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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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Related in: MedlinePlus

Analysis of delta3-transcript expression. a: Fragment analysis of Onestep RT-PCR products spanning exon 2 to 6. Semi-quantitative fragment analysis of Onestep RT-PCR products revealed the presence of the wild type transcripts at 477 bp and the delta3 alternative transcripts at 231 bp, as shown in the electrophoretograms of a control sample (wild type) and samples with the c.68-7T>A and c.316+3delA mutations. The percentages of delta3-transcript were, respectively: 6%, 22% and 61% for the sample illustrated. Left upper panels: agarose gel images of the same products after 33 cycles of PCR; the left wells contain the control, the right wells the samples with the respective mutation. b: Pyrosequencing histogram from the pyrogram for the c.-26A>G polymorphism with the primer set Ex1-2 F and Ex2-Ex3/4 R (only the full-length transcript). The sequence is C/T TTGGTAAATA. The proportion of C and T is directly computed by the pyrosequencing software. c: Pyrosequencing histogram from the pyrogram for the competitive QPCR using primer set exon 2/exon 3 and exon 2/exon 4 for the full-length transcript and delta3-transcripts, respectively. The sequence of the exon 2/3 transcript is CAGATTT and the sequence of the exon 2/4 transcript is CAGGAA. The proportion of GA (full-length transcript) and GGA (delta3-transcript) is directly computed on the pyrosequencing software.
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Figure 2: Analysis of delta3-transcript expression. a: Fragment analysis of Onestep RT-PCR products spanning exon 2 to 6. Semi-quantitative fragment analysis of Onestep RT-PCR products revealed the presence of the wild type transcripts at 477 bp and the delta3 alternative transcripts at 231 bp, as shown in the electrophoretograms of a control sample (wild type) and samples with the c.68-7T>A and c.316+3delA mutations. The percentages of delta3-transcript were, respectively: 6%, 22% and 61% for the sample illustrated. Left upper panels: agarose gel images of the same products after 33 cycles of PCR; the left wells contain the control, the right wells the samples with the respective mutation. b: Pyrosequencing histogram from the pyrogram for the c.-26A>G polymorphism with the primer set Ex1-2 F and Ex2-Ex3/4 R (only the full-length transcript). The sequence is C/T TTGGTAAATA. The proportion of C and T is directly computed by the pyrosequencing software. c: Pyrosequencing histogram from the pyrogram for the competitive QPCR using primer set exon 2/exon 3 and exon 2/exon 4 for the full-length transcript and delta3-transcripts, respectively. The sequence of the exon 2/3 transcript is CAGATTT and the sequence of the exon 2/4 transcript is CAGGAA. The proportion of GA (full-length transcript) and GGA (delta3-transcript) is directly computed on the pyrosequencing software.

Mentions: Semi-quantitative fragment analysis of Onestep PCR products spanning exons 2 to 6 was necessary, since products fractionated on agarose gels do not give reliable results. In particular, the abnormal shorter PCR product, corresponding to the delta3-transcript, was very faint and not always detectable (Figure 2a). This observation could explain why Machado et al. [11] and Peixoto et al. [12] report discordant results. Fragment analysis with a fluorophore on an automatic sequencer gave reliable results, which were confirmed with PCR products spanning exons 2 to 10 (data not shown). As shown in Table 2 and Figure 2a, the proportion of the delta3-transcript to the total transcript was variable according to the type of variant. The "control samples" (i.e. wild type or not mutated in exon 3), gave low levels of the delta3-transcript that was, on average, less than 10% of the total transcript. In contrast, with samples from cases carrying the Δ3-LR or the c.316+3delA, the PCR products corresponding to the delta3-transcript corresponded to 61% and 52% of the total. Samples with nucleotide variants in intron 2 also had increased levels of the delta3-transcript, which proportions ranging from 14% to 52% of the total. Heterogeneity within samples carrying the same variant was also observed. The discrepancy for c.68-7T>A could be associated with the nature and quality of the cDNA from lymphoblastoid cells (52% and 45%) or from PaxGene extraction (14% and 23%). Samples with nonsense mutations in exon 3 gave a level that was similar to control samples, with a range between 5% and 12%. The results were similar when samples were treated with puromycin. Thus, two classes of expression were detected, with low or high levels of delta3-transcripts. In this approach, competition in the PCR between the two amplicons with different sizes could result in preferential amplification of the lower sized delta3-transcript. To circumvent this problem, we developed two alternative methods: allele-specific RT-PCR and competitive quantitative RT-PCR (C-QPCR) combined with pyrosequencing.


