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Mutations which affect splicing are significant contributors to rare disease, but are frequently overlooked by diagnostic sequencing pipelines. Greater ascertainment of pathogenic splicing variants will increase diagnostic yields, ending the diagnostic odyssey for patients and families affected by rare disorders, and improving treatment and care strategies. Advances in sequencing technologies, predictive modeling, and understanding of the mechanisms of splicing in recent years pave the way for improved detection and interpretation of splice affecting variants, yet several limitations still prohibit their routine ascertainment in diagnostic testing. This review explores some of these advances in the context of clinical application and discusses challenges to be overcome before these variants are comprehensively and routinely recognized in diagnostics. [ABSTRACT FROM AUTHOR]

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). [ABSTRACT FROM AUTHOR]

The article discusses a study on the prediction of single-nucleotide substitutions that induce exon skipping in the breast cancer susceptibility gene BRCA1. Missense and silent mutations from BRCA1 mutation databases were tested to determine their effects on exon inclusion. The study identified a single exon-skipping mutation and a splicing regulatory element in BRCA1 exon 6. It found a link between some proteins and BRCA1 exon 6 inclusion changes in the messenger ribonucleic acid (mRA).

Many disease-causing splicing mutations described in the literature produce changes in splice sites (SS) or in exon-regulatory sequences. The delineation of these splice aberrations can provide important insights into novel regulation mechanisms. In this study, we evaluated the effect of patient variations in neurofibromatosis type 1 ( NF1) exon 29 and its 5′SS surrounding area on its splicing process. Only two of all nonsense, missense, synonymous and intronic variations analyzed in this study clearly altered exon 29 inclusion/exclusion levels. In particular, the intronic mutation +5g>a had the strongest effect, resulting in total exon exclusion. This finding prompted us to evaluate the exon 29 5′SS in relation to its ability to bind U1 snRNP. This was performed by direct analysis of the ability of U1 to bind to wild-type and mutant donor sites, by engineering an in vitro splicing system to directly evaluate the functional importance of U1 snRNA base pairing with the exon 29 donor site, and by coexpression of mutant U1 snRNP molecules to try to rescue exon 29 inclusion in vivo. The results revealed a low dependency on the presence of U1 snRNP, and suggest that exon 29 donor site definition may depend on alternative mechanisms of 5′SS recognition. [ABSTRACT FROM AUTHOR]

In disease-associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is ‘c.293-279 A>G’ mutation [Raponi M, Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294–295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclusion levels are strongly downregulated by polypyrimidine tract binding protein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cis-acting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons. [ABSTRACT FROM AUTHOR]

Abstract: We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting. [Copyright &y& Elsevier]

Abstract High-throughput next-generation sequencing technologies have led to a rapid increase in the number of sequence variants identified in clinical practice via diagnostic genetic tests. Current bioinformatic analysis pipelines fail to take adequate account of the possible splicing effects of such variants, particularly where variants fall outwith canonical splice site sequences, and consequently the pathogenicity of such variants may often be missed. The regulation of splicing is highly complex and as a result , in silico prediction tools lack sufficient sensitivity and specificity for reliable use. Variants of all kinds can be linked to aberrant splicing in disease and the need for correct identification and diagnosis grows ever more crucial as novel splice-switching antisense oligonucleotide therapies start to enter clinical usage. RT-PCR provides a useful targeted assay of the splicing effects of identified variants, while minigene assays, massive parallel reporter assays and animal models can also be used for more detailed study of a particular splicing system, given enough time and resources. However, RNA-sequencing (RNA-seq) has the potential to be used as a rapid diagnostic tool in genomic medicine. By utilising data science approaches and machine learning, it may prove possible to finally understand and interpret the 'splicing code' and apply this knowledge in human disease diagnostics. [ABSTRACT FROM AUTHOR]

Variable levels of gene expression between tissues complicates the use of RNA sequencing of patient biosamples to delineate the impact of genomic variants. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA sequencing. This overcomes limitations of using expression values alone as a metric to predict RNA-sequencing utility. We have derived a metric, minimum required sequencing depth (MRSD), that estimates the depth of sequencing required from RNA sequencing to achieve user-specified sequencing coverage of a gene, transcript, or group of genes. We applied MRSD across four human biosamples: whole blood, lymphoblastoid cell lines (LCLs), skeletal muscle, and cultured fibroblasts. MRSD has high precision (90.1%–98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that fibroblasts, of these four biosamples, are the optimum source of RNA for 63.1% of gene panels. Using this approach, up to 67.8% of the variants of uncertain significance in ClinVar that are predicted to impact splicing could be assayed by RNA sequencing in at least one of the biosamples. We demonstrate the utility and benefits of MRSD as a metric to inform functional assessment of splicing aberrations, in particular in the context of Mendelian genetic disorders to improve diagnostic yield. [ABSTRACT FROM AUTHOR]