glycoprotein receptor and transcription factor genes such as GP9 and IKZF5 already affect the MK transcriptome. RNA‐seq data from MKs at four stages of differentiation revealed widespread but distinct changes in expression over time between the GP9 ^−/− and the IKZF5 ^WT/Y121F genotypes. Dysregulated genes in GP9 ^−/− MKs were enriched for RNA metabolism and actin/tubulin folding pathways, whereas those in IKZF5 ^WT/Y121F MKs were enriched for cell cycle pathways. Most of these genes were also dysregulated in the platelets of patients with the corresponding diseases. Our results suggest that patients with inherited forms of thrombocytopenia present with specific transcriptomic changes during platelet formation.

Glanzmann Thrombasthenia (GT) is characterized by absent platelet aggregation in response to all agonists except ristocetin and is caused by recessive inactivating variants in ITGA2B or ITGB3. While GT patients typically are described as having normal platelet counts, autosomal dominant activating variants in ITGA2B or ITGB3 cause macrothrombocytopenia. Interestingly, in our cohort of 16 GT patients, eight consistently exhibited platelet counts at the lower end of the normal range. We studied the role of integrin αIIbβ3 in platelet formation using megakaryocytes (MKs) derived from genetically modified immortalized megakaryocyte cell lines (imMKCLs), focusing on two modifications of ITGB3: ITGB3-/- (inactivating) and ITGB3WT/D673_E713del (activating). In static differentiation cultures, ITGB3-/- and ITGB3WT/D673_E713del imMKCLs exhibited normal MK differentiation but reduced proplatelet formation. Platelet production was also impaired in a 3D silk-based bone marrow system and in shaking cultures, confirming a quantitative role for ITGB3 in platelet production, independent of the type of variant. While TRAP-activated in vitro-generated platelets lacking αIIbβ3 failed to bind the activation-dependent PAC-1 antibody, ITGB3WT/D673_E713del platelets bound PAC-1 prior to activation mimicking the patient's phenotype. Transcriptome profiling and metabolomic analyses of integrin αIIbβ3 deficient MKs revealed impaired serine metabolism and downregulation of SLC3A2 (CD98hc), an amino acid transporter chaperon known to interact with the β3 subunit. Flow cytometry confirmed decreased CD98hc in mutant MKs, while re-expression of wild-type ITGB3 in ITGB3-/- MKs restored αIIbβ3 and CD98hc expression, normalized proplatelet formation, and enhanced serine uptake. These results uncover a previously unrecognized role of integrin αIIbβ3 in coupling serine metabolism to platelet biogenesis.