Aberrant Splicing Research Articles

Clinical implementation of RNA sequencing for Mendelian disease diagnostics

BackgroundLack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.MethodsWe implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.ResultsWe detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.ConclusionTogether, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

A Novel Mutation c.3392G>T of COL2A1 Causes Spondyloepiphyseal Dysplasia Congenital by Affecting Pre-mRNA Splicing.

Spondyloepiphyseal dysplasia congenital (SEDC) is a rare chondrodysplasia caused by dominant pathogenic variants in COL2A1. Here, we detected a novel variant c.3392G > T (NM_001844.4) of COL2A1 in a Chinese family with SEDC by targeted next-generation sequencing. To confirm the pathogenicity of the variant, we generated an appropriate minigene construct based on HeLa and HEK293T cell lines. Splicing assay indicated that the mutated minigene led to aberrant splicing of COL2A1 pre-mRNA and produced an alternatively spliced transcript with a skipping of partial exon 48, which generated a predicted in-frame deletion of 15 amino acids (p. Gly1131_Pro1145del) in the COL2A1 protein. Due to the pathogenicity of the variation, we performed prenatal diagnosis on the proband’s wife, which indicated that the fetus carried the same mutation.

Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer

The dynamic regulation of alternative splicing requires coordinated participation of multiple RNA binding proteins (RBPs). Aberrant splicing caused by dysregulation of splicing regulatory RBPs is implicated in numerous cancers. Here, we reveal a frequently overexpressed cancer-associated protein, DAP3, as a splicing regulatory RBP in cancer. Mechanistically, DAP3 coordinates splicing regulatory networks, not only via mediating the formation of ribonucleoprotein complexes to induce substrate-specific splicing changes, but also via modulating splicing of numerous splicing factors to cause indirect effect on splicing. A pan-cancer analysis of alternative splicing across 33 TCGA cancer types identified DAP3-modulated mis-splicing events in multiple cancers, and some of which predict poor prognosis. Functional investigation of non-productive splicing of WSB1 provides evidence for establishing a causal relationship between DAP3-modulated mis-splicing and tumorigenesis. Together, our work provides critical mechanistic insights into the splicing regulatory roles of DAP3 in cancer development.

Nuclear Aurora kinase A switches m6A reader YTHDC1 to enhance an oncogenic RNA splicing of tumor suppressor RBM4

Aberrant RNA splicing produces alternative isoforms of genes to facilitate tumor progression, yet how this process is regulated by oncogenic signal remains largely unknown. Here, we unveil that non-canonical activation of nuclear AURKA promotes an oncogenic RNA splicing of tumor suppressor RBM4 directed by m6A reader YTHDC1 in lung cancer. Nuclear translocation of AURKA is a prerequisite for RNA aberrant splicing, specifically triggering RBM4 splicing from the full isoform (RBM4-FL) to the short isoform (RBM4-S) in a kinase-independent manner. RBM4-S functions as a tumor promoter by abolishing RBM4-FL-mediated inhibition of the activity of the SRSF1-mTORC1 signaling pathway. Mechanistically, AURKA disrupts the binding of SRSF3 to YTHDC1, resulting in the inhibition of RBM4-FL production induced by the m6A-YTHDC1-SRSF3 complex. In turn, AURKA recruits hnRNP K to YTHDC1, leading to an m6A-YTHDC1-hnRNP K-dependent exon skipping to produce RBM4-S. Importantly, the small molecules that block AURKA nuclear translocation, reverse the oncogenic splicing of RBM4 and significantly suppress lung tumor progression. Together, our study unveils a previously unappreciated role of nuclear AURKA in m6A reader YTHDC1-dependent oncogenic RNA splicing switch, providing a novel therapeutic route to target nuclear oncogenic events.

Aberrant RNA Splicing Is a Primary Link between Genetic Variation and Pancreatic Cancer Risk.

In pancreatic cancer, splicing quantitative trait loci analysis identifies a rs1785932 variant that contributes to decreased risk of disease by influencing ELP2 mRNA splicing and blocking the STAT3 oncogenic pathway.

