High-resolution oligonucleotide array-CGH applied to the detection and characterization of large rearrangements in the hereditary breast cancer gene BRCA1

Laboratoire d'Oncogénétique, Centre René Huguenin, St Cloud, France.
Clinical Genetics (Impact Factor: 3.93). 10/2007; 72(3):199-207. DOI: 10.1111/j.1399-0004.2007.00849.x
Source: PubMed


We have developed a new method for detecting and characterizing large rearrangements in the BRCA1 gene based on high-resolution oligonucleotide array-CGH technology. We designed a specific CGH array for the BRCA1 gene and its flanking regions. We then used this approach to analyze nine DNA samples known to contain large deletions and large duplications. When possible, the deleted or duplicated region was sequenced to identify the break point. All the large rearrangements were detected by the new method, and their size was estimated to be within 1--2 kb. This enabled us to develop a simple polymerase chain reaction screening test for other family members. A refined choice of oligonucleotides should improve the precision of the breakpoint determination. Finally, the high resolution of oligonucleotide array-CGH should help to detect new large rearrangements missed by other current methods.

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    • "To confirm and characterize large rearrangements of the BRCA2 gene, a zoom-in dedicated CGH-array was applied. An 11 kb-oligonucleotide microarray was specially designed using oligonucleotides designed in house and with validated oligonucleotides from Agilent (Agilent technology, USA), as described [9]. Of these, 9294 were located throughout the genome (4339 Agilent oligonucleotides and 3107 BRCA1 in-house oligonucleotides). "
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    ABSTRACT: 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. 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. 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. 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.
    BMC Medical Genetics 09/2011; 12(1):121. DOI:10.1186/1471-2350-12-121 · 2.08 Impact Factor
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    • "All rearrangements were confirmed on a second blood sample and using another technique e.g. long range PCR, transcript analysis or a dedicated array-CGH (Rouleau, et al., 2007) . Spurious single-exon deletions due to mispriming were therefore unambiguously excluded. "
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    ABSTRACT: The detection of unknown mutations remains a serious challenge and, despite the expected benefits for the patient's health, a large number of genes are not screened on a routine basis. We present the diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis(®) , Fluigent, Paris, France), a novel method based on heteroduplex analysis by capillary electrophoresis using innovative matrices. BRCA1 and BRCA2 were screened for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis) using a single analytical condition. Seven working days were needed for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing). A total of 137 mutations were found, including a BRCA2 duplication of exons 19 and 20, previously missed by Comprehensive BRACAnalysis(®) . The mutation detection rate was 11.9%, which is consistent with patient inclusions. This study therefore suggests that EMMA represents a valuable short-term and midterm option for many diagnostic laboratories looking for an easy, reliable, and affordable strategy, enabling fast and sensitive analysis for a large number of genes.
    Human Mutation 03/2011; 32(3):325-34. DOI:10.1002/humu.21414 · 5.14 Impact Factor
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    • "Tumor samples were analyzed with the Agilent Human Genome CGH Microarray 44K. DNA samples for array CGH were labeled as previously described [38]. Briefly, 1 μg each of breast tumor DNA and commercial pooled human normal genomic DNAs (Promega, Madison, WI) was digested with 5 μg of AluI (50 units) and 5 ml of RsaI (50 units) (Promega, Madison, WI) and labeled by random priming with CY3- and CY5-dUTP, respectively (Agilent Technologies, Massy, France). "
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    ABSTRACT: Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer. To obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples. The expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors. This study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.
    Molecular Cancer 02/2011; 10(1):23. DOI:10.1186/1476-4598-10-23 · 4.26 Impact Factor
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