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The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area.

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area. [ABSTRACT FROM AUTHOR]

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area. [ABSTRACT FROM AUTHOR]

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts. [ABSTRACT FROM AUTHOR]