A systematic assessment of the impact of rare canonical splice site variants on splicing using functional and in silico methods.

Canonical splice site variants (CSSVs) are often presumed to cause loss-of-function (LoF) and are assigned very strong evidence of pathogenicity (according to American College of Medical Genetics/Association for Molecular Pathology criterion PVS1). The exact nature and predictability of splicing effects of unselected rare CSSVs in blood-expressed genes are poorly understood. We identified 168 rare CSSVs in blood-expressed genes in 112 individuals using genome sequencing, and studied their impact on splicing using RNA sequencing (RNA-seq). There was no evidence of a frameshift, nor of reduced expression consistent with nonsense-mediated decay, for 25.6% of CSSVs: 17.9% had wildtype splicing only and normal junction depths, 3.6% resulted in cryptic splice site usage and in-frame insertions or deletions, 3.6% resulted in full exon skipping (in frame), and 0.6% resulted in full intron inclusion (in frame). Blind to these RNA-seq data, we attempted to predict the precise impact of CSSVs by applying in silico tools and the ClinGen Sequence Variant Interpretation Working Group 2018 guidelines for applying PVS1 criterion. The predicted impact on splicing using (1) SpliceAI, (2) MaxEntScan, and (3) AutoPVS1, an automatic classification tool for PVS1 interpretation of null variants that utilizes Ensembl Variant Effect Predictor and MaxEntScan, was concordant with RNA-seq analyses for 65%, 63%, and 61% of CSSVs, respectively. In summary, approximately one in four rare CSSVs did not show evidence for LoF based on analysis of RNA-seq data. Predictions from in silico methods were often discordant with findings from RNA-seq. More caution may be warranted in applying PVS1-level evidence to CSSVs in the absence of functional data.

SEPT-GD: A decision tree to prioritise potential RNA splice variants in cardiomyopathy genes for functional splicing assays in diagnostics.

BACKGROUND: Splice prediction algorithms currently used in routine DNA diagnostics have limited sensitivity and specificity, therefore many potential splice variants are classified as variants of uncertain significance (VUSs). However, functional assessment of VUSs to test splicing is labour-intensive and time-consuming. We developed a decision tree to prioritise potential splice variants for functional studies and functionally verified the outcome of the decision tree. MATERIALS AND METHODS: We built the decision tree, SEPT-GD, by setting thresholds for the splice prediction programs implemented in Alamut. A set of 343 variants with known effects on splicing was used as control for sensitivity and specificity. We tested SEPT-GD using variants from a Dutch cardiomyopathy cohort of 2002 patients that were previously classified as VUS and predicted to have a splice effect according to diagnostic rules. We then selected 12 VUSs ranked by SEPT-GD to functionally verify the predicted effect on splicing using a minigene assay: 10 variants predicted to have a strong effect and 2 with a weak effect. RT-PCR was performed for nine variants. Variant classification was re-evaluated based on the functional test outcome. RESULTS: Compared to similar individually tested algorithms, SEPT-GD shows higher sensitivity (91 %) and comparable specificity (88 %) for both consensus (dinucleotides at the start and end of the intron, GT at the 5' end and AG at the 3' end) and non-consensus splice-site variants (excluding middle of exon variants). Using clinical diagnostic criteria, 1295 unique variants in our cardiomyopathy cohort had originally been classified as VUSs, with 57 predicted by Alamut to have an effect on splicing. Using SEPT-GD, we prioritised 31 variants in 40 patients. In the minigene assay, all 12 variants showed results concordant with SEPT-GD predictions. RT-PCR confirmed the minigene results for two variants, TMEM43 c.1000 + 5G > T and TTN c.25922-6 T > G. Based on all outcomes, the SGCD c.4-1G > A and CSRP3 c.282-5_285del variants were reclassified as likely pathogenic. CONCLUSION: SEPT-GD outperforms the tools commonly used for RNA splicing prediction and improves prioritisation of variants in cardiomyopathy genes for functional splicing analysis in a diagnostic setting.

