Contribution of Noncanonical Splice Variants to TTN Truncating Variants Cardiomyopathy

Background: Heterozygous TTN truncating variants cause 10% to 20% of idiopathic dilated cardiomyopathy (DCM). Although variants which disrupt canonical splice signals (ie, invariant dinucleotide of splice donor site, invariant dinucleotide of the splice acceptor site) at exon-intron junctions are readily recognized as TTN truncating variants, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain. Methods: Rare variants of unknown significance located in the splice regions of highly expressed TTN exons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay. Results: Splice-altering variants of unknown significance were enriched in DCM cases over controls and present in 2% of DCM patients ( P =0.002). Application of this method to clinical variant databases demonstrated 20% of similar variants of unknown significance in TTN splice regions affect splicing. Noncanonical splice-altering variants were most frequently located at position +5 of the donor site ( P =4.4×10 7 ) and position -3 of the acceptor site ( P =0.002). SpliceAI, an emerging in silico prediction tool, had a high positive predictive value (86%–95%) but poor sensitivity (15%–50%) for the detection of splice-altering variants. Alternate exons spliced out of most TTN transcripts frequently lacked the consensus base at +5 donor and −3 acceptor positions. Conclusions: Noncanonical splice-altering variants in TTN explain 1-2% of DCM and offer a 10-20% increase in the diagnostic power of TTN sequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternate TTN exons.

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Efficacy of DNA versus RNA NGS-based Methods in MET Exon 14 skipping mutation detection.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.9036 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 9036-9036

Author(s):

Magdalena Jurkiewicz ◽

Anjali Saqi ◽

Mahesh M Mansukhani ◽

Vaishali Hodel ◽

Anamaria Krull ◽

...

Keyword(s):

Splice Site ◽

Donor Site ◽

Driver Mutations ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Acceptor Site ◽

Female Ratio ◽

Lung Carcinomas ◽

Rna Analysis ◽

Lung Adenocarcinomas

9036 Background: Exon 14 skipping mutations in the mesenchymal-epithelial transition ( MET) gene are reported in 2-5% of lung adenocarcinomas and are mutually exclusive of other driver mutations. Small-molecule MET tyrosine kinase inhibitors, capmatinib and tepotinib, showed durable responses in previously treated and treatment-naïve patients harboring MET-exon-14 skipping mutations. Studies suggest that for detection of MET-ex14 mutations, DNA-based assays alone may be sub-optimal when compared to RNA-based NGS assays. We compared the performance of DNA and RNA-based assays for detection of MET-ex14 variants. Methods: We examined NGS-based profiling data of lung adenocarcinomas (or when this diagnosis could not be excluded) to identify MET-ex14 mutations missed by DNA but identified by RNA analysis. The carcinomas were profiled by a DNA-based NGS panel that targets MET exons 2, 14, 16, 18 and 19. Cases without driver mutations were reflexed to an NGS-based RNA fusion panel (Archer’s Anchored Multiplex PCR). Results: Over a 21-month period, MET-ex14 skipping events were detected in 16/644 (2.5%) lung carcinomas by DNA profiling. RNA analysis on driver-negative cases identified 9 additional MET-ex14 mutations. All 16 MET-ex14 DNA variants occurred at or around the intron 14 splice donor site, as the assay did not include the intron 13 splice acceptor site. Clinical characteristics of the MET positive cohort include a male to female ratio of 0.8:1.0, an average age of 76.5 years and 52% non-smoker status. All tumors were adenocarcinomas (including one with a < 10% spindle/pleomorphic component) with the exception of 3 adenosquamous carcinomas and 1 squamous cell carcinoma. Conclusions: DNA based NGS-panels can potentially miss MET-ex14 skipping events in lung carcinomas, when the primers do not target both 3′ splice site of intron 13, and the 5′ splice site of intron 14. A reflex work flow interrogating RNA fusions can potentially capture such events. The clinical and molecular characterization of the variants detected only by RNA NGS assays warrants further exploration.

