cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer.
BACKGROUND
Recently published data showed discrepancies between P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.


METHODS
To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.


RESULTS
We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations.


CONCLUSIONS
In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.
Szybka
Malgorzata
M
Department of Oncological Pathology, Medical University of Lodz, Lodz, Poland. mszybka@wp.pl
Zakrzewska
Magdalena
M
Rieske
Piotr
P
Pasz-Walczak
Grazyna
G
Kulczycka-Wojdala
Dominika
D
Zawlik
Izabela
I
Stawski
Robert
R
Jesionek-Kupnicka
Dorota
D
Liberski
Pawel P
PP
Kordek
Radzislaw
R
D018389:Codon, Nonsense; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D016158:Genes, p53; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D019656:Loss of Heterozygosity; D009154:Mutation; D020125:Mutation, Missense; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D018389:Codon, Nonsense; D018076:DNA, Complementary; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53