Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194−12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977−7C>G induced in frame inclusion of 6 nt from the 3′ end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.     

AG‐exclusion zone revisited: Lessons to learn from 91 intronic NF1 3′ splice site mutations outside the canonical AG‐dinucleotides

Uncovering frequent motives of action by which variants impair 3′ splice site (3′ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini‐gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722‐11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG‐exclusion zone (AGEZ) between the authentic 3′ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3′ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single‐nucleotide variants upstream of positions ‐3 interrupt the AGEZ. Of these AGEZ‐interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3′ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3′ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position ‐3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice‐neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.     

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion‐Almeida type

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion‐Almeida type (MFDGA). MFDGA‐associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss‐of‐function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss‐of‐function are self‐evident, the mechanisms by which missense variants are disease‐causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss‐of‐function. Only four of 19 assessed missense variants cause EFTUD2 loss‐of‐function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre‐messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory‐based validation of bioinformatic predictions for EFTUD2 missense variants.     

U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prediction of Mutant mRNA Splice Isoforms by Information Theory‐Based Exon Definition

Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon‐skipping isoforms in mRNA produced by splicing mutations from the combined information contents (R_i, which measures binding‐site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (R_i,total) is the sum of the R_i values of its acceptor and donor splice sites, adjusted for the self‐information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (ΔR_i,total) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis ( http://splice.uwo.ca ) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations.     

RNA analysis reveals splicing mutations and loss of expression defects in MLH1 and BRCA1

The classical paradigm of mutation screening seeks to relate alterations in DNA sequence to their effect at the protein level. However, the majority of missense mutations are problematic as their pathological significance is often unclear. In order to test the hypothesis that many missense mutations primarily cause defects at the RNA rather than the protein level, we have performed retrospective RNA analysis of 12 individuals carrying missense mutations in the cancer predisposition genes APC, BRCA1, BRCA2, MLH1, and MSH2. RNA was extracted from peripheral blood samples and RT-PCR performed in order to assess the splicing and expression of the mutant allele in each case. Four of the 12 missense mutations analysed were associated with RNA defects. We detected two cases of exon skipping and one case of partial intron inclusion with activation of a cryptic intronic splice site in MLH1. A fourth case was associated with monoallelic expression of BRCA1. In addition, allele-specific analysis of common coding polymorphisms identified a further case of monoallelic BRCA1 expression in one of two individuals who had previously screened as mutation-negative. Although we were unable to identify the underlying cause of this loss of expression, it strongly suggests the presence of a pathogenic defect in BRCA1 in this case, highlighting the use of allelic expression studies as a method of mutation scanning. Finally, we used our dataset to test the ability of several in silico sequence analysis tools to identify splicing defects. Our results suggest that a significant number of missense mutations in cancer predisposition genes are associated with defects of RNA splicing, and that the use of gene- and splice site prediction software can aid in identifying such mutations.     

Leveraging splice‐affecting variant predictors and a minigene validation system to identify Mendelian disease‐causing variants among exon‐captured variants of uncertain significance

The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice‐affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT‐AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice‐disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines.     

The ETFDH c.158A>G Variation Disrupts the Balanced Interplay of ESE‐ and ESS‐Binding Proteins thereby Causing Missplicing and Multiple Acyl‐CoA Dehydrogenation Deficiency

Multiple acyl‐CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull‐down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease‐causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.     

Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene

Missense, iso‐semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5′ or 3′ splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5′ or 3′ splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.     

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.