Informing variant assessment using structured evidence from prior classifications (PS1, PM5, and PVS1 sequence variant interpretation criteria).

PURPOSE: This study aimed to explore whether evidence of pathogenicity from prior variant classifications in ClinVar could be used to inform variant interpretation using the American College of Medical Genetics and Genomics/Association for Molecular Pathology clinical guidelines. METHODS: We identified distinct single-nucleotide variants (SNVs) that are either similar in location or in functional consequence to pathogenic variants in ClinVar and analyzed evidence in support of pathogenicity using 3 interpretation criteria. RESULTS: Thousands of variants, including many in clinically actionable disease genes (American College of Medical Genetics and Genomics secondary findings v3.0), have evidence of pathogenicity from existing variant classifications, accounting for 2.5% of nonsynonymous SNVs within ClinVar. Notably, there are many variants with uncertain or conflicting classifications that cause the same amino acid substitution as other pathogenic variants (PS1, N = 323), variants that are predicted to cause different amino acid substitutions in the same codon as pathogenic variants (PM5, N = 7692), and loss-of-function variants that are present in genes in which many loss-of-function variants are classified as pathogenic (PVS1, N = 3635). Most of these variants have similar computational predictions of pathogenicity and splicing effect as their associated pathogenic variants. CONCLUSION: Broadly, for >1.4 million SNVs exome wide, information from previously classified variants could be used to provide evidence of pathogenicity. We have developed a pipeline to identify variants meeting these criteria that may inform interpretation efforts.

Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modulation Therapies.

Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of human mutations affecting pre-mRNA splicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy .

NucHMM: a method for quantitative modeling of nucleosome organization identifying functional nucleosome states distinctly associated with splicing potentiality.

We develop a novel computational method, NucHMM, to identify functional nucleosome states associated with cell type-specific combinatorial histone marks and nucleosome organization features such as phasing, spacing and positioning. We test it on publicly available MNase-seq and ChIP-seq data in MCF7, H1, and IMR90 cells and identify 11 distinct functional nucleosome states. We demonstrate these nucleosome states are distinctly associated with the splicing potentiality of skipping exons. This advances our understanding of the chromatin function at the nucleosome level and offers insights into the interplay between nucleosome organization and splicing processes.

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.

Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy.

RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.

The intronic BRCA1 c.5407-25T>A variant causing partly skipping of exon 23-a likely pathogenic variant with reduced penetrance?

Rare sequence variants in the non-coding part of the BRCA genes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1 c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant's effect on mRNA splicing. Mean age at breast (n = 12) and ovarian (n = 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1 c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1 c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1 c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.

Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Minigene splicing assessment of 20 novel synonymous and intronic glucokinase gene variants identified in patients with maturity-onset diabetes of the young.

The next-generation sequencing (NGS) has become a routine method for diagnostics of inherited disorders. However, assessment of the discovered variants may be challenging, especially when they are not predicted to change the protein sequence. Here we performed a functional analysis of 20 novel or rare intronic and synonymous glucokinase (GCK) gene variants identified by targeted NGS in 1,130 patients with maturity-onset diabetes of the young. Human Splicing Finder, ver 3.1 and a precomputed index of splicing variants (SPIDEX) were used for in silico prediction. In vitro effects of GCK gene variants on splicing were tested using a minigene expression approach. In vitro effect on splicing was shown for 9 of 20 variants, including two synonymous substitutions. In silico and in vitro results matched in about 50% of cases. The results demonstrate that novel or rare apparently benign GCK gene variants should be regarded as potential splicing mutations.

Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology.

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease.

PURPOSE: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

Constitutive splicing and economies of scale in gene expression.