Study of novel NARS2 variants in patient of combined oxidative phosphorylation deficiency 24.

NARS2 catalyzes the attachment of asparagine amino acids to mitochondrial tRNAAsn and is critical for efficient mitochondrial protein synthesis. Biallelic variants in NARS2 are associated with combined oxidative phosphorylation deficiency 24 (COXPD24) and autosomal recessive deafness-94. Patient information was obtained after recruitment. Genetic tests were performed using whole exome sequencing (WES) and Sanger sequencing. Structure prediction was based on the RaptorX and SWISS-MODEL platforms. The mRNA analysis of paternal variant was performed. Expression levels and dimerization of wild-type (WT) and mutant NARS2 were detected in human embryonic kidney (HEK) 293T cells. Mitochondrial localization of NARS2 variants was determined using immunofluorescence staining. The patient presented early onset generalized epilepsy, myoclonic seizures, severe bilateral hearing impairment and affected liver and heart. WES identified two compound heterozygous variants in NARS2: c.1141A>G/p.Asn381Asp and c.1290G>C/p.Trp430Cys. In silico analysis predicted that both variants would cause significant and pathogenic changes in secondary structure. NARS2 c.1290G>C is a variant at the first nucleotide of an exon, a location thought to affect mRNA splicing. Although transcriptional experiments did not identify aberrant splicing, we observed a lower proportion of transcripts from the NARS2 c.1290G>C variant. An in vitro experiment showed that both variants impaired NARS2 expression, while mitochondrial localization and dimerization remained unaffected. The patient was diagnosed with COXPD24 caused by novel NARS2 variations. The cardiac dysfunction is identified for the first time. In vitro study revealed impairment of variants on NARS2 expression. These data enrich our knowledge regarding the phenotypic and genotypic spectra of NARS2.

Identification of a Novel Deep Intronic Variant by Whole Genome Sequencing Combined With RNA Sequencing in a Chinese Patient With Menkes Disease

Background: Menkes disease (MD) is a rare X-linked connective tissue disorder of copper metabolism caused by pathogenic variant(s) in ATP7A gene. The aim of the present study is to determine the clinical characteristics and molecular basis of one patient with MD. Methods: One 10-month-old Chinese boy who met the clinical manifestations of MD was enrolled in this study. Whole genome sequencing (WGS) was performed in the patient in order to identify the variant(s), followed by Sanger sequencing. RNA sequencing (RNA-seq) from whole blood was subsequently applied to assess the effect of variant on transcription levels, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for further validation. In addition, X chromosome inactivation (XCI) status of the patient’s mother at the DNA level was measured by capillary electrophoresis. Results: The patient suffered from intermittent convulsions for more than 6 months, with psychomoto retardation and neurodegenerations. The patient also had curly hair, hypopigmented skin, cutis laxa, decreased muscle strength and hypotonia. MRI showed the intracranial arteries were tortuous with some “spiral” changes. The patient’s serum ceruloplasmin level was low. WGS revealed one novel hemizygous variant, c.2627-501C > T (NM_000,052.7), located in the deep intronic sequence of ATP7A gene. Sanger sequencing confirmed that the variant was inherited from his mother. RNA-seq confirmed the variant itself, and identified a pseudo-exon inserted between exons 12 and 13 in mRNA of ATP7A. The sequencing results of RT-PCR from the patient confirmed this finding, while neither of his parents detected aberrant splicing. The Capillary electrophoresis results showed that the patient’s mother had a skewed XCI. Conclusion: Our finding of the variant enlarges the variant spectrum in the ATP7A gene. This is a novel deep intronic variant which leads to the activation of a pseudo-exons in the ATP7A gene, and it demonstrates the usefulness of WGS combined with RNA-seq, in terms of revealing disease-causing variants in non-coding regions. Furthermore, the fact that the deep intronic variants cause disease by the activation of pseudo-exon inclusion indicates that in MD this might be an important mechanism.

CD55-deficiency in Jews of Bukharan descent is caused by the Cromer blood type Dr(a-) variant.

