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Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene., (© 2021 Wiley Periodicals LLC.)

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC., (© 2019 Wiley Periodicals, Inc.)

Tumors carrying hereditary mutations in BRCA1, which attenuate the BRCA1 DNA damage repair pathway, are more susceptible to dual treatment with PARP inhibitors and DNA damaging therapeutics. Conversely, breast cancer tumors with nonmutated functional BRCA1 are less sensitive to PARP inhibition. We describe a method that triggers susceptibility to PARP inhibition in BRCA1-functional tumor cells. BRCA1 exon 11 is a key for the function of BRCA1 in DNA damage repair. Analysis of the BRCA1 exon 11 splicing mechanism identified a key region within this exon which, when deleted, induced exon 11 skipping. An RNA splice-switching oligonucleotide (SSO) developed to target this region was shown to artificially stimulate skipping of exon 11 in endogenous BRCA1 pre-mRNA. SSO transfection rendered wild-type BRCA1 expressing cell lines more susceptible to PARP inhibitor treatment, as demonstrated by a reduction in cell survival at all SSO concentrations tested. Combined SSO and PARP inhibitor treatment increased γH2AX expression indicating that SSO-dependent skipping of BRCA1 exon 11 was able to promote DSBs and therefore synthetic lethality. In conclusion, this SSO provides a new potential therapeutic strategy for targeting BRCA1-functional breast cancer by enhancing the effect of PARP inhibitors., (© 2016 UICC.)

Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among disease-causing exonic substitutions is unknown. Here, we have tested missense and silent mutations deposited in the BRCA1 mutation databases of unclassified variants for their effects on exon inclusion. Analysis of 21 BRCA1 variants using minigene assays revealed a single exon-skipping mutation c.231G>T. Comprehensive mutagenesis of an adjacent 12-nt segment showed that this silent mutation resulted in a higher level of exon skipping than the 35 other single-nucleotide substitutions. Exon inclusion levels of mutant constructs correlated significantly with predicted splicing enhancers/silencers, prompting the development of two online utilities freely available at http://www.dbass.org.uk. EX-SKIP quickly estimates which allele is more susceptible to exon skipping, whereas HOT-SKIP examines all possible mutations at each exon position and identifies candidate exon-skipping positions/substitutions. We demonstrate that the distribution of exon-skipping and disease-associated substitutions previously identified in coding regions was biased toward top-ranking HOT-SKIP mutations. Finally, we show that proteins 9G8, SC35, SF2/ASF, Tra2, and hnRNP A1 were associated with significant alterations of BRCA1 exon 6 inclusion in the mRNA. Together, these results facilitate prediction of exonic substitutions that reduce exon inclusion in mature transcripts., (© 2011 Wiley-Liss, Inc.)

Genomic variations with no apparent effect ("neutral polymorphisms") may have a significant effect on splicing. The effect of this type of mutation is difficult to spot, unless a functional assay is undertaken. In our study, DNA sequencing of a patient with clinically defined neurofibromatosis type 1 (NF1) showed only a single polymorphism in intron 30 due to an A>G transition 279 nucleotides from the 3' splice site. Using a minigene splicing assay we conclusively show that this change produces a cryptic exon with a 3' SS defined by the nucleotide change and the unexpected activation of a very weak 5'SS. Further site directed mutagenesis studies aimed at identifying the signals involved in the cryptic exon inclusion were carried out. Interestingly we find that particular characteristics of the cryptic 5' SS are essential for its inclusion. Significantly an additional single nucleotide change disrupting the cryptic 5'ss consensus sequence rescues the effect of the pathogenetic mutation resulting in normal splicing., (2006 Wiley-Liss, Inc.)

A female patient of normal intelligence with short stature and Madelung deformity is reported with Léri-Weill dyschondrosteosis and a de novo pseudodicentric X;Y translocation chromosome. The phenotype is consistent with the observed deletion of the SHOX gene by FISH and molecular studies. The Y chromosome breakpoint was in the short arm but proximal to SRY, consistent with her phenotypic sex. X-inactivation studies have shown a skewed pattern in favour of the dic (X;Y) chromosome. The ARSE gene was also deleted on the dic (X;Y) chromosome but chondrodysplasia punctata was not expressed, as CDP is recessive and ARSE escapes inactivation on the normal X chromosome. Breakpoint mapping assisted in karyotype/phenotype correlation and reproductive counselling. In particular, molecular analysis showed that the putative MRX 49 gene for mental retardation is unlikely to be deleted in this case.