The Psuedogene Problem
It is known that the PI3 kinase gene has a pseudogene. Pseudogenes are believed to be relatives of functional genes due to their homology to real genes but lack of functionality compared to their relative, which has occurred over many evolutionary cycles. Pseudogenes occur as a result of two main processes: duplication and retrotransposition.
As natural selection affects individual genes, beneficial mutations in a gene that improve an organism’s fitness tend to be preserved, whereas a sequence change that impairs a gene’s function leads to it being discarded. As pseudogenes are classed as junk DNA and most commonly found in the intergenic regions, they are released from this selection pressure and are free to accumulate all kinds of mutations, including changes that would be deleterious to normal genes.
The activating mutations in the PI3 kinase oncogene promote cellular replication, so the cells bearing these mutations have a growth advantage whereas similar mutations in the pseudogene have no affect on cell fitness and viability.
In designing the ARMS and Scorpions primers for the PI3K assay, the pseudogene needed to be considered. In the region of mutations in exon 9 of the PI3 kinase gene, there is a high level of homolgy with the pseudogene making it difficult to design primers to selectively amplify the true gene over the pseudogene. The particular selectivity of ARMS allows specific yet sensitive amplification of only mutant variants of the true gene (Figure 3). In exon 20, the homology is much less significant and primer design to differentiate between true gene and pseudogene is less problematic.
DxS has designed an assay in kit form to detect the most common mutations seen in the PIK3CA gene that correlate with tumor progression in numerous types of cancer. Previously, an alternative method for detection of these mutations was sequencing, however, this technology has its limitations in that it is labor intensive and has a low sensitivity of 15–25%. The combination of ARMS and Scorpions technology in a real-time PCR assay has overcome the limitations of sequencing and the assay can detect down to 1% mutant in a background of wild-type DNA.
The PI3K-AKT pathway is an attractive target for anticancer therapies, and this assay allows detection of the most common mutations, which will aid in the development of targeted therapies for individuals affected by these cancers. Due to the presence of a pseudogene that shows a lot of homology with exon 9 of the PI3 kinase gene, ARMS and Scorpions primers have had to be specifically designed to ensure that they only detect the mutations seen in the actual gene and not the pseudogene. The DxS PI3K assay achieves this goal.