In eukaryotic cells, many introns are constitutively, rather than alternatively, spliced and therefore do not contribute to isoform diversification. It has remained unclear what functional roles such constitutive splicing provides. To explore this issue, we asked how splicing affects the efficiency with which individual pre-messenger RNA transcripts are productively processed across different gene expression levels. We developed a quantitative single-molecule fluorescence in situ hybridization-based method to quantify splicing efficiency at transcription active sites in single cells. We found that both natural and synthetic genes in mouse and human cells exhibited an unexpected 'economy of scale' behavior in which splicing efficiency increased with transcription rate. Correlations between splicing efficiency and spatial proximity to nuclear speckles could explain this counterintuitive behavior. Functionally, economy of scale splicing represents a non-linear filter that amplifies the expression of genes when they are more strongly transcribed. These results indicate that constitutive splicing plays an active functional role in modulating gene expression.

Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro.

Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2'-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2'-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2'-O-methyl (2'-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2'-F nucleotides induced efficient exon-23 skipping, with LNA/2'-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2'-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2'-OMe/2'-F chimeras. Overall, our findings certainly expand the scope of constructing 2'-F modified AOs in splice modulation by incorporating 2'-OMe and LNA modifications.

Alternative RNA Splicing as a Potential Major Source of Untapped Molecular Targets in Precision Oncology and Cancer Disparities.

Studies of alternative RNA splicing (ARS) have the potential to provide an abundance of novel targets for development of new biomarkers and therapeutics in oncology, which will be necessary to improve outcomes for patients with cancer and mitigate cancer disparities. ARS, a key step in gene expression enabling individual genes to encode multiple proteins, is emerging as a major driver of abnormal phenotypic heterogeneity. Recent studies have begun to identify RNA splicing-related genetic and genomic variation in tumors, oncogenes dysregulated by ARS, RNA splice variants driving race-related cancer aggressiveness and drug response, spliceosome-dependent transformation, and RNA splicing-related immunogenic epitopes in cancer. In addition, recent studies have begun to identify and test, preclinically and clinically, approaches to modulate and exploit ARS for therapeutic application, including splice-switching oligonucleotides, small molecules targeting RNA splicing or RNA splice variants, and combination regimens with immunotherapies. Although ARS data hold such promise for precision oncology, inclusion of studies of ARS in translational and clinical cancer research remains limited. Technologic developments in sequencing and bioinformatics are being routinely incorporated into clinical oncology that permit investigation of clinically relevant ARS events, yet ARS remains largely overlooked either because of a lack of awareness within the clinical oncology community or perceived barriers to the technical complexity of analyzing ARS. This perspective aims to increase such awareness, propose immediate opportunities to improve identification and analysis of ARS, and call for bioinformaticians and cancer researchers to work together to address the urgent need to incorporate ARS into cancer biology and precision oncology.

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the "shock and kill" approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

Mathematical modeling identifies potential gene structure determinants of co-transcriptional control of alternative pre-mRNA splicing.

The spliceosome catalyzes the removal of introns from pre-messenger RNA (mRNA) and subsequent pairing of exons with remarkable fidelity. Some exons are known to be skipped or included in the mature mRNA in a cell type- or context-dependent manner (cassette exons), thereby contributing to the diversification of the human proteome. Interestingly, splicing is initiated (and sometimes completed) co-transcriptionally. Here, we develop a kinetic mathematical modeling framework to investigate alternative co-transcriptional splicing (CTS) and, specifically, the control of cassette exons' inclusion. We show that when splicing is co-transcriptional, default splice patterns of exon inclusion are more likely than when splicing is post-transcriptional, and that certain exons are more likely to be regulatable (i.e. cassette exons) than others, based on the exon-intron structure context. For such regulatable exons, transcriptional elongation rates may affect splicing outcomes. Within the CTS paradigm, we examine previously described hypotheses of co-operativity between splice sites of short introns (i.e. 'intron definition') or across short exons (i.e. 'exon definition'), and find that models encoding these faithfully recapitulate observations in the fly and human genomes, respectively.