NEW YORK(GenomeWeb) — Researchers from Baylor College of Medicine and Baylor Miraca Genetic Laboratories have created a more accurate method to detect mutations in the PMS2 gene, they reported today in the online version of the Journal of Molecular Diagnostics.
Mutations in this gene are associated with hereditary nonpolyposis colorectal cancer (Lynch syndrome) and Turcot syndrome, and are a cause of supratentorial primitive neuroectodermal tumors.
Aiming to improve the detection accuracy for PMS2 gene mutations, the researchers performed multiplex ligation-dependent probe amplification (MLPA), target capture next-generation sequencing, and long-range PCR (LR-PCR). They performed their method on 32 clinical samples and 14 control samples (eight positive samples with known mutations and six negative samples).
Of the 140,000 cases of colorectal cancer that are diagnosed each year in the US, only three to five percent are caused by Lynch syndrome, according to the National Institutes of Health. However, the researchers involved in this study thought the PMS2 gene's role in developing Lynch syndrome had been underestimated based on the high frequency of negative staining in PMS2 immunohistochemistry in colorectal cancer without identifiable PMS2 mutations. They also said they believe that accurate molecular analysis of this gene has been hindered by highly homologous sequences in the genomic region and frequent sequence exchange between active and pseudogenes.
Previously, PMS2 mutations have been detected using Sanger sequencing and the analysis of the coding exons and flanking intron regions individually, which did not provide copy-number variation information. The researcher's new approach incorporated LR-PCR and NGS, which allowed them to detect sequence variants and CNVs, as well as distinguish the mutations in PMS2 from its pseudogene PMS2CL. They were also able to generate long-range, contiguous uniform coverage of the gene specific LR-PCR fragments containing multiple exons and introns, which is important for CNV differentiation among highly homologous regions such as the ones surrounding PMS2 gene regions.
"The combination of LR-PCR/NGS and target capture NGS resolves both allele dropout and homology problems," the researchers wrote in the JMD paper. "Thus, this approach would be particularly advantageous in testing patients with early-onset cancers with suspicion of PMS2 mutations."
The researchers analyzed each sample by running the three processes — MLPA, NGS, and LR-PCR — in parallel. The MLPA was performed using an MRC-Holland SALSA MLPA kit P008-C1, which included probes specific for PMS2 exons 1 to 11, exons 11 to 15, and paralogous sequence variants in exons 11 to 15 targeting both PMS2 and PMS2CL. For target capture NGS, the researchers used a Roche NimbleGen SeqCap probe library, which targeted all coding exons and 50 bp of flanking intronic regions of 197 hereditary cancer genes.
Then for LR-PCR, they used previously published primers to amplify three fragments — E1 to E5, E6 to E10, and E11 to E15 — for both the PMS2 gene and the PMS2CL pseudogene. The LR-PCR was performed using Clontech Laboratories' TaKaRa LA Taq DNA polymerase Hot-Start Version, and LR-PCR products of PMS2 were fragmented and indexed for the subsequent analysis by high-throughput sequencing on Illumina's HiSeq2000 platform.
"The results from the three methods serve as cross validation for enhanced accuracy and reduced turnaround time," they said.
Having a more accurate method of PMS2 gene mutation would allow for better preventative methods, diagnosis, and treatment options for people at risk for colorectal cancer. In addition, the researchers believe that it can be applied to the molecular analysis of other genes containing multiple copies of highly homologous sequences.