No abstract

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

Numerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild-type exons to be tested were PCR-amplified from patient genomic DNA together with approximately 150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE-dependent central exon of a three-exon splicing minigene and we showed in cell transfection experiments that the wild-type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases.

No abstract

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for "unclassified variant") found in genetic screenings represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patient genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing. This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA, for the identification of pathogenic splicing mutations.

Recent studies have revealed a significant proportion of BRCA1 exon deletions or duplications in breast-ovarian cancer families with high probability of BRCA1- or BRCA2-linked predisposition, in which mutations of these genes have not been found. The difficulty of detecting such heterozygous rearrangements has stimulated the development of several new screening methods. Quantitative fluorescent multiplex PCR is based on simultaneous amplification of multiple target sequences under conditions that allow rapid and reliable quantitative comparison of the fluorescence of each amplicon in test samples and in controls. The modified method described here, named quantitative multiplex PCR of short fluorescent fragments (QMPSF), is particularly well suited for large genes. All BRCA1 coding exons were analyzed using four multiplexes in 52 families without point mutations in the exons or splice-sites of BRCA1 and BRCA2, and selected because of high probability of a BRCA1- or BRCA2-linked genetic predisposition. Five distinct BRCA1 rearrangements were detected: a deletion of exons 8-13, a duplication of exons 3-8, a duplication of exons 18-20, a deletion of exons 15-16, and a deletion of exons 1-22-which is the largest deletion found so far within the BRCA1 gene. The method described here lends itself to rapid, sensitive, and cost-effective search of BRCA1 rearrangements and may be included into the routine molecular analysis of breast-ovarian cancer predispositions. Hum Mutat 20:218-226, 2002.

We show that functional analysis using a splicing reporter minigene is sensitive and specific, and should be used for initial screening of potential splicing defects, especially when patient RNA is not readily available.

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+3A4G and BRCA1 c.5194À12G4A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977À7C4G induced in frame inclusion of 6 nt from the 3¢ end of intron 17. The novel variants BRCA2 c.520C4T and BRCA2 c.7992T4A 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. Keywords: variants of unknown significance; splicing defects; splicing reporter minigene; breast and ovarian cancer; BRCA1 and BRCA2 INTRODUCTIONThe interpretation of variants of unknown significance (VUS) found in the molecular screenings of the breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, is essential for genetic counseling of patients and their families and for the implementation of new therapies targeted to carriers of BRCA mutations, such as those based on poly-(ADP-ribose) polymerase inhibitors. 1 The number of these variants already exceeds that of the reported pathogenic mutations, and is expected to increase rapidly with the use of new high throughput sequencing technologies that are based on massive parallel sequencing. 2 It is now widely accepted that RNA analyses should be used to improve the assessment of pathogenicity of sequence variation, because a large fraction of sequence variants, both intronic and exonic, may induce splicing defects. However, in many cases, patient RNA is either not available or it has been obtained in ways that do not ensure its stability. Moreover, it is sometimes difficult to detect the mRNA affected by a truncating mutation because of the activation of the nonsense-mediated mRNA decay pathway. 3 Functional assays of the effect of VUS on RNA splicing, using patient genomic DNA and a splicing reporter hybrid minigene,