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Previously published online at DOI: 10.1373/clinchem.2005.057695 Multiplex Tetra-Primer Amplification Refractory Muta- tion System PCR to Detect 6 Common Germline Muta- tions of the MUTYH Gene Associated with Polyposis and Colorectal Cancer, Patrizia Piccioli, 1† Martina Serra, 1† Viviana Gismondi, 2 Simona Pedemonte, 1 Fabrizio Loiacono, 1 Sonia Lastraioli, 1 Lucio Bertario, 3 Maria De Angioletti, 4 Lili- ana Varesco, 2 and Rosario Notaro 1* ( 1 Laboratory of Human Genetics, Medical Oncology C, and 2 Hereditary Tumors Unit, IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; 3 Preventive-Predictive Medicine Unit, Isti- tuto Nazionale Tumori, Milan, Italy; 4 Istituto di Genetica e Biofisica “Adriano Buzzati Traverso”, Consiglio Nazio- nale delle Ricerche, Naples, Italy; † these authors contrib- uted equally to this work; * address 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@istge.it) Background: We describe a simple tetra-primer ampli- fication refractory mutation system PCR (T-ARMS- PCR) for detecting MUTYH mutations, which are asso- ciated 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 pa- tients, and each mutation was confirmed by direct DNA sequencing. Conclusions: T-ARMS-PCR does not require any spe- cial equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 com- mon 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. © 2006 American Association for Clinical Chemistry MUTYH-associated polyposis (MAP) is an autosomal recessive syndrome associated with biallelic germline mutations in the base excision repair gene MUTYH (OMIM #608456) (1). MUTYH biallelic germline muta- tions have been found in 4%–33% (2, 3) of patients with multiple colorectal adenomas and in 7.5%–29% of patients who have attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations (2, 4–6). Population-based studies suggest that biallelic MUTYH germline mutations might be also responsible for 0.5% of unselected colorectal cancers (7, 8). At least 23 different putative pathogenic mutations have been identified as widespread in the MUTYH gene (9). Two of these mutations (Y165C in exon 7 and G382D in exon 13) account for at least 70% of the mutant MUTYH alleles (2, 6), and at least 1 of them is found in more than 80% of Caucasian MAP patients (2, 4–6, 10, 11). In addi- tion, these 2 mutations have been found in the general Caucasian population with a frequency of 0.5% (1, 2, 8, 12, 13). Other mutations may be frequent in pa- tients from some populations; e.g., the homozygous E466X (exon 14) mutation has been found in 3 patients from unrelated Indian families (3). Recently, we found that in Italian patients a 3-bp deletion in exon 14 (1395_ 7delGGA) is relatively frequent (5) and that each of the mutations Y90X (exon 3), 1103delC (exon 12), and R231H (exon 9) represents more than 6% of mutant MUTYH alleles (14 ). The identification of germline mutations in both MUTYH alleles in patients with multiple colorectal ade- nomas or colorectal cancer has clinical relevance because their siblings may also have a very high risk of cancer. Thus, genetic testing for MUTYH mutations should be offered, after appropriate counseling, to individuals with multiple colorectal adenomas and to members of their families; it may also be offered to individuals with early- onset colorectal cancer (9, 15). In addition, because some of the pathogenic MUTYH mutations have relatively high frequencies in the general population and heterozygotes may also have an increased risk of colorectal cancer (8, 12), more widespread genetic testing for MUTYH mutations, perhaps in any individual with colorectal cancer, may be advisable. Many methods, such as single-strand conformation polymorphism analysis and DNA HPLC (dHPLC), are suitable for MUTYH mutation detection. These methods, however, require specialized equipment and, most impor- Clinical Chemistry 52, No. 4, 2006 739 Downloaded from https://academic.oup.com/clinchem/article/52/4/739/5626896 by guest on 04 June 2023