Identification of <i>MLH1</i> exon 10 splicing mutations by using a pSPL3m-M1e10 minigene splicing reporter assay.

<p>(A) Distribution of all SNVs reported in <i>MLH1</i> exon 10 (n = 22). The diagram shows the nucleotide composition of <i>MLH1</i> exon 10 (c.791-c.884) and the corresponding amino-acid sequence (1-letter code, p.264-p.295), as well as the position and identity of each SNV. (B) Structure of the pSPL3m-M1e10 minigenes used in the splicing reporter assay. Boxes represent exons and lines in between indicate introns. The minigenes were generated by inserting a genomic fragment containing <i>MLH1</i> exon 10 and upstream/downstream flanking intronic sequences (168/187 nucleotides, respectively) into the intron of pSPL3m, as described under Materials and Methods. Arrows above the exons represent RT-PCR primers used in the splicing reporter assay. SV40, SV40 promoter; Poly A, polyadenylation site. (C) Analysis of the splicing pattern of pSPL3m-M1e10 minigenes containing the variants indicated in (A). Wild-type (WT) and mutant pSPL3m-M1e10 constructs were transfected into HeLa cells and then the minigenes’ transcripts were analyzed by RT-PCR as described under Materials and Methods. The top panel shows the RT-PCR products, obtained for WT and mutant constructs as indicated, separated on a 2.5% agarose gel stained with ethidium bromide. The identities of the two major RT-PCR products, with or without exon 10 (b and c, respectively), are indicated on the left. All the products (a, b, c, d, e and f) are described in detail in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005756#pgen.1005756.s001" target="_blank">S1 Fig</a>, f corresponding to RT-PCR products containing exon 10 deleted of the last 48 nucleotides. The bottom panel shows the quantification of the RT-PCR products. Results are shown as the average of three independent experiments and are expressed as percentage of exon inclusion ([exon inclusion products (a+b) x 100/total transcripts]). Error bars indicate individual standard deviation (SD) values, whereas the horizontal dashes delineate the limits of the SD bar obtained for WT (79±9; i.e. 70% and 88% exon inclusion). Variants producing exon inclusion levels outside this range were considered as splicing mutations.</p

Detection of aberrant splicing in the blood cells of a patient carrying <i>MLH1</i> c.793C>T variant.

<p>(A) Comparative RT-PCR analysis of fresh blood RNA samples obtained from 3 healthy control individuals (C1, C2 and C3) and from a patient carrying the heterozygous variant <i>MLH1</i> c.793C>T (P_793CT.1). RT-PCR reactions were performed with primers mapping to <i>MLH1</i> exon 8 (forward primer) and exon 12 (reverse primer), as described under Materials and Methods. The panel on the left shows the RT-PCR products separated on a 2% agarose gel, and is representative of 3 independent experiments. RT-PCR product identifiers are indicated on the left of the gel. The sequence on the right refers to the FL product obtained from patient P_793CT.1, with a vertical line indicating the junction between exons 9 and 10. FL, full-length; Δ, exon skipping. (B) Allele-specific expression of <i>MLH1</i> in the blood cells of patient P_793CT.1 carrying the heterozygous variant <i>MLH1</i> c.793C>T. PCR, RT-PCR and primer extension reactions were performed as described under Materials and Methods, and are schematically represented above the graphs. The discriminating nucleotides C and T are indicated. Boxes represent exons, lines indicate intronic sequences, and arrows symbolize reaction primers. The discriminating fluorophore-labeled ddG and ddA terminators, incorporated into the noncoding strand at the c.793 position, are represented by stars. Results from complementary DNA (cDNA) analysis and genomic DNA (gDNA) are shown on the left-hand and on the right-hand graphs, respectively. Results (peak areas) obtained with cDNA were normalized to those obtained with gDNA and are expressed, in the left-hand graph, as relative level (in percentage) of FL transcripts produced by each <i>MLH1</i> allele. (C) Relative contribution of each allele to the expression of full-length <i>MLH1</i> transcripts in the blood cells of patient P_793CT.1 carrying the heterozygous variant <i>MLH1</i> c.793C>T. The graph displays the expression level of the mutant allele (<i>MLH1</i> c.793C>T) relative to wild-type (WT). Results are shown as the average of 3 independent experiments performed as described in (B). Error bars indicate standard deviation values.</p

Characterization of <i>MLH1</i> exon 10 variants affecting potential ESRs by using an ESR-dependent minigene assay.

