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Published on: February 17, 2017
Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics
R Suspène1, V Caval1, M Henry1
1Molecular Retrovirology Unit, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, France.
Background:
The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5'TpC context. These can be copied as G->A transitions in the 5'GpA context.
Methods:
DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA.
Results:
Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5'GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR.
Conclusions:
Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10(4)-10(5) base substitutions per cancer genome, or 0.003-0.03 kb(-1). By contrast, the 3DPCR error rate is of the order of 4-20 kb(-1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5'GpC represent artefacts.
Insights
Differential DNA denaturation PCR (3D PCR) introduces artifacts, generating false C->T and G->A mutations. This technique is unsuitable for identifying true cancer genome mutations.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Cancer genomics reveals CG->TA transitions as dominant mutations, likely caused by APOBEC3 cytidine deaminases.
- APOBEC3A can access nuclear DNA, introducing numerous C->T mutations in a 5'TpC context, which are copied as G->A in a 5'GpA context.
Purpose of the Study:
- To evaluate the error rate of differential DNA denaturation PCR (3D PCR) for analyzing APOBEC3-edited DNA.
- To determine if 3D PCR can accurately identify C->T transitions in cancer genomes.
Main Methods:
- Utilized 3D PCR, a technique exploiting A+T richness in APOBEC3-edited DNA for recovery.
- Investigated 3D PCR errors using cloned DNA and compared results with standard PCR.
Main Results:
- 3D PCR exhibits a higher error rate than standard PCR.
- The technique generates DNA strands with both C->T and G->A mutations in a 5'GpCpR context, mimicking cancer mutations.
- Similar mutation patterns were observed in human tumor DNA analyzed via 3D PCR.
Conclusions:
- 3D PCR is not a reliable method for identifying fixed C->T transitions in cancer genomes due to significant artifact generation.
- The high error rate of 3D PCR (4-20 kb(-1)) surpasses the mutation frequency in cancer genomes, leading to artefactual findings.
- Sequences with mixed C->T and G->A mutations recovered by 3D PCR in a 5'GpC context are considered artefacts.
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