Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms

Leeneer, Kim De; Coene, Ilse; Poppe, Bruce; Paepe, Anne De; Claes, Kathleen

doi:10.1373/clinchem.2007.098764

Cited by 65 publications

(61 citation statements)

References 19 publications

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“…6 Although direct Sanger sequencing is considered the gold standard for the analysis of BRCA1 and BRCA2 mutations, their large size (5592 bp and 10257 bp, respectively), and lack of mutation hot spots (see Breast Cancer Information Core database: http://www.research.nhgri.nih.gov/bic/) mean useful prescreening strategies. [7][8][9] Moreover, large genomic rearrangements (LGRs) of these genes require the use of other complementary techniques. 10,11 The development of cost-effective BRCA mutation detection workflows will not only benefit the genetic counseling process for patients with HBOCS but will also enhance the process of selecting patients for personalized treatments, as could be the case of PARP inhibitors, for example.…”

Section: Introductionmentioning

confidence: 99%

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

et al. 2012

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Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

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Section: Introductionmentioning

confidence: 99%

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

et al. 2012

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“…High-resolution melting (HRM or HRMA) is an effective method to screen for sequence variation and has been effectively used to assess genes involving inborn errors of metabolism, cancer susceptibility, and other genes whose dysfunction is associated with disease [Bastien et al, 2008;De Leeneer et al, 2008;Dobrowolski et al, 2007aDobrowolski et al, , 2007bDobrowolski et al, , 2005Erali et al, 2008;Laurie and George, 2009]. HRM profiling identifies the presence of sequence variants by deviation in the shape of a post-PCR melting profile using probe-based and amplicon-based strategies [Dobrowolski et al, 2007a[Dobrowolski et al, , 2007bMontgomery et al, 2007;Zhou et al, 2004].…”

Section: Introductionmentioning

confidence: 99%

Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

Dobrowolski¹,

Hendrickx

Bosch

et al. 2009

Hum. Mutat.

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Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. Highresolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301-658 bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A4G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A4G, and m.1555A4G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1 hr.

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“…All the curves differed from wild type and from all other variants (example in online supplementary Figure 3). High-resolution melting is an effective method for variant detection in BRCA1 and BRCA2 genes and can be applied for rapid screening of samples in gene testing (54,55).…”

Section: Application Of High-resolution Meltingmentioning

confidence: 99%

New sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Croatia

Cvok¹,

Čretnik²,

Musani³

et al. 2008

Clinical Chemistry and Laboratory Medicine

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Background: Mutations in BRCA1 and BRCA2 genes are associated with family predisposition to breast and ovarian cancer. Novel screening methods are required for efficient and rapid detection of sequence variants in cancer patients and their family members. Methods: The screening for variants in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 in Croatia was performed by a high-resolution melting approach, which is based on differences in melting curves caused by variations in nucleotide sequence. This is the first screening in Croatia on elderly healthy women with no family history of cancer. BRCA1 screening was performed on 220 and BRCA2 screening on 115 samples.

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Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms

Cited by 65 publications

References 19 publications

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

New sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Croatia

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