<p>(A) Strategy for mapping potential splicing regulatory regions in <i>MLH1</i> exon 10. The horizontal bars under the sequence of <i>MLH1</i> exon 10 represent the ~30 bp-long exonic fragments tested in the ESR-dependent minigene reporter assay (R1 to R4, nucleotide coordinates as indicated). Variants selected for ESR-dependent analysis are indicated above the exon. (B) Structure of the pcDNA-Dup-M1e10-R minigenes used in the ESR-dependent reporter assay. Minigenes were prepared by inserting individual wild-type or mutant <i>MLH1</i> exon 10 fragments (M1e10-R as indicated) into the middle exon of pcDNA-Dup, a three-exon vector with a central exon particularly sensitive to ESRs. Boxes indicate exons and lines in between indicate introns. Arrows above the exons represent RT-PCR primers used in the ESR-dependent reporter assay. CMV, CMV promoter; Poly A, polyadenylation site; ßg1 and ßg2, ß-globin exons 1 and 2. (C) Analysis of the splicing pattern of pcDNA-Dup-M1e10-R minigenes containing different wild-type (WT) <i>MLH1</i> exon 10 fragments, as indicated. After transfection into HeLa cells the minigenes’ transcripts were analyzed by RT-PCR as described under Materials and Methods. The image shows the RT-PCR products separated on a 2.5% agarose gel stained with ethidium bromide and is representative of 3 independent experiments. The identities of the RT-PCR products are indicated on the left. Empty, BR11, SC35 and SF2ASF refer to previously described negative and positive control pcDNA-Dup minigenes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005756#pgen.1005756.ref013" target="_blank">13</a>]. (D) Comparative analysis of the splicing pattern of pcDNA-Dup-M1e10-R minigenes containing either wild-type or mutant <i>MLH1</i> exon 10 fragments, as indicated. The assay was performed as described in (C). The image shows the RT-PCR products separated on a 2.5% agarose gel stained with ethidium bromide and is representative of 3 independent experiments. WT, wild-type.</p

Correlation analysis between exon 10 inclusion levels and results obtained from new ESR-dedicated bioinformatics tools.

<p>(A), (B) and (C) refer to results obtained with ΔtESRseq-, ΔHZ_EI- and ΔΨ-based bioinformatics approaches, respectively, as described under Materials and Methods. Only <i>MLH1</i> exon 10 variants located outside the sequences that define the reference splice sites were retained for this analysis as already mentioned in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005756#pgen.1005756.g003" target="_blank">Fig 3</a>. The precise correspondence between each Δ value (ΔtESRseq, ΔHZ_EI or ΔΨ), the level of exon inclusion observed in the pSPL3m-M1e10 minigene assay, and the identity of the corresponding <i>MLH1</i> exon 10 variant, is indicated on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005756#pgen.1005756.s008" target="_blank">S1 Table</a>. Correlation coefficients (r) and p-values were determined by performing a Pearson correlation analysis, as described under Materials and Methods.</p

Comparison of pSPL3m-M1e10 minigene data with results obtained from new ESR-dedicated bioinformatics tools.

<p>Top, middle and bottom panels refer to results obtained with ΔtESRseq-, ΔHZ_EI- and ΔΨ-based bioinformatics approaches, respectively, as described under Materials and Methods. <i>MLH1</i> exon 10 variants located within the sequences that define the reference splice sites were eliminated from this analysis. Retained variants were separated into 3 groups depending on their impact on splicing as determined on the pSPL3m-M1e10 minigene assay and indicated above the graphs. Dashed lines indicate the thresholds used in this study. P-values were calculated by using the one-way ANOVA test, as described under Materials and Methods.</p

Additional file 1: of Identification of genetic variants for clinical management of familial colorectal tumors

Table S1. Primers used in the pCAS2 minigene splicing assay. (DOCX 15 kb

Additional file 1: of Genetic variants of prospectively demonstrated phenocopies in BRCA1/2 kindreds

The concentration in a 10 ml PCR was 1xThermopol Reaction Buffer with 2 mM MgS04, 0.3 μM “reverse” primers, 0.15 μM “forward” primer, 0.1 μM, 6-Carboxyfluorescein-GC clamp primer, 600 μM dNTP, 100 μg Bovine Serum Albumine (Sigma-Aldrich, Oslo, Norway) and 0.75 U Taq DNA polymerase. Plates were sealed with two strips of electrical tape (Clas Ohlson, Oslo, Norway). The temperature cycling was repeated 35 times; 94 °C for 30 s, annealing temperature held for 30 s and extension at 72 °C for 60 s (Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany)). Table S1. primers used to amplify PCR product to be analysed by cycling temperature capillary electrophoresis. (DOCX 16 kb