The complement system regulator CD55 was initially found to carry the Cromer blood group system antigens, and its complete loss of function was subsequently revealed to cause a severe monogenic gastrointestinal syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis. Here we present homozygosity to the CD55 c.596C>T; p.Ser199Leu variant, which was previously described as the Cromer Dr(a−) genotype, in two Bukharan Jewish CD55-deficiency patients with variable disease severity. We confirm that this missense variant causes aberrant splicing and deletion of 44 bp in exon 5, leading to premature termination and low expression of the CD55 protein. Furthermore, Patient 1 exhibited a mildly abnormal B cell immunophenotyping profile. By population screening we established that this variant is highly prevalent in the Bukharan Jewish population, with a carrier frequency of 1:17, suggesting that many similar patients are un- or mis-diagnosed. The phenotypic variability, ranging from abdominal pain when eating a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. Establishing the diagnosis of CD55-deficiency in a timely manner, even in patients with milder symptoms, may have a critical effect on their management and quality-of-life since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations. Awareness of founder mutations within certain populations can further guide genetic testing and prevent a diagnostic odyssey, by placing this CD55 variant high on the differential diagnosis.

Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing.

BackgroundThe incorrect interpretation of missense and synonymous variants can lead to improper molecular diagnosis and subsequent faulty genetic counselling. The aim of this study was to evaluate the pathogenicity of presumed COL4A3/COL4A4 missense and synonymous variants detected by next-generation sequencing to provide evidence for diagnosis and genetic counselling.MethodsPatients' clinical findings and genetic data were analysed retrospectively. An in vitro minigene assay was conducted to assess the effect of presumed COL4A3/COL4A4 missense and synonymous variants on RNA splicing.ResultsFive unclassified COL4A3/COL4A4 variants, which were detected in five of 343 patients with hereditary kidney diseases, were analysed. All of them were predicted to affect splicing by Human Splicing Finder. The presumed COL4A3 missense variant c.4793T > G [p. (Leu1598Arg)] resulted in a loss of alternative full-length transcript during the splicing process. The COL4A3 transcript carried synonymous variant c.765G > A [p. (Thr255Thr)], led to an in-frame deletion of exon 13. Nevertheless, variants c.3566G > A [p. (Gly1189Glu)] in COL4A3 and c.3990G > A [p. (Pro1330Pro)], c.4766C > T [p. (Pro1589Leu)] in COL4A4 exhibited no deleterious effect on splicing. Among the five patients harbouring the abovementioned COL4A3/COL4A4 variants, three patients were genetically diagnosed with autosomal recessive Alport syndrome, one patient was highly suspected of having thin basement membrane nephropathy, and the other patient was clinically diagnosed with Alport syndrome.ConclusionsCOL4A3 presumed missense variant p. (Leu1598Arg) and synonymous variant p. (Thr255Thr) affect RNA splicing, which highlights the prime importance of transcript analysis of unclassified exonic sequence variants for better molecular diagnosis and genetic counselling. Meanwhile, the reliability of splicing predictions by predictive tools for exonic substitutions needs to be improved.

Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysms.

Intracranial aneurysms (IAs) cause subarachnoid hemorrhage, which has high rates of mortality and morbidity when ruptured. Recently, the role of rare variants in the genetic background of complex diseases has been increasingly recognized. The aim of this study was to identify rare variants for susceptibility to IA. Whole-exome sequencing was performed on seven members of a Japanese pedigree with highly aggregated IA. Candidate genes harboring co-segregating rare variants with IA were re-sequenced and tested for association with IA using additional 500 probands and 323 non-IA controls. Functional analysis of rare variants detected in the pedigree was also conducted. We identified two gene variants shared among all four affected participants in the pedigree. One was the splicing donor c.1515+1G>A variant in NPNT (Nephronectin), which was confirmed to cause aberrant splicing by a minigene assay. The other was the missense p.P83T variant in CBY2 (Chibby family member 2). Overexpression of p.P83T CBY2 fused with red fluorescent protein tended to aggregate in the cytoplasm. Although Nephronectin has been previously reported to be involved in endothelial angiogenic functions, CBY2 is a novel molecule in terms of vascular pathophysiology. We confirmed that CBY2 was expressed in cerebrovascular smooth muscle cells in an isoform2-specific manner. Targeted CBY2 re-sequencing in additional case-control samples identified three deleterious rare variants (p.R46H, p.P83T, and p.L183R) in seven probands, showing a significant enrichment in the overall probands (8/501) compared to the controls (0/323) (p = 0.026, Fisher's extract test). NPNT and CBY2 were identified as novel susceptibility genes for IA. The highly heterogeneous and polygenic architecture of IA susceptibility can be uncovered by accumulating extensive analyses that focus on each pedigree with a high incidence of IA.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B

IntroductionHemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. Patient and methodsThe patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. ResultsA 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. ConclusionA novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking.

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.

The Molecular Basis of FIX Deficiency in Hemophilia B.

Coagulation factor IX (FIX) is a vitamin K dependent protein and its deficiency causes hemophilia B, an X-linked recessive bleeding disorder. More than 1000 mutations in the F9 gene have been identified in hemophilia B patients. Here, we systematically summarize the structural and functional characteristics of FIX and the pathogenic mechanisms of the mutations that have been identified to date. The mechanisms of FIX deficiency are diverse in these mutations. Deletions, insertions, duplications, and indels generally lead to severe hemophilia B. Those in the exon regions generate either frame shift or inframe mutations, and those in the introns usually cause aberrant splicing. Regarding point mutations, the bleeding phenotypes vary from severe to mild in hemophilia B patients. Generally speaking, point mutations in the F9 promoter region result in hemophilia B Leyden, and those in the introns cause aberrant splicing. Point mutations in the coding sequence can be missense, nonsense, or silent mutations. Nonsense mutations generate truncated FIX that usually loses function, causing severe hemophilia B. Silent mutations may lead to aberrant splicing or affect FIX translation. The mechanisms of missense mutation, however, have not been fully understood. They lead to FIX deficiency, often by affecting FIX’s translation, protein folding, protein stability, posttranslational modifications, activation to FIXa, or the ability to form functional Xase complex. Understanding the molecular mechanisms of FIX deficiency will provide significant insight for patient diagnosis and treatment.

Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.

RAS family members are the most frequently mutated oncogenes in human cancers. Although KRAS(G12C)-specific inhibitors show clinical activity in patients with cancer1-3, there are no direct inhibitors of NRAS, HRAS or non-G12C KRAS variants. Here we uncover the requirement of the silent KRASG60G mutation for cells to produce a functional KRAS(Q61K). In the absence of this G60G mutation in KRASQ61K, a cryptic splice donor site is formed, promoting alternative splicing and premature protein termination. A G60G silent mutation eliminates the splice donor site, yielding a functional KRAS(Q61K) variant. We detected a concordance of KRASQ61K and a G60G/A59A silent mutation in three independent pan-cancer cohorts. The region around RAS Q61 is enriched in exonic splicing enhancer (ESE) motifs and we designed mutant-specific oligonucleotides to interfere with ESE-mediated splicing, rendering the RAS(Q61) protein non-functional in a mutant-selective manner. The induction of aberrant splicing by antisense oligonucleotides demonstrated therapeutic effects in vitro and in vivo. By studying the splicing necessary for a functional KRAS(Q61K), we uncover a mutant-selective treatment strategy for RASQ61 cancer and expose a mutant-specific vulnerability, which could potentially be exploited for therapy in other genetic contexts.

Using long-read RNA sequencing to decipher the role of RNA isoforms in disease

Accurate quantification of genes and their mRNA products is essential to understanding health and disease. In humans, processes such as alternative splicing cause almost all genes to express multiple mRNA products (isoforms), which can have different functions. In addition, aberrant splicing is a common cause of disease. Standard short-read RNA sequencing (RNA-seq) methodologies have limitations in identifying isoforms. In contrast, long-read RNA-seq can address this challenge by covering the entire mRNA sequence in a single read and so identify and quantify the isoforms present.

EP407: Aberrant KCNQ1 splicing as an emerging mechanism underlying the pathogenesis of familial Beckwith-Wiedemann syndrome with reduced penetrance

Beckwith-Wiedemann Syndrome (BWS) is a phenotypically and genetically heterogeneous congenital imprinting disorder characterized by macroglossia, omphalocele, hemihypertrophy and a predisposition to embryonal tumors. BWS is caused by genetic or epigenetic alterations in the chromosomal region 11p15.5, which consists of genes that are involved in the regulation of cell growth and proliferation. BWS is generally considered a sporadic disease, as approximately 85% of BWS cases occur without family history.

In This Issue

See article, p. 95.Shree et al. find that up to a year after anti-CD20 treatment, most patients with lymphoma do not generate neutralizing antibody responses to COVID-19 vaccination. However, if patients are vaccinated and mount an antibody response prior to anti-CD20 treatment, they tend to maintain these antibody responses through subsequent anti-CD20 lymphoma therapy, suggesting a strategy of vaccinating first and treating later. These results also call to weigh the risks of maintenance therapy with rituximab or other B cell–depleting antibodies during the COVID-19 era.See article, p. 103.Downregulation of surface epitopes, including CD22, causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Zheng et al. report that CD22 mRNA is pervasively misspliced in B-ALL cell lines and primary samples. Some misspliced isoforms still encode plasma membrane–bound CD22 variants, while others lack the start codon in exon 2 and are noncoding. These exon 2–skipping isoforms are prevalent in high-risk B-ALL patients treated with the CD22-directed antibody–drug conjugate inotuzumab, where they correlate with low CD22 surface expression and are likely to account for treatment failure in two cases. Thus, aberrant CD22 splicing is an important mechanism of resistance to immunotherapy.See article, p. 116.Polycomb repressive epigenetic complexes are recurrently dysregulated in cancer. The mechanisms by which genetic alterations affect polycomb regulatory function to drive oncogenesis are poorly understood. By combining human genomics with biochemical and functional approaches, Schaefer et al. demonstrate that highly recurrent mutations in leukemia of the PRC1.1 subunits BCOR and BCORL1 dissociate DNA binding from enzymatic subunits. Uncoupling of the PRC1.1-repressive function from target genes alters epigenetic and signaling states, including FGFR derepression. BCOR deletion or truncation confers ibrutinib resistance, which is abrogated by FGFR inhibition. This epigenetic mechanism explains PRC1.1 tumor-suppressive activity and informs therapeutic approaches in PRC1.1-mutated cancer.See article, p. 136.IFNγ production has long been considered necessary for antitumor T-cell response. IFNγ also fuels macrophage activation in cytokine release syndrome (CRS), a significant toxicity of chimeric antigen receptor (CAR) T-cell therapy. Bailey et al. have discovered that either blockade or genetic knockout of IFNγ does not impair the anticancer activity of CAR T cells in preclinical models of hematologic malignancies. Further, there is reduced expression of immune checkpoint receptors on the CAR T cells and less activation of macrophages. Thus, it may be possible to separate the toxicity from the efficacy of CAR T cells for safer yet effective use.See article, p. 154.Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with very poor clinical outcomes. Mundy-Bosse et al. demonstrate that ENKTL cells are arrested at early stages of NK-cell maturation, characterized by broad epigenetic silencing of genes involved in normal NK-cell development. Treatment with the DNA hypomethylating agent 5-azacytidine led to a reduction of DNA hypermethylation, reexpression of normal NK-cell developmental genes, phenotypic NK-cell differentiation, and significant prolongation of survival in recipient mice. These studies lay the foundation for epigenetic-directed therapy targeting the developmental blockade in ENKTL.

Comprehensive Transcriptome Analysis of Patients With Keratoconus Highlights the Regulation of Immune Responses and Inflammatory Processes

