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Letters to the Editor |
1 Laboratory of Human Genetics, Medical Oncology C
2 Laboratory of Translational Research A
3 Medical Oncology A, IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
4 Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
5 Medical Oncology, Ospedale Sacro Cuore – Don Calabria, Verona, Italy
6 Istituto di Genetica e Biofisica, "Adriano Buzzati Traverso" (IGB-CNR), Naples, Italy
7 Laboratory of Genetics and Gene Transfer, Core Research Laboratory – Istituto, Toscano Tumori (CRL-ITT), Florence, Italy
aAddress correspondence to this author at: Laboratory of Genetics and Gene Transfer, Core Research Laboratory – Istituto, Toscano Tumori (ITT-CRL), Viale Pieraccini 6 (Cubo), 50139 Florence, Italy, Fax 39-055-427-1280, e-mail rosario.notaro{at}ittumori.it
To the Editor:
Tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) is a simple and inexpensive genotyping method for differentiating both alleles of a polymorphism/mutation (both single-nucleotide polymorphisms and small insertions/deletions) with a single-tube PCR (1). In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific control amplicon and in combination with 2 allele-specific (inner) primers (designed to anneal in the opposite orientation) produces 2 allele-specific amplicons. These allele-specific amplicons have different sizes because the polymorphism/mutation is asymmetrically located with respect to the common primers. Thus, the amplicons can be separated by standard gel electrophoresis. T-ARMS-PCR has also been designed in a multiplex fashion to genotype more than one polymorphism/mutation by a single-tube PCR (2).
We describe a modified multiplex T-ARMS-PCR, the hexaprimer ARMS-PCR (H-ARMS-PCR), which is for when 2 polymorphisms are close in the sequence. H-ARMS-PCR uses only 6 primers and provides direct information about haplotype structure.
The CTLA4 gene (cytotoxic T-lymphocyte–associated protein 4; also known as CD152) is a negative regulator of T-cell function (3). The CTLA4 polymorphisms –318 C>T (rs5742909) and +49 A>G (rs231775) are associated with susceptibility to autoimmune diseases and cancer (3)(4). To genotype these 2 polymorphisms, which are only 365 bp apart in the 5' region of the CTLA4 gene, we designed an H-ARMS-PCR that combines a single pair of common primers and 2 pairs of allele-specific primers in the same tube (Fig. 1A
).
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The PCR reaction (25 µL) contained 200 µmol/L deoxynucleoside triphosphates, 100 ng of genomic DNA, 1.25 U of HotStarTaq DNA Polymerase with its buffer (Qiagen), and the following primers at the indicated concentrations (deliberate mismatches from the reference sequence, GenBank no. M74363, are in boldface italics): –318fo, 5'-CAATGAAATGAATTGGACTGGATG-3' (0.5 µmol/L); +49ro, 5'-TACAGAGCCAGCCAAGCCAGATT-3' (0.8 µmol/L); –318fi(c), 5'-CTCCACTTAGTTATCCAGATCGTC-3' (2.5 µmol/L); –318ri(t), 5'-ACTGAAGCTTCATGTTCACTCTA-3' (0.5 µmol/L); +49fi(a), 5'-GCACAAGGCTCAGCTGAACCTGGATA-3' (0.1 µmol/L); and +49ri(g), 5'-ACAGGAGAGTGCAGGGCCAGGTCCTAGC-3' (0.5 µmol/L).
Cycling conditions were 12 min at 95 °C, followed by 5 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 45 s; 30 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s; and a final cycle at 72 °C for 10 min.
We genotyped both the –318 C>T and +49 A>G CTLA4 polymorphisms with this newly designed H-ARMS-PCR (Fig. 1A
) for samples from 80 individuals that had previously been characterized with 3 established methods [restriction fragment length polymorphism (RFLP) PCR, direct DNA sequencing, and T-ARMS-PCR] (4) and observed no discrepancies. Moreover, H-ARMS-PCR provides both an additional internal control and direct information about the haplotype structure by generating an amplicon that is specific for 1 of the 4 haplotypes that 2 polymorphisms can theoretically have. In this case, the haplotype-specific amplicon is observed when the alleles –318C and +49G are on the same chromosome (Fig. 1A
).
We further tested this approach by implementing H-ARMS-PCR in the genotyping of 2 polymorphisms that are 142 bp apart in the 3' untranslated region (UTR) of the CYP19A1 gene (cytochrome P450, family 19, subfamily A, polypeptide 1): rs10046 (C>T) and rs4646 (G>T). CYP19A1 encodes aromatase, the enzyme responsible for the final step of estrogen biosynthesis. CYP19A1 polymorphisms have been associated with estrogen concentrations in woman and with susceptibility to breast and prostate cancers (5).
We used the PCR to analyze these 2 CYP19A1 polymorphisms in the 3' UTR (Fig. 1B
; see legend for PCR conditions) in 100 individuals already characterized by RFLP PCR and/or direct DNA sequencing (P.P. and R.N., unpublished data) and observed no discrepancies. A haplotype-specific amplicon is present when alleles rs10046C and rs4646T are on the same chromosome. This H-ARMS-PCR is currently being used in a multicenter Italian clinical trial (GIM5) to test the possible association of CYP19A1 polymorphisms with the efficacy of adjuvant therapy with an aromatase inhibitor (letrozole) after tamoxifen treatment in postmenopausal patients with early breast cancer.
In addition, we genotyped a series of 40 individuals for both CTLA4 and CYP19A1 polymorphisms by H-ARMS-PCR analysis with different thermal cyclers (iCycler® by Bio-Rad, Px2® by Hybaid, and PTC-100® by MJ Research) and in different laboratories, and we obtained fully concordant results, demonstrating the robustness and reproducibility of this approach. All human samples were collected after informed consent was obtained according to institutional procedure.
We have shown that H-ARMS-PCR is an effective and robust technique for genotyping 2 close polymorphisms (in a range of about 100–400 bp) with only 6 primers. With appropriate long-range Taq DNA polymerase, H-ARMS-PCR may work for genotyping even more distantly separated polymorphisms, likely within a distance of about 5 kb. In addition, by providing direct information about one defined haplotype, H-ARMS-PCR, may be useful for the identification of potentially ambiguous haplotypes in individuals who are double heterozygotes.
Acknowledgments
Grant/funding Support: This work is supported in part by grants from Ministero della Salute (Italy), Compagnia di San Paolo (Torino, Italy), and CARIGE (Genova, Italy). P.P. is supported by Oncotech, Naples, Italy.
Financial Disclosures: None declared.
Footnotes
1 These authors contributed equally to this work. 
References
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