p53 Mutation analysis in breast tumors by a DNA microarray method.
The p53 gene acts as a regulator of cell growth and DNA repair in normal cells; inactivation of the gene seems to lead to cancer. It is the most commonly mutated gene in human cancers, and a high-throughput sequencing method is needed for cancer etiology studies using large sample sets. In our population-based case-control study of breast cancer, the p53 gene was amplified by PCR for 392 subjects from seven hospitals in Western New York using the Affymetrix GeneChip technology. One hundred thirty-eight (35%) of the breast tumors had p53 mutations, of which 88% were located in exons 5 to 8. New hotspots were identified at codons 179, 195, 196, 213, 217, 249, 254, 278, 281, and 298, and previously reported hotspots were found at codons 175, 248, and 273. Manual sequencing for exons 5 to 9 of the p53 gene was done for 139 tumors to validate the Affymetrix assay. The two methods had 100% concordance for mutations detectable by the Affymetrix assay. We also successfully assayed paraffin-embedded breast and lung tumors from as early as 1958 and employed a nested PCR strategy to improve weak PCR amplification. To have statistical power, the investigation of gene environment interactions and cancer requires a large number of tumor analyses, which are frequently only available from archived tissue from multiple sources. We have shown the utility of the Affymetrix GeneChip method under these challenging conditions and provided new data for the mutational spectra of breast cancer in a population-based study.
Tennis
Meredith
M
Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057-1465, USA. pgs2@georgetown.edu
Krishnan
Shiva
S
Bonner
Matthew
M
Ambrosone
Christine B
CB
Vena
John E
JE
Moysich
Kirsten
K
Swede
Helen
H
McCann
Susan
S
Hall
Per
P
Shields
Peter G
PG
Freudenheim
Jo L
JL
D001943:Breast Neoplasms / Q000235:genetics*; D016022:Case-Control Studies; D003062:Codon; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis*; D005091:Exons; D005260:Female; D016158:Genes, p53; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D003062:Codon; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53