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Control of retroviral RNA splicing through maintenance of suboptimal processing signals

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.696-704.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 696-704 ◽

Cited By ~ 2

Author(s):

R A Katz ◽

A M Skalka

Keyword(s):

Rna Splicing ◽

Donor Site ◽

Protein S ◽

Full Length ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Insertion Mutation ◽

Acceptor Site ◽

Splice Acceptor

The full-length retroviral transcript serves as genomic RNA for progeny virions, as an mRNA for structural proteins and enzymes, and as a pre-mRNA substrate for splicing that yields subgenomic mRNAs that encode other essential proteins. Thus, RNA splicing to form subgenomic mRNAs must be incomplete or regulated in order to preserve some of the full-length transcripts. We have used the avian sarcoma virus system to delineate the viral functions that are required in the regulation of the splicing event that forms the envelope glycoprotein (env) subgenomic mRNA. We observed previously that a specific insertion mutation just 5' of the env splice acceptor site resulted in nearly complete splicing to form env mRNA and a concomitant replication defect which is presumably due to a deficit of the full-length transcript. Replication-competent pseudorevertants contained second-site mutations that restored splicing control, and these mapped either just upstream or downstream of the env splice acceptor site. In this report, we show that splicing control at this site does not require expression of any known viral replication protein(s), nor does it appear to require the viral splice donor site. From these results and analysis of additional splicing mutations obtained by in vivo selection, we conclude that splicing is controlled through the maintenance of suboptimal cis-acting signals in the viral RNA that alter the efficiency of recognition by the cellular splicing machinery.

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Human Cytomegalovirus 5-Kilobase Immediate-Early RNA Is a Stable Intron

Journal of Virology ◽

10.1128/jvi.78.23.13182-13189.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13182-13189 ◽

Cited By ~ 35

Author(s):

Caroline A. Kulesza ◽

Thomas Shenk

Keyword(s):

Viral Infection ◽

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Donor Site ◽

Immediate Early ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Viral Gene ◽

Acceptor Site ◽

Splice Donor

ABSTRACT Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5′ end of the 5-kb RNA to a consensus splice donor site and localized the 3′ end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5′ splice donor site that defines the 5′ end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV.

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Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

Journal of Endocrinology ◽

10.1677/joe.0.1710397 ◽

2001 ◽

Vol 171 (3) ◽

pp. 397-402 ◽

Cited By ~ 13

Author(s):

HH Lee ◽

SF Chang

Keyword(s):

Donor Site ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Acceptor Site ◽

Site Mutation ◽

Hydroxylase Deficiency ◽

Exon 2 ◽

Eukaryotic Genes ◽

21 Hydroxylase Deficiency ◽

Intron 2

Maturation of primary RNA transcripts of eukaryotic genes often involves the removal of introns and joining of exons. The fidelity of RNA splicing is dependent on the identity of the nucleotide (nt) sequences at exon/intron boundaries. Most importantly, the highly conserved intronic 5'GT and 3'AG sequences are essential for correct splicing. Substitution of GT by any other nt leads to incomplete mRNA and a disruption of protein structure. We describe here the results of our transfection experiments in COS-1 cells with a CYP21 genomic construct that contained an IVS 2+1G-->A mutation. Analysis of the transcripts by RT-PCR revealed that two different transcripts were generated by this mutant genome. In all the splicing products, we found that the entire exon 2 was deleted. Surprisingly, 30% of the transcripts from this mutant CYP21 genome were accompanied by an inclusion of 3' intron 2 sequences due to the use of a different splice acceptor site. This is the first report of the molecular characterization of a splice donor site mutation in CYP21 via transcription in COS-1 cells.

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iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition

BioMed Research International ◽

10.1155/2014/623149 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 60

Author(s):

Wei Chen ◽

Peng-Mian Feng ◽

Hao Lin ◽

Kuo-Chen Chou

Keyword(s):

Structural Properties ◽

Rna Splicing ◽

Donor Site ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Acceptor Site ◽

Splice Sites ◽

Success Rates ◽

Eukaryotic Genes ◽

Dinucleotide Composition

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called “iSS-PseDNC” was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called “pseudo dinucleotide composition” (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing.

