2462 Letters

structural issues and can be resolved. are small. In fact, it actually supports tations generate the most interest,
The p53 resequencing microarray is our conclusion that a combination of the recently described A1298C (3 )
currently undergoing a redesign both microarray and sequencing is and the most-characterized C677T
(Roche Molecular Diagnostics, per- required to identify p53 alterations, (4 ). The A1298C polymorphism in
sonal communication). Given the as they would have missed five poly- the MTHFR gene encodes for a glu-
cost-effectiveness of chip technology, morphisms. We also look forward to tamate to alanine substitution and
it is reasonable to expect a next- being able to use the next-generation leads to a decrease in enzyme activ-
generation Chip in which most of p53 microarray, as we stated in our ity. Combined heterozygosity for the
these limitations have been ad- report (1 ), because this methodology C677T/A1298C polymorphisms in
dressed. Such improvements could definitely has a role to play in se- some studies (5 ) is associated with
lead to a more useful and productive quencing of the p53 gene. higher homocysteine concentrations
tool in cancer research and diagnos- and decreased plasma folate.
tics. Reference Amplification Refractory Mutation
1. Cooper M, Li S-Q, Bhardwhaj T, Rohan T, Kandel System (ARMS) PCR determination
RA. Evaluation of oligonucleotide arrays for se- of the MTHFR C677T mutation has
References quencing of the p53 gene in DNA from formalin-
1. Cooper M, Li S-Q, Bhardwhaj T, Rohan T, Kandel fixed, paraffin-embedded breast cancer speci- been described by Hessner et al. (6 ).
RA. Evaluation of oligonucleotide arrays for se- mens. Clin Chem 2004;50:500 – 8. To determine the frequency of the
quencing of the p53 gene in DNA from formalin-
fixed, paraffin-embedded breast cancer speci-
A1298C mutation in the Irish popu-
mens. Clin Chem 2004;50:500 – 8. Rita Kandel1* lation, we developed a reliable and
2. Ahrendt SA, Halachmi S, Chow JT, Wu L, Hala- Thomas Rohan2 rapid ARMS PCR method. We com-
machi N, Yang SC, et al. Rapid p53 sequence pared the results with those obtained
analysis in primary lung cancer using an oligonu- 1
cleotide probe array. Proc Natl Acad Sci U S A
University of Toronto with the standard method for detec-
1999;96:7382–7. Toronto, Canada tion, PCR followed by restriction
2
fragment length polymorphism (RFLP)
Antonette C.P. Allen* Albert Einstein College of Medicine analysis (3 ).
Francis A. Chiafari Bronx, NY Our cohort consisted of 120 blood
donors, none of whom had experi-
BRT Laboratories, Inc. enced any past or current thrombotic
400 West Franklin St. * Address correspondence to this au-
thor at: Pathology and Laboratory Medi- events or had a family history of
Baltimore, MD 21201 cine, Mt. Sinai Hospital, 600 University thrombosis. Informed consent was
Ave., Toronto, ON M5G1X5 Canada. Fax obtained from all study participants.
416-586-8628; e-mail [email protected]. Total genomic DNA was isolated
*Author for correspondence. Fax 410- ca.
383-0938; e-mail [email protected]. from blood leukocytes, and MTHFR
DOI: 10.1373/clinchem.2004.043117 A1298C was analyzed by PCR-RFLP
Previously published online at DOI: (3 ).
10.1373/clinchem.2004.038158
ARMS PCR was also used to de-
termine the frequency of this muta-
Drs. Kandel and Rohan respond: tion. A typical ARMS PCR set-up for
Increased Frequency of the MTHFR the wild-type reaction consisted of
To the Editor: A1298C Mutation in an Irish 200 ng of genomic DNA, 2.5 mM
We would like to thank Drs. Allen Population MgCl2, 0.4 mM each deoxynucleo-
and Chiafari for their comments. tide triphosphate (Invitrogen, Bio-
They reiterate many of the points we To the Editor: Sciences), 2.5 ␮L of 10⫻ buffer [200
made in our report (1 ). We are glad The enzyme methylenetetrahydrofo- mM Tris-HCl (pH 8.4), 500 mM KCl;
that they also had similar findings. late reductase (MTHFR) catalyzes Invitrogen], 1.5 U of Platinum Taq
However, they suggest that we differ the reduction of methylene tetrahy- polymerase (Invitrogen), and 50
in the detection of polymorphisms. drofolate to 5-methyltetrahydrofo- mL/L dimethyl sulfoxide (Sigma-
In the Discussion section of our re- late, the cosubstrate required for Aldrich). ARMS PCR primers used
port (1 ), we speculated that our cut- the remethylation of homocysteine in the wild-type reaction were as
off score might be too high because to methionine. Mutations in the follows: A1298C forward consensus
we observed that a polymorphism in MTHFR enzyme are reported as primer (5⬘-CCTTTGGGGAGCTGA-
exon 6 had a score of 6. In their causes of hyperhomocysteinemia (1 ). AGGACTACTAC-3⬘); A1298C wild-
study, the GeneChip detected one of Hyperhomocysteinemia is generally, type reverse primer (5⬘-CAAAGAC-
six exon 4 (codon 72) polymor- although not universally, seen as an TTCAAAGACAGTC-3⬘); cystic fi-
phisms, whereas we were unable to independent and graded risk factor brosis 22 (CF22) forward primer
detect any in 12 cases from which we for venous thrombosis and neural (5⬘-AAACGCTGAGCCTCACAAGA-
obtained a PCR product. We would tube defects (2 ). Several polymor- 3⬘), and CF22 reverse primer (5⬘-
argue that this was not a significant phisms have been reported in the TGTCACCATGAAGCAGGCAT-3⬘;
difference because both sample sizes MTHFR gene, but two particular mu- Sigma-Aldrich). The mutant reaction
 Clinical Chemistry 50, No. 12, 2004 2463

Fig. 1. ARMS PCR for MTHFR C677T and A1298C polymorphisms in the
A1298C. methylenetetrahydrofolate reductase gene on
homocysteine levels in elderly men and women
Reactions are shown in pairs. from the British regional heart study. Athero-
Products of the wild-type reaction sclerosis 2001;154:659 – 66.
are analyzed in the first lane of 8. De Marco P, Grazia Calevo M, Moroni A, Arata
each pair, products of mutant re- L, Merello E, Finnell RH, et al. Study of MTHFR
action are analyzed in the second and MS polymorphisms as risk factors for NTD
lane of each pair. Each pair con- in the Italian population. J Hum Genet 2002;
tains the CF22 internal control, 47:319 –24.
which generates a 578-bp prod-
uct. Lane 0, 100-bp DNA ladder 9. Hanson NQ, Aras O, Yang F, Tsai MY. C677T
(Invitrogen, Bio-Sciences); lanes and A1298C polymorphisms of the methyl-
1 and 2, presence of the 120-bp enetetrahydrofolate reductase gene: incidence
product in lane 1 indicates wild and effect of combined genotypes on plasma
type for the mutation; lanes 3 and fasting and post-methionine load homocysteine
4, presence of 120-bp product in in vascular disease. Clin Chem 2001;47:
lane 3 and 127-bp product in lane 661– 6.
4 indicate heterozygous carrier of 10. Sharp L, Little J, Schofield AC, Pavlidou E,
the mutation; lanes 5 and 6, pres- Cotton SC, Miedzybrodzka Z, et al. Folate and
ence of the 127-bp product in breast cancer: the role of polymorphisms in
lane 6 indicates a homozygous methylenetetrahydrofolate reductase (MTHFR).
carrier of the mutation. Cancer Lett 2002;181:65–71.
11. Sharp L, Little J, Brockton N, Cotton SC, Haites
NE, Cassidy J. Dietary intake of folate and
related micronutrients, genetic polymorphisms
had the same components as above, results obtained by standard PCR- in MTHFR and colorectal cancer: a population-
with the replacement of A1298C RFLP. The ability of the ARMS PCR based case-control study in Scotland [Ab-
wild-type reverse primer with to distinguish between genotypes for stract]. J Nutr 2002;132(Suppl 11):3542S.
A1298C mutant reverse primer, the A1298C mutation is much higher
which had the following sequence: than that of the standard method. Cathal Mc Carthy*
5⬘-GGTAAAGAACAAAGACTTCA- Fergus Ryan
AAGACACTGTG-3⬘ (Sigma-Aldrich). Joseph Vaughan
ARMS PCR conditions were 95 °C We thank Dr. A Davern, Dublin
for 5 min; 40 cycles of 95 °C for 30 s, Blood Transfusion Centre, for the Department of Biological Sciences
57 °C for 30 s, and 72 °C for 30 s; and blood samples. We would like to Dublin Institute of Technology
72 °C for 5 min. The wild-type reac- also thank Drs. Valeria Capra (Labo- Dublin, Ireland
tion produces a 120-bp product, ratorio del Servizio di Neurochiru-
whereas the mutant reaction pro- gia, Instituto Scientifico G. Gaslin, *Address correspondence to this au-
duces a 127-bp fragment (Fig. 1). The Genova, Italy) and Nurit Rosenberg thor at: Department of Biological Sci-
CF22 primers were used as internal (Department of Hematology, Insti- ences, Dublin Institute of Technology,
control primers and produce a tute of Thrombosis and Hemostasis, Kevin St., Dublin 8, Ireland. Fax 353-1-
The Chaim Sheba Medical Center, 402-2833; e-mail cathal_mccarthy@yahoo.
578-bp fragment in each reaction. com.
Statistical analysis was performed Tel-Hashomer, Israel) for the A1298C
using the Z-test for two independent positive controls. DOI: 10.1373/clinchem.2004.041517
proportions. Statistical significance
References
was set at Z ⬎1.96. 1. Lievers KJA, Kluijtmans LJA, Blom HJ. Genetics
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riers, giving a genotype frequency of bosis. Clin Lab Haematol 2000;22:133– 43.
46.7%, whereas 11 (14.2%) were 3. Van der Put NJM, Gabreels F, Stevens EMB, Potassium Caused by Bacterial
homozygous for A1298C. Of the Smeitink JAM, Trijbels FJM, Eskes TKAB, et al. Contamination of an Analyzer
A second common mutation in the methyl-
MTHFR C677T/A1298C genotype enetetrahydrofolate reductase gene: an addi-
combinations, 28 participants (23.3%) tional risk factor for neural tube defects? Am J To the Editor:
Hum Genet 1998;62:1044 –51.
were double heterozygotes. The 4. Frosst P, Blom HJ, Milos R. A candidate genetic
We encountered a problem with po-
prevalence of 1298CC homozygotes risk factor for vascular disease, a common tassium measurement after periods
in this Irish study is significantly mutation in methylenetetrahydrofolate reduc- of standby on a Beckman Synchron
tase. Nat Genet 1995;10:111–3.
higher than that reported for most 5. Weisberg I, Tran P, Christensen B, Sibani S, LX20 Pro. Initial potassium measure-
European populations, including a Rozen R. A second genetic polymorphism in ments were rejected because of ex-
UK study (Z ⫽ 1.97) (7 ), an Italian MTHFR associated with decreased enzyme ac- cessive reference drift, and potas-
tivity. Mol Genet Metab 1998;64:169 –72.
cohort (Z ⫽ 2.51) (8 ), and an Ameri- 6. Hessner MJ, Luhm RA, Pearson SL, Endean DJ, sium results for quality-control (QC)
can study (Z ⫽ 1.99) (9 ). Only two Friedman KD, Montgomery RR. Prevalence of samples were as much as 2.5
prothrombin G202010A, factor V G1691A (Lei-
studies from Northern Scotland den), and methylenetetrahydrofolate reductase
mmol/L above the previous mean.
(10, 11 ) have reported a higher prev- (MTHFR) C677T in seven different populations Replicate analyses of control material
alence. determined by multiplex allele-specific PCR. immediately after standby revealed a
Thromb Haemost 1999;81:733– 8.
The results of the ARMS PCR were 7. Dekou V, Whincup P, Papacosta O, Ebrahim S, decreasing trend in potassium con-
in complete concordance with the Lennon L, Ueland PM, et al. The effect of the centration, whereas simultaneously