901. Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2
- Author
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Olga M. Sinilnikova, Florence Le Calvez-Kelm, Melissa C. Southey, Lars Petter Jordheim, Sandrine McKay-Chopin, Jocelyne Michelon, Tu Nguyen-Dumont, Sean V. Tavtigian, Fabienne Lesueur, Nathalie Forey, Section Génétique - Groupe Prédispositions génétiques au cancer, Centre International de Recherche contre le Cancer (CIRC), Oncogénèse et progression tumorale, Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Léon Bérard [Lyon]-Hospices Civils de Lyon (HCL), Department of Pathology, University of Melbourne, Department of Oncological Sciences, University of Utah-Huntsman Cancer Institute, kConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. We gratefully acknowledge Arnaud Dumont for his help on developing the R script. TN-D was the recipient of a fellowship from Fondation de France and a Special Trainee Award from the International Agency for Research on Cancer. LPJ was the recipient of a fellowship from Ligue Contre le Cancer - Comité du Rhône. MCS is a National Health and Medical Research Council Senior Research Fellow and Victorian Breast cancer Research Consortium (VBCRC) Group Leader. This work was also supported by the Canadian Institutes of Health Research team grant CRN-87521-IC089832 and by the National Cancer Institute, National Institutes of Health (NIH) grant R01 CA121245., for Kathleen Cuningham Foundation Consortium for Research into Familial Aspects of Breast Cancer (kConFab), and BMC, Ed.
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Candidate gene ,RNA Stability ,DNA Mutational Analysis ,Allelic Imbalance ,medicine.disease_cause ,Nucleic Acid Denaturation ,MESH: Genotype ,0302 clinical medicine ,Coding region ,Genetics(clinical) ,MESH: Codon, Nonsense ,MESH: DNA Mutational Analysis ,skin and connective tissue diseases ,Genetics (clinical) ,MESH: Heterozygote ,Regulation of gene expression ,Genetics ,0303 health sciences ,Mutation ,MESH: Polymorphism, Single Nucleotide ,MESH: Genetic Predisposition to Disease ,MESH: RNA Stability ,MESH: Gene Expression Regulation, Neoplastic ,Exons ,Gene Expression Regulation, Neoplastic ,Codon, Nonsense ,030220 oncology & carcinogenesis ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Female ,Research Article ,lcsh:Internal medicine ,Heterozygote ,MESH: Cell Line, Tumor ,lcsh:QH426-470 ,Genotype ,Breast Neoplasms ,MESH: Nucleic Acid Denaturation ,Biology ,Protein Serine-Threonine Kinases ,Polymorphism, Single Nucleotide ,MESH: Protein-Serine-Threonine Kinases ,03 medical and health sciences ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Cell Line, Tumor ,medicine ,Humans ,Genetic Predisposition to Disease ,lcsh:RC31-1245 ,Gene ,CHEK2 ,MESH: Genes, Neoplasm ,Alleles ,030304 developmental biology ,MESH: Humans ,MESH: Alleles ,MESH: Allelic Imbalance ,lcsh:Genetics ,Checkpoint Kinase 2 ,Human genome ,MESH: Exons ,MESH: Female ,MESH: Breast Neoplasms ,Genes, Neoplasm - Abstract
BackgroundThe geneCHEK2encodes a checkpoint kinase playing a key role in the DNA damage pathway. ThoughCHEK2has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whetherCHEK2was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation inBRCA1orBRCA2had been identified.MethodsWe implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment.ResultsWe observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element ofCHEK2that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs.ConclusionsOur results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms.
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- 2011