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Technical Briefs |
1
Laboratoire de biochimie et biologie moléculaire, Hôpital R. Poincaré, F92380 Garches, France;
2
Laboratoires d'hématologie du centre hospitalier de Poissy-Saint Germain en Laye, F78303 Poissy, France;
3
Service de Médecine Interne, centre hospitalier de Poissy-Saint Germain en Laye, F78303 Poissy, France;
4
Faculté de Médecine Paris Ouest, CJF 9402, Université Paris, V, F92380 Garches, France;
a author for correspondence: fax 331 47 10 79 23
The thermolabile variant of the methylene tetrahydrofolate reductase (MTHFR) in the homozygous state has been shown to be responsible for mild hyperhomocystinemia, hypomethioninemia, and hyperhomocystinuria (1). This variant is responsible for an increased risk for recurrent early pregnancy loss and neural-tube defects (2)(3). The presence of hyperhomocystinemia is also predictive of both arterial and venous thromboembolic disease (4)(5)(6)(7) and is a risk factor for coronary artery stenosis, independent of other risk factors such as age, smoking, hypercholesterolemia, and hypertension (8). Four to 6% of the Caucasian population (9) and 13–20% of the thrombosis-prone patients are homozygous for the thermolabile variant of MTHFR, which is caused by a C-to-T substitution at nucleotide 677 of the cDNA, resulting in the substitution of a valine for an alanine (8). A simple molecular diagnosis is of particular interest because this risk factor is quite common, the biochemical assay requires a methionine load, and folate supplementation is likely to prevent some of the complications (10)(11). Thus, the exploration has been recommended in the management of premature venous and arterial occlusive diseases (4). We report here an improvement of the method described previously to assess the thermolabile variant of MTHFR, based on multiplex amplification of MTHFR and an internal control.
We studied 30 healthy control volunteers and 30 patients with personal or familial history of thrombosis or phlebitis. Informed written consent was obtained in all cases.
DNA extraction was performed from frozen blood either by phenol-chloroform extraction according to McIndoe et al. (12) or with DNAzol (Life Technologies, Inc.) as recommended by the manufacturer. In most of the control subjects, DNA was extracted with DNAzol from the cell pellet of saliva after two washes in 9 g/L NaCl.
Amplification of MTHFR was adapted from the method described by Frosst
et al. (1) as follows. Initial denaturation step was for 4
min at 94 °C followed by 30 cycles of denaturation for 30 s at
94 °C, annealing for 30 s at 62 °C, and elongation for
90 s at 72 °C. The final elongation step was for 12 min at
72 °C. Primers used were: primer A, 5'-TGA AGG AGA AGG TGT CTG CGG
GA; primer B, 5'-AGG ACG GTG CGG TGA GAG TG; primer C, 5'-CTC CCT TCA
CTT TCA GAA CTA CA; and primer D, 5' GAC CTC TCA GTT TTC ACC TTT A for
MTHFR (1) and fibrinogen A
exon III coamplification
(HUMFIBRA, positions 1723 and 2252). Each primer was used at 0.25
µmol/L in a 25-µL final volume in the presence of standard PCR
master mix (Boehringer-Mannheim). Final concentrations were 10 mmol/L
Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L each
deoxynucleotide triphosphate, 0.5 mL/L Brij 35, and 0.625 U Taq DNA
polymerase. Amplifications were performed with a Perkin-Elmer 2400
thermocycler. Amplicons were then digested for at least 2 h
(usually 4 h) with HinfI (Euromedex; 1 IU/10 µL
amplicon) in the buffer supplied by the manufacturer, and the products
were visualized by ethidium bromide staining (0.5 mg/L) of a 3%
agarose gel.
When using only the A and B primers as in the reference method, the elongation time in the amplification can be shortened to 60 s, and the final elongation step at 72 °C to 7 min. However, in the adaptation we describe above, shortening the elongation steps resulted in insufficient amplification of the fibrinogen fragment and poor signal ratio for MTHFR and fibrinogen amplicons, hampering the detection of fibrinogen digestion fragments.
When coamplified with the primers A, B, C, and D as described above (90
s at 72 °C for the elongation steps and 12 min for the final
elongation), DNA samples generated two DNA fragments of 552 and 198 bp
for the fibrinogen A and MTHFR fragments, respectively (Fig. 1
, lane 1). Digestion of the fibrinogen fragments for 2–4 h at
37 °C with 2 U HinfI generated three fragments of 56,
136, and 360 bp for the A fragments. The 56-bp fragment was not seen
under our experimental conditions. As can be seen from Fig. 1
, lanes 2
to 4, the 136- and 360-bp fragments did not overlap with the MTHFR
amplification and digestion products. Digestion of the MTHFR fragment
generated a 175-bp fragment when an allele coding for the thermolabile
variant of MTHFR was present (Fig. 1
, lanes 3 and 4). For homozygous
patients for the thermolabile variant of MTHFR, the 198-bp fragment was
absent (Fig. 1
, lane 4). For heterozygous patients, both the 198- and
175-bp fragments were present (Fig. 1
, lane 3), and for unaffected
patients, the 175-bp fragment was absent (Fig. 1
, lane 2).
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For strategies based on digestion of unique sites, incomplete digestion of PCR products from homozygous patients leads to a pattern similar to heterozygous patients, and failure of digestion of PCR product from heterozygous patients leads to a pattern similar to unaffected patients. Some nonorganic DNA extraction procedures carryover marked amounts of enzyme inhibitors (13). The addition of homozygous, heterozygous, and unaffected controls in the series does not avoid tube-to-tube variability in inhibitor contamination. A first possibility is to amplify a larger DNA fragment including a second site, as reported for the inclusion of a second MstII site downfield of the sickle cell mutation (14). For rare restriction sites, this is sometimes impossible. With the method we describe here, the control fragment carries HinfI sites generating fragments that do not interfere with the detection of the MTHFR fragments. Should any incomplete digestion occur, the digestion of the control fragment would leave part of the 552-bp fragment, avoiding misdiagnosis due to incomplete digestion. This method is simple, reliable, can be set up in any laboratory, and requires only another set of primers and prolongation of the elongation steps in the PCR.
References
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