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An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition.

Muller D, Rouleau E, Schultz I, Caputo S, Lefol C, Bièche I, Caron O, Noguès C, Limacher JM, Demange L, Lidereau R, Fricker JP, Abecassis J - BMC Med. Genet. (2011)

Bottom Line: Deletion of exon 3, which is in phase, does not alter the reading frame.Exclusive expression of the delta3 transcript by the mutant allele and segregation data provide evidence for a causal effect of the exon 3 deletion.In addition, our findings suggest that, to interpret the pathogenic effect of any variants of exon 3, both accurate transcript quantification and co-segregation analysis are required.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of oncogenetic, Department of Biology and Pathology, Regional Cancer Centre Paul Strauss, BP30042, 67065 Strasbourg, France. dmuller@strasbourg.fnclcc.fr

ABSTRACT

Background: Germ-line mutations in the BRCA1 and BRCA2 genes are major contributors to hereditary breast/ovarian cancer. Large rearrangements are less frequent in the BRCA2 gene than in BRCA1. We report, here, the first total deletion of exon 3 in the BRCA2 gene that was detected during screening of 2058 index cases from breast/ovarian cancer families for BRCA2 large rearrangements. Deletion of exon 3, which is in phase, does not alter the reading frame. Low levels of alternative transcripts lacking exon 3 (Δ3 delta3 transcript) have been reported in normal tissues, which raises the question whether deletion of exon 3 is pathogenic.

Methods: Large BRCA2 rearrangements were analysed by QMPSF (Quantitative Multiplex PCR of Short Fluorescent Fragments) or MLPA (Multiplex Ligation-Dependent Probe Amplification). The exon 3 deletion was characterized with a "zoom-in" dedicated CGH array to the BRCA2 gene and sequencing. To determine the effect of exon 3 deletion and assess its pathogenic effect, three methods of transcript quantification were used: fragment analysis of FAM-labelled PCR products, specific allelic expression using an intron 2 polymorphism and competitive quantitative RT-PCR.

Results: Large rearrangements of BRCA2 were detected in six index cases out of 2058 tested (3% of all deleterious BRCA2 mutations). This study reports the first large rearrangement of the BRCA2 gene that includes all of exon 3 and leads to an in frame deletion of exon 3 at the transcriptional level. Thirty five variants in exon 3 and junction regions of BRCA2 are also reported, that contribute to the interpretation of the pathogenicity of the deletion. The quantitative approaches showed that there are three classes of delta3 BRCA2 transcripts (low, moderate and exclusive). Exclusive expression of the delta3 transcript by the mutant allele and segregation data provide evidence for a causal effect of the exon 3 deletion.

Conclusion: This paper highlights that large rearrangements and total deletion of exon 3 in the BRCA2 gene could contribute to hereditary breast and/or ovarian cancer. In addition, our findings suggest that, to interpret the pathogenic effect of any variants of exon 3, both accurate transcript quantification and co-segregation analysis are required.

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Genomic analysis of the Δ3 BRCA2 large rearrangement. a: Dedicated BRCA2 CGH array. The gene is represented at the top, with vertical boxes that indicate exon positions and sizes. Black plots are considered to be within the diploidy range (the y axis gives the log2 intensity ratios). The green dots indicate signals that were below the threshold for deletion (-0.4 log2 ratio). b: Sequence analysis of the smaller PCR product obtained by long-range PCR of proband DNA with the exon 3 large rearrangement in BRCA2 (Hg18/build36, 2006). The sequence crosses the breakpoint that begins in intron 2 and ends in intron 3.
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Figure 1: Genomic analysis of the Δ3 BRCA2 large rearrangement. a: Dedicated BRCA2 CGH array. The gene is represented at the top, with vertical boxes that indicate exon positions and sizes. Black plots are considered to be within the diploidy range (the y axis gives the log2 intensity ratios). The green dots indicate signals that were below the threshold for deletion (-0.4 log2 ratio). b: Sequence analysis of the smaller PCR product obtained by long-range PCR of proband DNA with the exon 3 large rearrangement in BRCA2 (Hg18/build36, 2006). The sequence crosses the breakpoint that begins in intron 2 and ends in intron 3.

Mentions: Zoom-in dedicated CGH-array to the BRCA2 gene described the deleted region to measure from 3.6 to 6.7 kb (Figure 1a). The breakpoints and precise size of the deletion were established by long-range PCR. The primer set used gave rise to the expected normal PCR product of 4,572 bp in the control DNA. In the mutant DNA, a lower product of approximately 500 bp was observed in addition to the normal PCR product. Sequencing of the lower PCR product (Figure 1b) showed that the deletion spans 4,063 bp and includes 925 bp of intron 2, the entire exon 3 (249 bp) and 2,889 bp of intron 3. The rearrangement (Δ3-LR) is formally described as g.31.790.289_31.794.351del, and the deletion c.68-925_316+2889del, p.Asp23_Leu105del.


An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition.

