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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, and² 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", Consiglio Nazionale delle Ricerche, Naples, Italy;

^aaddress correspondence to this author at: Laboratory of Human Genetics, Medical Oncology C, IST, Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi, 10, 16132 Genova, Italy; fax 39-010-560-0066, e-mail rosario.notaro{at}istge.it)

Abstract

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: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing.

Conclusions: T-ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 common MUTYH mutations with use of as few as 3 single tube PCR reactions. It could be useful to carry out large population-based epidemiologic studies.

The following articles in journals at HighWire Press have cited this article:

				P. Piccioli, M. Serra, S. Pedemonte, G. Balbi, F. Loiacono, S. Lastraioli, L. Gargiulo, A. Morabito, D. Zuccaro, L. Del Mastro, et al. Hexaprimer Amplification Refractory Mutation System PCR for Simultaneous Single-Tube Genotyping of 2 Close Polymorphisms Clin. Chem., January 1, 2008; 54(1): 227 - 229. [Full Text] [PDF]