Document Detail


A new assay for functional screening of BRCA2 linker region mutations identifies variants that alter chemoresistance to cisplatin.
MedLine Citation:
PMID:  21741379     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Variants of unknown significance (VUS) complicate the assignment of risk to new DNA sequence variants found in at-risk populations. This study focused on the poorly studied linker region of the cancer-associated BRCA2 protein encoded by exons twelve through fourteen of BRCA2. To develop a new method to characterize VUS in this region of BRCA2, we first chose to study 4 reported VUS occurring on evolutionarily conserved residues within the linker region. To determine if these VUS represent neutral changes or if they impact the function of the BRCA2 protein, we stably transfected expression plasmids encoding wild-type or each mutant peptide into T47D breast cancer cells, which are wild-type for BRCA2. Four mutant peptide expressing cell lines and a wild-type linker region expressing cell line next were studied by challenging transfected cell lines with the DNA crosslinking compound cisplatin (10μM) for 5days. Expression of the wild-type linker region and certain mutant linker peptides (N2452D and I2285V) decreased apoptosis (as demonstrated by cell death detection assay) in transfected cell lines, indicating that the linker region peptide directly or indirectly affects the DNA damage repair pathway. By determining the cell survival and assaying the apoptotic index of treated cell lines, one could potentially use this screen to determine that a particular VUS has a functional impact on BRCA2 function, and hence is of functional significance. We conclude that this method is useful for screening the effect of linker region VUS on BRCA2 function, and to identify mutations for further testing. We also conclude that mutations in the linker region may have heretofore unappreciated roles in BRCA2 function.
Authors:
Curtis R Warren; Zohra Ali-Khan Catts; Mary C Farach-Carson
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-06-29
Journal Detail:
Title:  Experimental cell research     Volume:  317     ISSN:  1090-2422     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  2011 Sep 
Date Detail:
Created Date:  2011-08-01     Completed Date:  2011-10-21     Revised Date:  2014-09-08    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2099-109     Citation Subset:  IM    
Copyright Information:
Copyright © 2011 Elsevier Inc. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Antineoplastic Agents / pharmacology*
BRCA2 Protein / genetics*
Base Sequence
Cell Line, Tumor
Cisplatin / pharmacology*
DNA Mutational Analysis
Drug Resistance, Neoplasm / genetics
Genetic Predisposition to Disease
Genetic Testing / methods*
Genetic Variation*
Humans
Molecular Sequence Data
Mutation
Grant Support
ID/Acronym/Agency:
P01 CA098912/CA/NCI NIH HHS; P01 CA098912/CA/NCI NIH HHS; P01 CA098912-07/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Antineoplastic Agents; 0/BRCA2 Protein; Q20Q21Q62J/Cisplatin
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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