Validation of predicted mRNA splicing mutations using high-throughput transcriptome data

Viner, Coby; Dorman, Stephanie; Shirley, Ben C.; Rogan, Peter K.

doi:10.12688/f1000research.3-8.v2

Cited by 18 publications

(23 citation statements)

References 40 publications

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“…Hollestelle et al. [] argue that a more stringent statistical standard must be applied (i.e., P ‐values of 0.01 should be used as opposed to 0.05) to underreported variants (namely in moderate‐risk alleles), because of failure to replicate pathogenic variants, which we have also found [Viner et al., ]. In the same way that we use IT‐based analysis to justify prioritizing variants for further investigation, variants that are disregarded as lower priority (and that are likely not disease causing) have been subjected to the same thresholds and criteria.…”

Section: Discussionmentioning

confidence: 67%

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking KnownBRCAMutations

et al. 2016

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BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including noncoding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize noncoding variants of uncertain significance in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common framework to evaluate potential affinity changes in transcription factor (TFBSs), splicing regulatory (SRBSs), and RNA-binding protein (RBBSs) binding sites following mutation. We prioritized variants affecting the strengths of 10 splice sites (four natural, six cryptic), 148 SRBS, 36 TFBS, and 31 RBBS. Three variants were also prioritized based on their predicted effects on mRNA secondary (2°) structure and 17 for pseudoexon activation. Additionally, four frameshift, two in-frame deletions, and five stop-gain mutations were identified. When combined with pedigree information, complete gene sequence analysis can focus attention on a limited set of variants in a wide spectrum of functional mutation types for downstream functional and co-segregation analysis.

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Section: Discussionmentioning

confidence: 67%

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking KnownBRCAMutations

et al. 2016

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“…Exon-based expression microarrays and q-RT-PCR techniques were initially used to confirm the predicted impact of common and rare SNPs on splicing. Results were subsequently confirmed using RNAseq data for some of these SNPs (Dorman et al, 2014;Viner et al, 2014;Shirley et al, 2019). However, exon skipping due to rs1893592 was not consistently seen in all carriers.…”

Section: Discussionmentioning

confidence: 82%

“…This facilitates prediction of phenotypic severity (Rogan and Schneider, 1995;von Kodolitsch et al, 1999;von Kodolitsch et al, 2006). The effects of splicing mutations can be predicted in silico by information theory (Rogan and Schneider, 1995;Kannabiran et al, 1998;Rogan et al, 1998;Svojanovsky et al, 2000;Rogan et al, 2003;Caminsky et al, 2014;Dorman et al, 2014;Viner et al, 2014;Caminsky et al, 2016;Mucaki et al, 2016;Shirley et al, 2019), and these predictions can be confirmed by in vitro experimental studies (Vockley et al, 2000;Lamba et al, 2003;Rogan et al, 2003;Khan et al, 2004;Susani et al, 2004;Hobson et al, 2006;Caux-Moncoutier et al, 2009;Olsen et al, 2014;Vemula et al, 2014;Peterlongo et al, 2015). Strengths of one or more splice sites may be altered and, in some instances, concomitant with amino acid changes in coding sequences (Rogan et al, 1998;Peterlongo et al, 2015).…”

Section: Introductionmentioning

confidence: 98%

“…Large transcriptome studies have suggested that a large fraction of genome-wide association studies (GWAS) signals for disease and complex traits are due to single nucleotide polymorphisms (SNPs) affecting mRNA splicing (Park et al, 2018). ValidSpliceMut (Shirley et al, 2019) presents evidence of altered splicing (Viner et al, 2014;Dorman et al, 2014) for 309,848 validated genome splice-variant predictions (Shirley et al, 2013). The majority of mutations were associated with exon skipping, cryptic site use, or intron retention, and in these cases ValidSpliceMut assigns a molecular phenotype classification to all variants as either aberrant, likely aberrant or inducing alternative isoforms.…”

Section: Introductionmentioning

confidence: 99%

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Expression Changes Confirm Genomic Variants Predicted to Result in Allele-Specific, Alternative mRNA Splicing

Mucaki

Shirley

2020

Front. Genet.

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Splice isoform structure and abundance can be affected by either noncoding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these nonconstitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays and custom designed q-RT-PCR or TaqMan assays. The majority of these SNPs (15 of 22) as well as an independent set of 24 variants were then subjected to RNAseq analysis using the ValidSpliceMut web beacon (http:// validsplicemut.cytognomix.com), which is based on data from the Cancer Genome Atlas and International Cancer Genome Consortium. SNPs from different genes analyzed with gene expression microarray and q-RT-PCR exhibited significant changes in affected splice site use. Thirteen SNPs directly affected exon inclusion and 10 altered cryptic site use. Homozygous SNP genotypes resulting in stronger splice sites exhibited higher levels of processed mRNA than alleles associated with weaker sites. Four SNPs exhibited variable expression among individuals with the same genotypes, masking statistically significant expression differences between alleles. Genome-wide information theory and expression analyses (RNAseq) in tumor exomes and genomes confirmed splicing effects for 7 of the HapMap SNP and 14 SNPs identified from tumor genomes. q-RT-PCR resolved rare splice isoforms with read abundance too low for statistical significance in ValidSpliceMut. Nevertheless, the web-beacon provides evidence of unanticipated splicing outcomes, for example, intron retention due to compromised recognition of constitutive splice sites. Thus, ValidSpliceMut and q-RT-PCR represent complementary resources for identification of allele-specific, alternative splicing.

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“…separate transfections on different weeks) were converted to log‐ratios for calculation of averages and standard errors for each variant. This log transformation was sufficient to produce approximately normally distributed ratios in this dataset with relatively large read counts (not shown); however for a dataset with small read counts, a transformation of the counts would be required (Viner, Dorman, Shirley, & Rogan, ).…”

Section: Methodsmentioning

confidence: 99%

Characterizing variants of unknown significance in rhodopsin: A functional genomics approach

et al. 2019

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Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r 2 = 0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders. K E Y W O R D S functional genomics, inherited retinal diseases, next-generation sequencing, retinitis pigmentosa, rhodopsin, variant library, variant pathogenicity, variant of unknown significance, VUS

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Validation of predicted mRNA splicing mutations using high-throughput transcriptome data

Cited by 18 publications

References 40 publications

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking KnownBRCAMutations

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking KnownBRCAMutations

Expression Changes Confirm Genomic Variants Predicted to Result in Allele-Specific, Alternative mRNA Splicing

Characterizing variants of unknown significance in rhodopsin: A functional genomics approach

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