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)

Analysis of delta3-transcript expression. a: Fragment analysis of Onestep RT-PCR products spanning exon 2 to 6. Semi-quantitative fragment analysis of Onestep RT-PCR products revealed the presence of the wild type transcripts at 477 bp and the delta3 alternative transcripts at 231 bp, as shown in the electrophoretograms of a control sample (wild type) and samples with the c.68-7T>A and c.316+3delA mutations. The percentages of delta3-transcript were, respectively: 6%, 22% and 61% for the sample illustrated. Left upper panels: agarose gel images of the same products after 33 cycles of PCR; the left wells contain the control, the right wells the samples with the respective mutation. b: Pyrosequencing histogram from the pyrogram for the c.-26A>G polymorphism with the primer set Ex1-2 F and Ex2-Ex3/4 R (only the full-length transcript). The sequence is C/T TTGGTAAATA. The proportion of C and T is directly computed by the pyrosequencing software. c: Pyrosequencing histogram from the pyrogram for the competitive QPCR using primer set exon 2/exon 3 and exon 2/exon 4 for the full-length transcript and delta3-transcripts, respectively. The sequence of the exon 2/3 transcript is CAGATTT and the sequence of the exon 2/4 transcript is CAGGAA. The proportion of GA (full-length transcript) and GGA (delta3-transcript) is directly computed on the pyrosequencing software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Analysis of delta3-transcript expression. a: Fragment analysis of Onestep RT-PCR products spanning exon 2 to 6. Semi-quantitative fragment analysis of Onestep RT-PCR products revealed the presence of the wild type transcripts at 477 bp and the delta3 alternative transcripts at 231 bp, as shown in the electrophoretograms of a control sample (wild type) and samples with the c.68-7T>A and c.316+3delA mutations. The percentages of delta3-transcript were, respectively: 6%, 22% and 61% for the sample illustrated. Left upper panels: agarose gel images of the same products after 33 cycles of PCR; the left wells contain the control, the right wells the samples with the respective mutation. b: Pyrosequencing histogram from the pyrogram for the c.-26A>G polymorphism with the primer set Ex1-2 F and Ex2-Ex3/4 R (only the full-length transcript). The sequence is C/T TTGGTAAATA. The proportion of C and T is directly computed by the pyrosequencing software. c: Pyrosequencing histogram from the pyrogram for the competitive QPCR using primer set exon 2/exon 3 and exon 2/exon 4 for the full-length transcript and delta3-transcripts, respectively. The sequence of the exon 2/3 transcript is CAGATTT and the sequence of the exon 2/4 transcript is CAGGAA. The proportion of GA (full-length transcript) and GGA (delta3-transcript) is directly computed on the pyrosequencing software.
Mentions: Semi-quantitative fragment analysis of Onestep PCR products spanning exons 2 to 6 was necessary, since products fractionated on agarose gels do not give reliable results. In particular, the abnormal shorter PCR product, corresponding to the delta3-transcript, was very faint and not always detectable (Figure 2a). This observation could explain why Machado et al. [11] and Peixoto et al. [12] report discordant results. Fragment analysis with a fluorophore on an automatic sequencer gave reliable results, which were confirmed with PCR products spanning exons 2 to 10 (data not shown). As shown in Table 2 and Figure 2a, the proportion of the delta3-transcript to the total transcript was variable according to the type of variant. The "control samples" (i.e. wild type or not mutated in exon 3), gave low levels of the delta3-transcript that was, on average, less than 10% of the total transcript. In contrast, with samples from cases carrying the Δ3-LR or the c.316+3delA, the PCR products corresponding to the delta3-transcript corresponded to 61% and 52% of the total. Samples with nucleotide variants in intron 2 also had increased levels of the delta3-transcript, which proportions ranging from 14% to 52% of the total. Heterogeneity within samples carrying the same variant was also observed. The discrepancy for c.68-7T>A could be associated with the nature and quality of the cDNA from lymphoblastoid cells (52% and 45%) or from PaxGene extraction (14% and 23%). Samples with nonsense mutations in exon 3 gave a level that was similar to control samples, with a range between 5% and 12%. The results were similar when samples were treated with puromycin. Thus, two classes of expression were detected, with low or high levels of delta3-transcripts. In this approach, competition in the PCR between the two amplicons with different sizes could result in preferential amplification of the lower sized delta3-transcript. To circumvent this problem, we developed two alternative methods: allele-specific RT-PCR and competitive quantitative RT-PCR (C-QPCR) combined with pyrosequencing.

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