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Obstetrics & Gynecology 2003;101:762-766
© 2003 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

The 677 C-T Methylenetetrahydrofolate Reductase Mutation Does Not Predict Increased Maternal Homocysteine During Pregnancy

Robert W. Powers, PhD, Michael S. Dunbar, Marcia J. Gallaher and James M. Roberts, MD

From the Magee-Womens Research Institute and Department of Obstetrics, Gynecology, and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

Address reprint requests to: Robert W. Powers, PhD, Magee-Womens Research Institute, 204 Craft Avenue, Room 620, Pittsburgh, PA 15213; E-mail: rsirwp{at}mail.magee.edu.


   ABSTRACT
TOP
ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE: To test the hypothesis that, regardless of the presence of the 677 C-T methylenetetrahydrofolate reductase (MTHFR) mutation, maternal homocysteine concentrations will not be significantly different in women who are taking prenatal vitamins containing folic acid, and to test this relationship in preeclampsia because homocysteine concentrations are higher in preeclamptic pregnancies.

METHODS: Fifty-seven pregnant white women (control and preeclamptic) with and without the 677 C-T MTHFR mutation were studied. Total plasma homocysteine and plasma folic acid were analyzed.

RESULTS: Homocysteine concentrations were not different by MTHFR genotype (wild type 677 CC 8.7 ± 5.6 µM versus mutant 677 TT 9.0 ±5.7 µM, P = .84) in preeclamptic or normal pregnancies. However, mean homocysteine concentrations were significantly increased in preeclamptic pregnancies compared with those in normal pregnancies (10.6 ± 7.3 µM versus 7.2 ± 3.0 µM, P < .03) as previously reported.

CONCLUSION: The 677 C-T MTHFR polymorphism does not significantly affect maternal homocysteine concentrations in most women taking prenatal vitamins including women with preeclampsia. The increase in plasma folic acid likely affects maternal homocysteine more than the MTHFR genotype. If homocysteine is considered a thrombophilia risk factor, the concentration of the amino acid and not a particular genotype should be determined.

Elevated circulating homocysteine is an independent risk factor for peripheral vascular disease and coronary artery disease.1–4 Homocysteine is a demethylated metabolite of the essential amino acid methionine and is associated with thrombosis and several pregnancy complications including preeclampsia.5–10 The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) is responsible for converting 5,10 methyl tetrahydrofolate to 5-methyl tetrahydrofolate, which is used as a methyl donor in the conversion of homocysteine to methionine.11,12 One polymorphism of the MTHFR gene results in a cytosine to thymine conversion at nucleotide 677, and causes an alanine to valine missense mutation at amino acid 222 in the MTHFR protein.13,14 The 677 C-T missense polymorphism in the MTHFR gene is associated with decreased enzyme activity and therefore increased homocysteine.14 Because of the association of increased homocysteine with thrombosis and adverse pregnancy outcomes, it has been recommended to screen for the MTHFR mutation in at-risk pregnancies.15,16 However, increased folic acid overcomes the reduced MTHFR activity resulting in normal homocysteine concentrations.17,18 Results from our previous studies indicate that folic acid concentrations are significantly higher in pregnant women taking folic acid-containing vitamins compared with those in nonpregnant women (pregnant 23.9 ±8.8 ng/mL versus nonpregnant 10.± 4.9 ng/mL, P < .001). Therefore, the use of one particular genetic polymorphism (677 C-T MTHFR) as a diagnostic indicator of plasma homocysteine is indirect and subject to error because homocysteine is readily affected by other mediators, particularly folic acid. Furthermore, if one is interested in plasma homocysteine concentration as an indicator of thrombotic risk, homocysteine should be measured instead of relying on one particular genotype as a surrogate marker. Therefore, the focus of this study was to estimate whether the presence of the 677 C-T MTHFR polymorphism would affect maternal homocysteine concentrations in women during pregnancy. We also investigated whether this relationship would affect homocysteine concentrations in women with the pregnancy complication preeclampsia, which is also associated with increased homocysteine.


   MATERIALS AND METHODS
TOP
ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients were recruited at the time of admission to labor and delivery at Magee-Womens Hospital as part of the ongoing investigation of preeclampsia (1997–2001, with 80% successful enrollment). This is a retrospective, case-control study, which was approved by the hospital Institutional Review Board. Fifty-seven nulliparous women were chosen for this study, 30 with a normal pregnancy outcome and 27 with preeclampsia. Within these subjects, each group was comprised of women who either had both alleles affected by the MTHFR polymorphism (677 TT) or both alleles were normal (677 CC). Therefore, 15 women with a normal pregnancy outcome had the TT genotype, 15 normal pregnant women had the CC genotype, 13 women with preeclampsia had the TT genotype, and 14 women with preeclampsia had the CC genotype. All patients were white to ensure homogeneity of ethnic background to reduce genetic variability. Preeclampsia was defined using the criteria of gestational hypertension, proteinuria, hyperuricemia, and the reversal of hypertension and proteinuria after pregnancy. