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Letters to the Editor |
1 Department of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland
aAddress correspondence to this author at: Department of Biological Sciences, Dublin Institute of Technology, Kevin St., Dublin 8, Ireland. Fax 353-1-402-2833; e-mail cathal_mccarthy{at}yahoo.com.
To the Editor:
The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylene tetrahydrofolate to 5-methyltetrahydrofolate, the cosubstrate required for the remethylation of homocysteine to methionine. Mutations in the MTHFR enzyme are reported as causes of hyperhomocysteinemia (1). Hyperhomocysteinemia is generally, although not universally, seen as an independent and graded risk factor for venous thrombosis and neural tube defects (2). Several polymorphisms have been reported in the MTHFR gene, but two particular mutations generate the most interest, the recently described A1298C (3) and the most-characterized C677T (4). The A1298C polymorphism in the MTHFR gene encodes for a glutamate to alanine substitution and leads to a decrease in enzyme activity. Combined heterozygosity for the C677T/A1298C polymorphisms in some studies (5) is associated with higher homocysteine concentrations and decreased plasma folate.
Amplification Refractory Mutation System (ARMS) PCR determination of the MTHFR C677T mutation has been described by Hessner et al. (6). To determine the frequency of the A1298C mutation in the Irish population, we developed a reliable and rapid ARMS PCR method. We compared the results with those obtained with the standard method for detection, PCR followed by restriction fragment length polymorphism (RFLP) analysis (3).
Our cohort consisted of 120 blood donors, none of whom had experienced any past or current thrombotic events or had a family history of thrombosis. Informed consent was obtained from all study participants Total genomic DNA was isolated from blood leukocytes, and MTHFR A1298C was analyzed by PCR-RFLP (3).
ARMS PCR was also used to determine the frequency of this mutation. A typical ARMS PCR set-up for the wild-type reaction consisted of 200 ng of genomic DNA, 2.5 mM MgCl2, 0.4 mM each deoxynucleotide triphosphate (Invitrogen, Bio-Sciences), 2.5 µL of 10x buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl; Invitrogen], 1.5 U of Platinum Taq polymerase (Invitrogen), and 50 mL/L dimethyl sulfoxide (Sigma-Aldrich). ARMS PCR primers used in the wild-type reaction were as follows: A1298C forward consensus primer (5'-CCTTTGGGGAGCTGAAGGACTACTAC-3'); A1298C wild-type reverse primer (5'-CAAAGACTTCAAAGACAGTC-3'); cystic fibrosis 22 (CF22) forward primer (5'-AAACGCTGAGCCTCACAAGA-3'), and CF22 reverse primer (5'-TGTCACCATGAAGCAGGCAT-3'; Sigma-Aldrich). The mutant reaction had the same components as above, with the replacement of A1298C wild-type reverse primer with A1298C mutant reverse primer, which had the following sequence: 5'-GGTAAAGAACAAAGACTTCAAAGACACTGTG-3' (Sigma-Aldrich).
ARMS PCR conditions were 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s; and 72 °C for 5 min. The wild-type reaction produces a 120-bp product, whereas the mutant reaction produces a 127-bp fragment (Fig 1
). The CF22 primers were used as internal control primers and produce a 578-bp fragment in each reaction. Statistical analysis was performed using the Z-test for two independent proportions. Statistical significance was set at Z >1.96.
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Of the 120 healthy Irish participants, 56 were heterozygous carriers, giving a genotype frequency of 46.7%, whereas 11 (14.2%) were homozygous for A1298C. Of the MTHFR C677T/A1298C genotype combinations, 28 participants (23.3%) were double heterozygotes. The prevalence of 1298CC homozygotes in this Irish study is significantly higher than that reported for most European populations, including a UK study (Z = 1.97) (7), an Italian cohort (Z = 2.51) (8), and an American study (Z = 1.99) (9). Only two studies from Northern Scotland (10)(11) have reported a higher prevalence.
The results of the ARMS PCR were in complete concordance with the results obtained by standard PCR-RFLP. The ability of the ARMS PCR to distinguish between genotypes for the A1298C mutation is much higher than that of the standard method.
Acknowledgments
We thank Dr. A Davern, Dublin Blood Transfusion Centre, for the blood samples. We would like to also thank Drs. Valeria Capra (Laboratorio del Servizio di Neurochirugia, Instituto Scientifico G. Gaslin, Genova, Italy) and Nurit Rosenberg (Department of Hematology, Institute of Thrombosis and Hemostasis, The Chaim Sheba Medical Center, Tel-Hashomer, Israel) for the A1298C positive controls.
References
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