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Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis.

Takano EA, Mitchell G, Fox SB, Dobrovic A - BMC Cancer (2008)

Bottom Line: Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population.In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results.This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria 8006, Australia. alexander.dobrovic@petermac.org

ABSTRACT

Background: Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions.

Methods: We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine.

Results: We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile.

Conclusion: HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

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Difference plot showing different mutations relative to the wild type controls. In difference plots, the melting profile of a wild type control is chosen as a horizontal base line and the relative differences in the melting of all the other samples are plotted relative to this baseline. The figure shows difference plots for each of the 3 exons containing one of the common Ashkenazi mutations (185delAG, 5382insC and 6174delT). Each trace represents the amplicon from a different individual's DNA sample. All mutations were clearly distinct from the wild type controls. a. BRCA1 exon 2: Melt curves of each mutation (pink: 188del11, red: 185delAG and green: 185insA) were plotted against the wild type (blue). b. BRCA1 exon 20: Melt curves of each mutation (pink: 5396+1G>A, red: 5331G>A and green: 5382insC) were plotted against melt curve of the wild type (blue). c. BRCA2 exon 11: Melt curves of each mutation (pink: 6024delTA, red: 6293C>G and green: 6174delT) were plotted against melt curve of the wild type (blue).
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Figure 1: Difference plot showing different mutations relative to the wild type controls. In difference plots, the melting profile of a wild type control is chosen as a horizontal base line and the relative differences in the melting of all the other samples are plotted relative to this baseline. The figure shows difference plots for each of the 3 exons containing one of the common Ashkenazi mutations (185delAG, 5382insC and 6174delT). Each trace represents the amplicon from a different individual's DNA sample. All mutations were clearly distinct from the wild type controls. a. BRCA1 exon 2: Melt curves of each mutation (pink: 188del11, red: 185delAG and green: 185insA) were plotted against the wild type (blue). b. BRCA1 exon 20: Melt curves of each mutation (pink: 5396+1G>A, red: 5331G>A and green: 5382insC) were plotted against melt curve of the wild type (blue). c. BRCA2 exon 11: Melt curves of each mutation (pink: 6024delTA, red: 6293C>G and green: 6174delT) were plotted against melt curve of the wild type (blue).

Mentions: For BRCA1 exon 2 (Figure 1a), we tested DNA from 4 individuals with the Ashkenazi mutation 185delAG as well as 1 individual with the mutation 188del11 and 2 individuals with the mutation 185insA. The mutations were all readily differentiated from the wild type. They were also readily differentiated from each other giving characteristic melting curves as is readily seen from the figure. It can also readily be seen that biological replicates had near identical melting patterns.


Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis.

Takano EA, Mitchell G, Fox SB, Dobrovic A - BMC Cancer (2008)

Difference plot showing different mutations relative to the wild type controls. In difference plots, the melting profile of a wild type control is chosen as a horizontal base line and the relative differences in the melting of all the other samples are plotted relative to this baseline. The figure shows difference plots for each of the 3 exons containing one of the common Ashkenazi mutations (185delAG, 5382insC and 6174delT). Each trace represents the amplicon from a different individual's DNA sample. All mutations were clearly distinct from the wild type controls. a. BRCA1 exon 2: Melt curves of each mutation (pink: 188del11, red: 185delAG and green: 185insA) were plotted against the wild type (blue). b. BRCA1 exon 20: Melt curves of each mutation (pink: 5396+1G>A, red: 5331G>A and green: 5382insC) were plotted against melt curve of the wild type (blue). c. BRCA2 exon 11: Melt curves of each mutation (pink: 6024delTA, red: 6293C>G and green: 6174delT) were plotted against melt curve of the wild type (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266761&req=5

Figure 1: Difference plot showing different mutations relative to the wild type controls. In difference plots, the melting profile of a wild type control is chosen as a horizontal base line and the relative differences in the melting of all the other samples are plotted relative to this baseline. The figure shows difference plots for each of the 3 exons containing one of the common Ashkenazi mutations (185delAG, 5382insC and 6174delT). Each trace represents the amplicon from a different individual's DNA sample. All mutations were clearly distinct from the wild type controls. a. BRCA1 exon 2: Melt curves of each mutation (pink: 188del11, red: 185delAG and green: 185insA) were plotted against the wild type (blue). b. BRCA1 exon 20: Melt curves of each mutation (pink: 5396+1G>A, red: 5331G>A and green: 5382insC) were plotted against melt curve of the wild type (blue). c. BRCA2 exon 11: Melt curves of each mutation (pink: 6024delTA, red: 6293C>G and green: 6174delT) were plotted against melt curve of the wild type (blue).
Mentions: For BRCA1 exon 2 (Figure 1a), we tested DNA from 4 individuals with the Ashkenazi mutation 185delAG as well as 1 individual with the mutation 188del11 and 2 individuals with the mutation 185insA. The mutations were all readily differentiated from the wild type. They were also readily differentiated from each other giving characteristic melting curves as is readily seen from the figure. It can also readily be seen that biological replicates had near identical melting patterns.

Bottom Line: Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population.In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results.This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria 8006, Australia. alexander.dobrovic@petermac.org

ABSTRACT

Background: Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions.

Methods: We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine.

Results: We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile.

Conclusion: HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

Show MeSH
Related in: MedlinePlus