Mutation analysis and characterization of ATR sequence variants in breast cancer cases from high-risk French Canadian breast/ovarian cancer families.
BACKGROUND
Ataxia telangiectasia-mutated and Rad3-related (ATR) is a member of the PIK-related family which plays, along with ATM, a central role in cell-cycle regulation. ATR has been shown to phosphorylate several tumor suppressors like BRCA1, CHEK1 and TP53. ATR appears as a good candidate breast cancer susceptibility gene and the current study was designed to screen for ATR germline mutations potentially involved in breast cancer predisposition.


METHODS
ATR direct sequencing was performed using a fluorescent method while widely available programs were used for linkage disequilibrium (LD), haplotype analyses, and tagging SNP (tSNP) identification. Expression analyses were carried out using real-time PCR.


RESULTS
The complete sequence of all exons and flanking intronic sequences were analyzed in DNA samples from 54 individuals affected with breast cancer from non-BRCA1/2 high-risk French Canadian breast/ovarian families. Although no germline mutation has been identified in the coding region, we identified 41 sequence variants, including 16 coding variants, 3 of which are not reported in public databases. SNP haplotypes were established and tSNPs were identified in 73 healthy unrelated French Canadians, providing a valuable tool for further association studies involving the ATR gene, using large cohorts. Our analyses led to the identification of two novel alternative splice transcripts. In contrast to the transcript generated by an alternative splicing site in the intron 41, the one resulting from a deletion of 121 nucleotides in exon 33 is widely expressed, at significant but relatively low levels, in both normal and tumoral cells including normal breast and ovarian tissue.


CONCLUSIONS
Although no deleterious mutations were identified in the ATR gene, the current study provides an haplotype analysis of the ATR gene polymorphisms, which allowed the identification of a set of SNPs that could be used as tSNPs for large-scale association studies. In addition, our study led to the characterization of a novel Delta33 splice form, which could generate a putative truncated protein lacking several functional domains. Additional studies in large cohorts and other populations will be needed to further evaluate if common and/or rare ATR sequence variants can be associated with a modest or intermediate breast cancer risk.
Durocher
Francine
F
Cancer Genomics Laboratory, Oncology and Molecular Endocrinology Research Centre, Centre Hospitalier Universitaire de Québec and Laval University, Québec, G1V 4G2, Canada. Francine.durocher@crchul.ulaval.ca
Labrie
Yvan
Y
Soucy
Penny
P
Sinilnikova
Olga
O
Labuda
Damian
D
Bessette
Paul
P
Chiquette
Jocelyne
J
Laframboise
Rachel
R
Lépine
Jean
J
Lespérance
Bernard
B
Ouellette
Geneviève
G
Pichette
Roxane
R
Plante
Marie
M
Tavtigian
Sean V
SV
Simard
Jacques
J
D000328:Adult; D000368:Aged; D017398:Alternative Splicing; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D001943:Breast Neoplasms / Q000208:ethnology / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics*; D005006:Ethnicity / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005602:France / Q000208:ethnology; D020022:Genetic Predisposition to Disease; D018095:Germ-Line Mutation; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D015810:Linkage Disequilibrium; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D009386:Neoplastic Syndromes, Hereditary / Q000208:ethnology / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000208:ethnology / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011792:Quebec / Q000453:epidemiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D012306:Risk; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D004247:DNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases