Novel causative variants of VEXAS in UBA1 detected through whole genome transcriptome sequencing in a large cohort of hematological malignancies.

UBA1 is an X-linked gene and encodes an ubiquitin-activating enzyme. Three somatic mutations altering the alternative start codon (M41) in UBA1 in hematopoietic precursor cells have recently been described, resulting in a syndrome of severe inflammation, cytopenias, and the presence of intracellular vacuoles in hematopoietic precursors - termed VEXAS syndrome, a predominantly male disease. Here we present a patient with clinical features of VEXAS who harbored two novel somatic variants in UBA1 (I894S and N606I). To better understand the clinical relevance and biological consequences of non-M41 (UBA1non-M41) variants, we analyzed the whole genome and transcriptome data of 4168 patients with hematological malignancies and detected an additional 16 UBA1non-M41 putative somatic variants with a clear sex-bias in patients with myeloid malignancies. Patients diagnosed with myeloid malignancies carrying UBA1non-M41 putative somatic variants either had vacuoles or immunodysregulatory symptoms. Analysis of the transcriptome confirmed neutrophil activation in VEXAS patients compared to healthy controls but did not result in a specific transcriptomic signature of UBA1M41 patients in comparison with MDS patients. In summary, we have described multiple putative novel UBA1non-M41 variants in patients with various hematological malignancies expanding the genomic spectrum of VEXAS syndrome.

Hereditary Hemorrhagic Telangiectasia Presenting with Asymptomatic Liver Lesions and a History of Early-onset Myocardial Infarction and Multiple Intracranial Aneurysms.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder of the vasculature, characterized by epistaxis, telangiectasia and arteriovenous malformations in multiple organs. We herein report a 49-year-old woman with a history of early-onset myocardial infarction and intracranial aneurysms, in whom we incidentally detected multiple hepatic vascular abnormalities. We subsequently diagnosed her with HHT after discovering gastrointestinal telangiectases and a pulmonary arteriovenous fistula along with a history of recurrent epistaxis. Whole-exome sequencing revealed a novel pathogenic variant in SMAD4, a relatively rare causative gene for HHT. This case highlights the fact that HHT patients may present with asymptomatic liver lesions.

RBM24 promotes U1 snRNP recognition of the mutated 5' splice site in the IKBKAP gene of familial dysautonomia.

The 5' splice site mutation (IVS20+6T>C) of the inhibitor of κ light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene in familial dysautonomia (FD) is at the sixth intronic nucleotide of the 5' splice site. It is known to weaken U1 snRNP recognition and result in an aberrantly spliced mRNA product in neuronal tissue, but normally spliced mRNA in other tissues. Aberrantly spliced IKBKAP mRNA abrogates IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1) expression and results in a defect of neuronal cell development in FD. To elucidate the tissue-dependent regulatory mechanism, we screened an expression library of major RNA-binding proteins (RBPs) with our mammalian dual-color splicing reporter system and identified RBM24 as a regulator. RBM24 functioned as a cryptic intronic splicing enhancer binding to an element (IVS20+13-29) downstream from the intronic 5' splice site mutation in the IKBKAP gene and promoted U1 snRNP recognition only to the mutated 5' splice site (and not the wild-type 5' splice site). Our results show that tissue-specific expression of RBM24 can explain the neuron-specific aberrant splicing of IKBKAP exon 20 in familial dysautonomia, and that ectopic expression of RBM24 in neuronal tissue could be a novel therapeutic target of the disease.

Deciphering targeting rules of splicing modulator compounds: case of TG003.

BACKGROUND: Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the disease by altering the exon combination of transcripts to reduce the undesired effect of genetic mutations. However, the knowledge and tools to understand factors contributing to splicing modulator compound sensitivity have been lacking. Our goal was to establish a method to characterize sequence features found in compound sensitive exons. RESULTS: Here we developed a comparative transcriptomic approach to explore features that make an exon sensitive to a chemical compound. In this study, we chose TG003, a potential drug for Duchenne muscular dystrophy, and performed RNA-sequencing on samples from human and mouse skeletal muscle cells, with and without TG003 treatments. We compared TG003 responsiveness between homologous exon pairs and identified 21 pairs in which human exons were skip-enhanced but not mouse exons. We compared the sequence features; splice site scores, number of splicing factor binding sites, and properties of branch sequence and polypyrimidine tracts, and found that polypyrimidine tracts were stronger (longer stretches and richer content of consecutive polypyrimidine) in the mouse TG003 insensitive exons. We also compared the features between TG003 skip-enhanced and insensitive exons within the species, and discovered that human TG003 skip-enhanced exons were shorter and had less splicing factor binding sites than the group of human TG003 insensitive exons. Mouse insensitive exons homologous to human TG003 skip-enhanced exons shared these properties. Our results suggested that these features are prerequisites for TG003 skip-enhanced exons and weak polypyrimidine tracts are defining features, which were supported by a decision tree analysis on all cassette exons in human. CONCLUSIONS: In this study we established a comparative transcriptomic approach, which shed lights on how small chemical compounds modulate RNA splicing. The results described here was the first attempt to decipher the targeting rules of a splicing modulator compound. We expect that this approach would contribute to the precise understanding of the mechanism of TG003-induced splicing modulation, expand target diseases of splicing modulators in general, as well as the development of new splicing modulators.