Muller D, Rouleau E, Schultz I, Caputo S, Lefol C, Bièche I, Caron O, Noguès C, Limacher JM, Demange L, Lidereau R, Fricker JP, Abecassis J - BMC Med. Genet. (2011)

Genomic analysis of the Δ3 BRCA2 large rearrangement. a: Dedicated BRCA2 CGH array. The gene is represented at the top, with vertical boxes that indicate exon positions and sizes. Black plots are considered to be within the diploidy range (the y axis gives the log2 intensity ratios). The green dots indicate signals that were below the threshold for deletion (-0.4 log2 ratio). b: Sequence analysis of the smaller PCR product obtained by long-range PCR of proband DNA with the exon 3 large rearrangement in BRCA2 (Hg18/build36, 2006). The sequence crosses the breakpoint that begins in intron 2 and ends in intron 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198910&req=5

Figure 1: Genomic analysis of the Δ3 BRCA2 large rearrangement. a: Dedicated BRCA2 CGH array. The gene is represented at the top, with vertical boxes that indicate exon positions and sizes. Black plots are considered to be within the diploidy range (the y axis gives the log2 intensity ratios). The green dots indicate signals that were below the threshold for deletion (-0.4 log2 ratio). b: Sequence analysis of the smaller PCR product obtained by long-range PCR of proband DNA with the exon 3 large rearrangement in BRCA2 (Hg18/build36, 2006). The sequence crosses the breakpoint that begins in intron 2 and ends in intron 3.
Mentions: Zoom-in dedicated CGH-array to the BRCA2 gene described the deleted region to measure from 3.6 to 6.7 kb (Figure 1a). The breakpoints and precise size of the deletion were established by long-range PCR. The primer set used gave rise to the expected normal PCR product of 4,572 bp in the control DNA. In the mutant DNA, a lower product of approximately 500 bp was observed in addition to the normal PCR product. Sequencing of the lower PCR product (Figure 1b) showed that the deletion spans 4,063 bp and includes 925 bp of intron 2, the entire exon 3 (249 bp) and 2,889 bp of intron 3. The rearrangement (Δ3-LR) is formally described as g.31.790.289_31.794.351del, and the deletion c.68-925_316+2889del, p.Asp23_Leu105del.

Bottom Line: Deletion of exon 3, which is in phase, does not alter the reading frame.Exclusive expression of the delta3 transcript by the mutant allele and segregation data provide evidence for a causal effect of the exon 3 deletion.In addition, our findings suggest that, to interpret the pathogenic effect of any variants of exon 3, both accurate transcript quantification and co-segregation analysis are required.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of oncogenetic, Department of Biology and Pathology, Regional Cancer Centre Paul Strauss, BP30042, 67065 Strasbourg, France. dmuller@strasbourg.fnclcc.fr

ABSTRACT

Background: Germ-line mutations in the BRCA1 and BRCA2 genes are major contributors to hereditary breast/ovarian cancer. Large rearrangements are less frequent in the BRCA2 gene than in BRCA1. We report, here, the first total deletion of exon 3 in the BRCA2 gene that was detected during screening of 2058 index cases from breast/ovarian cancer families for BRCA2 large rearrangements. Deletion of exon 3, which is in phase, does not alter the reading frame. Low levels of alternative transcripts lacking exon 3 (Δ3 delta3 transcript) have been reported in normal tissues, which raises the question whether deletion of exon 3 is pathogenic.

Methods: Large BRCA2 rearrangements were analysed by QMPSF (Quantitative Multiplex PCR of Short Fluorescent Fragments) or MLPA (Multiplex Ligation-Dependent Probe Amplification). The exon 3 deletion was characterized with a "zoom-in" dedicated CGH array to the BRCA2 gene and sequencing. To determine the effect of exon 3 deletion and assess its pathogenic effect, three methods of transcript quantification were used: fragment analysis of FAM-labelled PCR products, specific allelic expression using an intron 2 polymorphism and competitive quantitative RT-PCR.

Results: Large rearrangements of BRCA2 were detected in six index cases out of 2058 tested (3% of all deleterious BRCA2 mutations). This study reports the first large rearrangement of the BRCA2 gene that includes all of exon 3 and leads to an in frame deletion of exon 3 at the transcriptional level. Thirty five variants in exon 3 and junction regions of BRCA2 are also reported, that contribute to the interpretation of the pathogenicity of the deletion. The quantitative approaches showed that there are three classes of delta3 BRCA2 transcripts (low, moderate and exclusive). Exclusive expression of the delta3 transcript by the mutant allele and segregation data provide evidence for a causal effect of the exon 3 deletion.

Conclusion: This paper highlights that large rearrangements and total deletion of exon 3 in the BRCA2 gene could contribute to hereditary breast and/or ovarian cancer. In addition, our findings suggest that, to interpret the pathogenic effect of any variants of exon 3, both accurate transcript quantification and co-segregation analysis are required.

Show MeSH
Related in: MedlinePlus