A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Blakes, Alexander J M; Wai, Htoo A.; Davies, Ian; Moledina, Hassan E; Ruiz, April; Thomas, Tessy; Bunyan, David J.; Thomas, N. Simon; Burren, Christine P; Greenhalgh, Lyn; Lees, Melissa; Pichini, Amanda; Smithson, Sarah; Tavares, Ana Lisa Taylor; O′Donovan, Peter; Douglas, Andrew G.L.; Whiffin, Nicola; Baralle, Diana; Lord, Jenny

doi:10.1101/2022.01.28.22270002

Cited by 5 publications

(4 citation statements)

References 41 publications

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“…For each variant, four predicted probabilities were obtained: probabilities of introducing a splice Acceptor Gain (AG), Acceptor Loss (AL), Donor Gain (DG), or Donor Loss (DL) accompanied by a predicted position of such change relative to each variant. We computed an aggregate SpliceAI score to incorporate contributions from each of the 4 predicted categories using a previously described formula 20 : We considered a high likelihood of disrupting splicing to be greater than 0.80, and a low likelihood less than 0.25.…”

Section: Methodsmentioning

confidence: 99%

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

O’Neill

Wada

Hall

et al. 2022

Preprint

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Background: Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the congenital Long QT Syndrome (LQTS). While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. Objective: We implemented two functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance (VUS) and 1 likely pathogenic (LP) variant without functional data observed in BrS and LQTS probands. Methods: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells (iPSCs) by CRISPR genome editing. We differentiated cells into iPSC-derived cardiomyocytes (iPSC-CMs) and studied splicing outcomes by reverse transcription-polymerase chain reaction (RT-PCR). We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. Results: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in iPSC-CMs. We reclassified 9 VUS to LP, 1 VUS to Likely Benign, and 1 LP variant to pathogenic. Conclusions: Functional assays reclassified splice-altering variants outside canonical splice sites in BrS- and LQTS-associated genes.

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Section: Methodsmentioning

confidence: 99%

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

O’Neill

Wada

Hall

et al. 2022

Preprint

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“…The branch point is a cis-acting intronic sequence that lies about 18 to 40 nucleotides upstream of the 3′ end of an intron and is crucial during the splicing reaction [26]. Variants at or around the branch point can also influence canonical splicing and cause disease (Figure 5) [27].…”

Section: Branch Point Variantsmentioning

confidence: 99%

Practical Recommendations for the Selection of Patients for Individualized Splice-Switching ASO-Based Treatments

Zardetto,

Lauffer,

van Roon-Mom

et al. 2024

Human Mutation

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Although around 6% of the world’s population is affected by rare diseases, only a small number of disease-modifying therapies are available. In recent years, antisense oligonucleotides (ASOs) have emerged as one option for the development of therapeutics for orphan diseases. In particular, ASOs can be utilized for individualized genetic treatments, addressing patients with a known disease-causing genetic variant, who would otherwise not be able to receive therapy. Careful prioritization of genetic variants amenable to an ASO approach is crucial to increase chances for successful treatments and reduce costs and time for drug development. At present, there is no consensus on how to systematically approach this selection procedure. Here, we present practical guidelines to evaluate disease-causing variants and standardize the process of selecting n-of-1 cases. We focus on variants leading to a loss of function in monogenic disorders and consider which splice-switching ASO-mediated treatments are applicable in each case. To ease the understanding and application of our guidelines, we created a hypothetical transcript covering different pathogenic variants and explained their evaluation in detail. We support our recommendations with real-life examples and add further considerations to be applied to specific cases to provide a comprehensive framework for selecting eligible variants.

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“…While this type of approach has been applied to identify genetic variation under strong selection in non-coding regions such as 5’ UTRs (Whiffin et al 2020) and introns at splice sites (Blakes et al 2022; Lord et al 2019), it has not yet been applied to 3’ UTRs, suggesting that signals of negative selection may be challenging to uncover in these regions. Instead, the few instances where negative selection in 3’ UTRs has been inferred have been limited in scope, did not adjust for mutability, and were secondary to other efforts (Zhang et al 2020; Park et al 2021; Kainov et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Quantifying negative selection in human 3’ UTRs uncovers constrained targets of RNA-binding proteins

Findlay

Romo

Burge

2022

Preprint

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Many non-coding variants associated with phenotypes occur in 3’ untranslated regions (3’ UTRs) and may affect interactions with RNA-binding proteins (RBPs) to regulate post-transcriptional gene expression. However, identifying functional 3’ UTR variants has proven difficult. We used allele frequencies from the Genome Aggregation Database (gnomAD) to identify classes of 3’ UTR variants under strong negative selection in humans. We developed intergenic mutability-adjusted proportion singleton (iMAPS), a generalized measure related to MAPS, to quantify negative selection in non-coding regions. This approach, in conjunction within vitroandin vivobinding data, identifies precise RBP binding sites, miRNA target sites, and polyadenylation signals (PASs) under strong selection. For each class of sites, we identified thousands of gnomAD variants under selection comparable to missense coding variants, and found that sites in core 3’ UTR regions upstream of the most-used PAS are under strongest selection. Together, this work improves our understanding of selection on human genes and validates approaches for interpreting genetic variants in human 3’ UTRs.

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A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Cited by 5 publications

References 41 publications

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

Practical Recommendations for the Selection of Patients for Individualized Splice-Switching ASO-Based Treatments

Quantifying negative selection in human 3’ UTRs uncovers constrained targets of RNA-binding proteins

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