Received on ,
Accepted on ,

Technical Briefs

Multiplex Tetra-Primer Amplification Refractory Mutation System PCR to Detect 6 Common Germline Mutations of the MUTYH Gene Associated with Polyposis and Colorectal Cancer

¹ Laboratory of Human Genetics, Medical Oncology C, IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
² Hereditary Tumors Unit, IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
³ Preventive-Predictive Medicine Unit, Istituto Nazionale Tumori, Milan, Italy
⁴ Istituto di Genetica e Biofisica "Adriano Buzzati Traverso" (Istituto di Genetica e Biofisica-Consiglio Nazionale delle Ricerche), Naples, Italy

^* To whom correspondence should be addressed. E-mail: rosario.notaro{at}istge.it.

Background: We describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) for detecting MUTYH mutations, which are associated with colorectal adenomas and colorectal cancer.

Methods: We designed specific T-ARMS-PCR assays for 6 mutations (Y165C, G382D, 1395_7delGGA, Y90X, 1103delC, and R231H) selected on the basis of the frequency of their occurrence. We also designed a set of 3 multiplex T-ARMS PCR assays, each for detection of 2 mutations. We tested DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing.

Results: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays.

Conclusions: T-ARMS-PCR can detect not only nucleotide substitutions but also small deletions; it can also detect 2 different mutations simultaneously. This method provides rapid, reproducible, and cost-effective detection of common MUTYH mutations.