I’m a researcher on BRCA1 and BRCA2 genes and I have a translational activity. I work at Curie Institute hospital curating the UMD-BRCA1/BRCA2 database (http://www.umd.be/) (new name BRCAShare). In France I coordinate a project whose main objective is to classify a maximum of BRCA1/2 VUS of this database by co-segregation studies: the COVAR study (https://clinicaltrials.gov/ct2/show/NCT01689584). In my laboratory, I have already implemented several tools to evaluate BRCA1/2 VUS at the functional level. Their current project is the classification of BRCA1/2 variants.'
Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Varian... more Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Variants selected in BRCA2 exon 12 and its flanking intronic regions. Table S3. Overview of bioinformatics predictions and experimental data obtained for the 40 selected BRCA2 exon 12 variants. Table S4. Clinical and family data of patients carrying BRCA2 exon 12 spliceogenic variants.
Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary... more Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of presumed LoF variants; Figure S3. Bioinformatics predictions of 3'/5' ss alterations for variants located at position IVS{plus minus}1/2 of BRCA2 exon 12; Figure S4. Bioinformatics analysis of variants predicted to alter 3'/5' ss of BRCA2 exon 12 (â^†MES {less than or equal to} -15%); Figure S5. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of variants predicted to alter 3'/5' ss (A) or ESR (B); Figure S6. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in control and patient lymphoblastoid cell lines; Figure S7. RT-PCR analysis of BRCA2 exon 12 splicing patterns in puromycin- or mock-treated lymphoblastoid cell lines of control individuals and patients carrying the c.6844G>T or c.6901G>T nonsense variants; Figure S8. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in variant-expressing mESC; Figure S9. Quantitation of BRCA2 protein expression in mESC; Figure S10. Sensitivity of BRCA2 variants to cisplatin and PARP inhibitors.
Western blot analysis of the expression of BRCA1 and I-SceI measured after expression of either W... more Western blot analysis of the expression of BRCA1 and I-SceI measured after expression of either WT BRCA1 or VUS in the HR assays. BRCA1 was revealed using an anti-BRCA1 (mouse, ab16780, Abcam) and I-SceI with an anti-HA (mouse, sc-7392, Santa Cruz Biotechnology Inc.).
Supplementary table I: List of 78 BRCA1 BRCT missense VUS from the UMD-BRCA1 database. Bioinforma... more Supplementary table I: List of 78 BRCA1 BRCT missense VUS from the UMD-BRCA1 database. Bioinformatics in silico protein and 3D prediction scores, classification from BRCAShare (ex-UMD-BRCA1), BIC, kConfab and ClinVar databases. Supplementary table II: Classification of the BRCA1 VUS C1697R on the basis of multifactorial information Supplementary table III: Quantification of the percentage of fluorescence corresponding to nuclear BRCA1. The yes/no results extracted from this quantification to obtain Figure 3B were deduced from a statistical analysis using a t-test calculation.
Isothermal Titration Calorimetry curves obtained by adding the different phosphopeptides at (A-B)... more Isothermal Titration Calorimetry curves obtained by adding the different phosphopeptides at (A-B) 200 mM onto the WT BRCT domains at 20 mM and (C-D) 100 mM onto the WT BRCT domains at 10 mM .
Size Exclusion Chromatography profiles obtained on BRCA1 BRCT domains free (red curve) or in comp... more Size Exclusion Chromatography profiles obtained on BRCA1 BRCT domains free (red curve) or in complex with BACH1-P (purple), AB-1P (green), AB-2P (blue). The experiment was performed using a Superdex-75 10/300 GL column (GE Healthcare) pre-equilibrated with 50mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol and protease inhibitors (Roche).
Germline nonsense and canonical splice site variants identified in disease-causing genes are gene... more Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications.Significance:This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.
Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Varian... more Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Variants selected in BRCA2 exon 12 and its flanking intronic regions. Table S3. Overview of bioinformatics predictions and experimental data obtained for the 40 selected BRCA2 exon 12 variants. Table S4. Clinical and family data of patients carrying BRCA2 exon 12 spliceogenic variants.
Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary... more Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of presumed LoF variants; Figure S3. Bioinformatics predictions of 3'/5' ss alterations for variants located at position IVS{plus minus}1/2 of BRCA2 exon 12; Figure S4. Bioinformatics analysis of variants predicted to alter 3'/5' ss of BRCA2 exon 12 (â^†MES {less than or equal to} -15%); Figure S5. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of variants predicted to alter 3'/5' ss (A) or ESR (B); Figure S6. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in control and patient lymphoblastoid cell lines; Figure S7. RT-PCR analysis of BRCA2 exon 12 splicing patterns in puromycin- or mock-treated lymphoblastoid cell lines of control individuals and patients carrying the c.6844G>T or c.6901G>T nonsense variants; Figure S8. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in variant-expressing mESC; Figure S9. Quantitation of BRCA2 protein expression in mESC; Figure S10. Sensitivity of BRCA2 variants to cisplatin and PARP inhibitors.
Western blot analysis of the expression of BRCA1 and I-SceI measured after expression of either W... more Western blot analysis of the expression of BRCA1 and I-SceI measured after expression of either WT BRCA1 or VUS in the HR assays. BRCA1 was revealed using an anti-BRCA1 (mouse, ab16780, Abcam) and I-SceI with an anti-HA (mouse, sc-7392, Santa Cruz Biotechnology Inc.).
Supplementary table I: List of 78 BRCA1 BRCT missense VUS from the UMD-BRCA1 database. Bioinforma... more Supplementary table I: List of 78 BRCA1 BRCT missense VUS from the UMD-BRCA1 database. Bioinformatics in silico protein and 3D prediction scores, classification from BRCAShare (ex-UMD-BRCA1), BIC, kConfab and ClinVar databases. Supplementary table II: Classification of the BRCA1 VUS C1697R on the basis of multifactorial information Supplementary table III: Quantification of the percentage of fluorescence corresponding to nuclear BRCA1. The yes/no results extracted from this quantification to obtain Figure 3B were deduced from a statistical analysis using a t-test calculation.
Isothermal Titration Calorimetry curves obtained by adding the different phosphopeptides at (A-B)... more Isothermal Titration Calorimetry curves obtained by adding the different phosphopeptides at (A-B) 200 mM onto the WT BRCT domains at 20 mM and (C-D) 100 mM onto the WT BRCT domains at 10 mM .
Size Exclusion Chromatography profiles obtained on BRCA1 BRCT domains free (red curve) or in comp... more Size Exclusion Chromatography profiles obtained on BRCA1 BRCT domains free (red curve) or in complex with BACH1-P (purple), AB-1P (green), AB-2P (blue). The experiment was performed using a Superdex-75 10/300 GL column (GE Healthcare) pre-equilibrated with 50mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol and protease inhibitors (Roche).
Germline nonsense and canonical splice site variants identified in disease-causing genes are gene... more Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications.Significance:This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.
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