Keratoconus (KTCN), characterized by the steeper curvature of the cornea and the thinner central corneal thickness, was a degenerative corneal ectasia with ambiguous etiology and mechanism. We aim to reveal underlying pathological mechanisms of KTCN by multi-level transcriptomic, integrative omics analyses. We performed RNA-sequencing on corneal epithelial samples from seven patients and seven control donors, as well as peripheral matched blood samples from three of the patients and three control donors. After RNA extraction, library construction, and sequencing, differentially expressed genes and splicing events were identified, followed by over-representation analysis. In total, 547 differential expressed genes were identified in KTCN corneal epithelial samples, which were mainly enriched in immune responses and inflammatory processes. WGCNA module analysis, the transcriptomic analysis of peripheral blood samples, multiple omics data, and the meta-analysis of GEO samples confirmed the involvement of immune and inflammatory factors. Besides, 190 and 1,163 aberrant splicing events were identified by rMATS combined with CASH methods in corneal epithelial and blood samples with KCTN. In conclusion, this comprehensive transcriptome analysis of KTCN patients based on patients’ tissue and blood samples uncovered a significant association between immune-inflammatory genes and pathways with KCTN, highlighting the contribution of the perturbed immune signaling to the pathogenesis of KCTN. Our study suggested the importance of measures to control inflammation in the treatment of KCTN.

Novel Intronic Mutations of TBK1 Promote Aberrant Splicing Modes in Amyotrophic Lateral Sclerosis.

TANK-binding kinase 1 (TBK1) has been identified as a causative gene of amyotrophic lateral sclerosis (ALS) in the Caucasian population in 2015. Here, we sequenced for TBK1 variants in a cohort of 15 familial ALS (fALS) and 275 sporadic ALS (sALS) of Chinese origin by targeted next-generation sequencing. We identified one likely benign missense variant (p. Ser398Pro), two missense variants of uncertain significance (p. Ile37Leu and p. Tyr677Asn), and two novel heterozygous variants in introns of TBK1, c.1522-3T > G and c.2066 + 4A > G. We performed splicing assays through minigene plasmids and RNA pull-down assay to determine that the two substitutions of nucleotides disrupted the binding of the important splicing regulator hnRNPA1 and promoted aberrant pre-mRNA splicing modes. The c.1522-3T > G variant promoted nearly 50.0% of abnormal transcripts (3 different types of insertions and deletions (indels) in junction of intron 13-exon 14) and the c.2066 + 4A > G variant inhibited about 75.0% inclusion of exon 19, both causing premature stop codon and producing TBK1 protein without CCD2. Immunofluorescence analysis showed that the expression of TBK1 with intronic variants was lower since less TBK1 distribution was observed in HEK293T cells. Both patients carrying TBK1 c.1522-3T > G and c.2066 + 4A > G variants developed a rapidly progressive ALS, with a survival of 31 and 10 months, respectively. The frequency of loss of function (LoF) variants in TBK1 was 0.73% in sALS in our cohort. We emphasize that intronic sequencing and pre-mRNA splicing analysis cannot be ignored to demonstrate the complex mutational spectrum and pathogenesis of ALS.

The Splicing of the Mitochondrial Calcium Uniporter Genuine Activator MICU1 Is Driven by RBFOX2 Splicing Factor during Myogenic Differentiation.

Alternative splicing, the process by which exons within a pre-mRNA transcript are differentially joined or skipped, is crucial in skeletal muscle since it is required both during myogenesis and in post-natal life to reprogram the transcripts of contractile proteins, metabolic enzymes, and transcription factors in functionally distinct muscle fiber types. The importance of such events is underlined by the numerosity of pathological conditions caused by alternative splicing aberrations. Importantly, many skeletal muscle Ca2+ homeostasis genes are also regulated by alternative splicing mechanisms, among which is the Mitochondrial Ca2+ Uniporter (MCU) genuine activator MICU1 which regulates MCU opening upon cell stimulation. We have previously shown that murine skeletal muscle MICU1 is subjected to alternative splicing, thereby generating a splice variant—which was named MICU1.1—that confers unique properties to the mitochondrial Ca2+ uptake and ensuring sufficient ATP production for muscle contraction. Here we extended the analysis of MICU1 alternative splicing to human tissues, finding two additional splicing variants that were characterized by their ability to regulate mitochondrial Ca2+ uptake. Furthermore, we found that MICU1 alternative splicing is induced during myogenesis by the splicing factor RBFOX2. These results highlight the complexity of the alternative splicing mechanisms in skeletal muscle and the regulation of mitochondrial Ca2+ among tissues.