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Technical Briefs |
(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.
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 mutations 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)(5)(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)(5)(6)(10)(11). In addition, 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 patients 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 adenomas 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 importantly, are not designed for the screening of known pathogenic mutations in a large number of samples.
Here we describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) (16)(17) for screening the more frequent MUTYH mutations that does not require specialized equipment. Conventional ARMS-PCR amplifies the 2 alleles in 2 different PCR reactions (18)(19). In contrast, T-ARMS-PCR amplifies both wild-type and mutant alleles, together with a control fragment, in a single tube PCR reaction. The region flanking the mutation is amplified by 2 common (outer) primers, producing a non–allele-specific control amplicon (Fig. 1A
). Two allele-specific (inner) primers are designed in opposite orientation (Fig. 1A
) and, in combination with the common primers, can simultaneously amplify both the wild-type and the mutant amplicons. The 2 allele-specific amplicons have different lengths and can be easily separated by standard gel electrophoresis (Fig. 1A
) because the mutation is asymmetrically located with respect to the common (outer) primers (Fig. 1A
). Because the control amplicon and at least 1 of the 2 allele-specific amplicons are always present, T-ARMS-PCR provides an internal control with respect to false negatives as well as amplification failure. In addition, the presence of wild-type and mutant allelic amplicons allows easy interpretation of PCR results.
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We designed a T-ARMS-PCR for each of the following 6 mutations selected on the basis of their frequency in the literature (1)(2)(4)(10)(20) and in our patient series (5)(14): Y165C, G382D, 1395_7delGGA, Y90X, 1103delC, and R231H (Table 1
; also see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol52/issue4/). According to reports on systematic mutation analysis of the entire MUTYH gene (2)(3)(6)(10)(11)(21)(22), screening for these 6 mutations would identify at least 85% of patients with biallelic MUTYH mutations.
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To carry out even more rapid genotyping, we designed a set of 3 multiplex T-ARMS-PCRs for the detection of these 6 frequent MUTYH mutations: (a) the relatively frequent Y165C and G382D mutations; (b) the 1103delC and 1395_7delGGA mutations; and (c) the Y90X and R231H mutations (Table 1
). Multiplexes with different combinations of mutations can be designed for the screening of populations in which mutations have different frequencies. For example, we have set up a multiplex PCR for the Y90X and Y165C mutations (Table 1
).
We obtained DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations. Written informed consent was obtained from each patient according to institutional procedures. Genomic DNA was extracted from blood samples by use of the QIAamp DNA Blood Midi Kit (Qiagen S.p.A) and from paraffin tissue inclusions by phenol–chloroform extraction.
We designed the primers on the basis of the published MUTYH genomic sequence (GenBank accession no. U63329). The specificity of allele-specific primers is conferred by the match of the terminal 3' nucleotide with either the wild-type or the mutant allele, and it is enhanced by the introduction of a deliberate mismatch near the primer 3' end, usually at the third position (18)(19). The PCR reaction (total volume, 20 µL) contained 200 µM deoxynucleoside triphosphates, 1 U of hot-start DNA polymerase AmpliTaq Gold with its buffer (Applera Europe B.V.), 100 ng of genomic DNA, and the appropriate concentration of each primer (see Table 1
; also see the online Data Supplement). PCR amplification was carried out in both an I-cycler® (Bio-Rad) and a Px2® (Hybaid) thermal cycler. After activation of the AmpliTaq Gold DNA polymerase for 10 min at 95 °C, the cycling conditions were as indicated in Table 1
and in the online Data Supplement. PCR products were separated by standard electrophoresis on 2.5% agarose gels containing ethidium bromide.
The method was first tested on samples previously analyzed by direct sequencing: at least 1 homozygous and 2 heterozygous samples were tested for each mutation (for 1103delC, no homozygous samples were available). All mutations were easily detected by both the T-ARMS-PCR specific for one mutation (see Fig. 1 in the online Data Supplement) and the multiplex T-ARMS-PCR (Fig. 1
, B–D; also see Fig. 2 in the online Data Supplement).
The T-ARMS-PCR procedures for single-mutation detection were validated by analysis of samples from a new series of 54 patients with polyposis and no detectable APC germline mutations. In this series, 7.5%–29% of patients were expected to carry MUTYH mutations (2)(4)(5)(6), and we identified 3 Y165C homozygotes, 4 compound heterozygotes (2 Y165C/G382D, 1 Y165C/1395_7delGGA, and 1 R231H/G382D), 1 Y90X heterozygote, and 1 G382D heterozygote. These results were confirmed by dHPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing.
The multiplex T-ARMS-PCR procedures were validated by the analysis of DNA samples from a previously characterized group of 22 patients without and 31 patients with MUTYH mutations. We identified 7 Y90X alleles, 14 Y165C alleles, 7 R231H alleles, 4 1103delC alleles, 10 G382D alleles, and 8 1395_7delGGA alleles. Our results were identical to those obtained with direct DNA sequencing.
Overall, these data indicate the reliability of the T-ARMS-PCR method. Repeat analyses demonstrated the reproducibility of the procedure. In addition, an independent laboratory (MDA) that uses different thermal cycler models (PTC-100® from MJ Research and PCR express® from Hybaid) obtained the same results using established PCR conditions. These results confirm the robustness and the reproducibility of the T-ARMS-PCR procedure and show for the first time that T-ARMS-PCR can be been used to detect not only nucleotide substitutions but also small deletions (1395_7delGGA and 1103delC).
This method provides rapid, reproducible, and cost-effective detection of common MUTYH mutations without the use of any special equipment. In addition, it is the first time that T-ARMS-PCR has been designed to detect 2 different mutations simultaneously. It is unlikely that this method could test more than 2 mutations in the same reaction; nevertheless, the described multiplex T-ARMS-PCR allows investigation of 6 common MUTYH mutations with as few as 3 single-tube PCR reactions. T-ARMS-PCR can be easily adapted for local mutation frequencies. Most importantly, given the short lengths of the amplicons, the method may be used to genotype archival material from paraffin-embedded tissues.
This simple, inexpensive, and accurate method could be used to genotype relatives of patients with known MUTYH mutations, to optimize the strategy for identification of MUTYH mutations in a diagnostic setting, and to carry out the large population-based epidemiologic studies needed to investigate the possible role of mono-allelic MUTYH mutations in predisposing to colorectal cancer.
Acknowledgments
We thank Lucio Luzzatto for much support and for helpful suggestions in reviewing the manuscript. This work was supported in part by grants from the Fondazione CARIGE (Genova, Italy), the Ministero della Salute (Italy), the Fondo Investimenti Ricerca di Base-MIUR (Italy), and the Alleanza contro il Cancro: Project 11 (Italy).
Footnotes
1 these authors contributed equally to this work; 
References
T:A mutations in colorectal tumors. Nat Genet 2002;30:227-232.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]T:A mutations. Hum Mol Genet 2002;11:2961-2967.The following articles in journals at HighWire Press have cited this article:
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  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]
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