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Functional Mapping of the Human Papillomavirus Type 16 E1 Cistron

Journal of Virology ◽

10.1128/jvi.00921-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10724-10734 ◽

Cited By ~ 20

Author(s):

Michael J. Lace ◽

James R. Anson ◽

Lubomir P. Turek ◽

Thomas H. Haugen

Keyword(s):

Human Papillomavirus ◽

Donor Site ◽

Dna Amplification ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Acceptor Site ◽

Mrna Levels ◽

Viral Dna ◽

Hpv 16 ◽

Hpv Dna

ABSTRACT Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1− mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5′ mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1− genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.

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Control of retroviral RNA splicing through maintenance of suboptimal processing signals.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.696 ◽

1990 ◽

Vol 10 (2) ◽

pp. 696-704 ◽

Cited By ~ 57

Author(s):

R A Katz ◽

A M Skalka

Keyword(s):

Rna Splicing ◽

Donor Site ◽

Protein S ◽

Full Length ◽

Splice Acceptor Site ◽

Splice Donor Site ◽

Insertion Mutation ◽

Acceptor Site ◽

Splice Acceptor

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Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine ◽

10.1186/s13073-021-00850-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

William L. Macken ◽

Annie Godwin ◽

Gabrielle Wheway ◽

Karen Stals ◽

Liliya Nazlamova ◽

...

Keyword(s):

Genome Sequencing ◽

Donor Site ◽

Xenopus Tropicalis ◽

Splice Donor Site ◽

Homologous Region ◽

Disease Genes ◽

Coat Proteins ◽

Splice Donor ◽

Severe Intellectual Disability

Abstract Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

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Nova Regulates GABAA Receptor γ2 Alternative Splicing via a Distal Downstream UCAU-Rich Intronic Splicing Enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.23.13.4687-4700.2003 ◽

2003 ◽

Vol 23 (13) ◽

pp. 4687-4700 ◽

Cited By ~ 79

Author(s):

B. Kate Dredge ◽

Robert B. Darnell

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Splicing Regulation ◽

Globin Genes ◽

Enhancer Element ◽

Intronic Splicing Enhancer ◽

Splicing Enhancer

ABSTRACT Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABAA receptor γ2 (GABAARγ2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABAARγ2 alternative splicing, we systematically screened minigenes derived from the GABAARγ2 and human β-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABAARγ2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABAARγ2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.

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Novel somatic mutations in UBA1 as a cause of VEXAS syndrome

Blood ◽

10.1182/blood.2020010286 ◽

2021 ◽

Cited By ~ 2

Author(s):

James A Poulter ◽

Jason Charles Collins ◽

Catherine Cargo ◽

Ruth M de Tute ◽

Paul Evans ◽

...

Keyword(s):

Bone Marrow ◽

Somatic Mutations ◽

Clinical Phenotype ◽

Splice Acceptor Site ◽

Acceptor Site ◽

The Novel ◽

Disease Mechanism ◽

Bone Marrow Biopsies ◽

Novel Variant

Somatic mutations at methionine 41 (Met41) in UBA1, encoding the major E1 enzyme responsible for initiating ubiquitylation, were recently identified as the cause of a novel autoinflammatory disease, named VEXAS (Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). We sought to determine the prevalence of UBA1 mutations in a UK cohort of patients matching the VEXAS clinical phenotype. We identified 10 new patients with somatic mutations in UBA1, but only 8 had altered p.Met41. A novel variant, c.167C>T; p.Ser56Phe was identified, which was present in myeloid, and not lymphoid lineages and led to preferential loss of the catalytic activity of cytoplasmic UBA1. An additional novel variant, c.118-1G>C was identified at the splice acceptor site of exon 3 leading to altered splicing in vitro. Bone marrow biopsies from two patients with a Met41 substitution and the novel splice site variant were consistent with previously reported features of VEXAS. The bone marrow of the patient with the p.Ser56Phe variant was less similar, likely driven by a distinct but overlapping disease mechanism. Our study therefore confirms somatic p.Met41 substitutions in UBA1 as a major cause of VEXAS syndrome and identifies two new disease causing mutations.

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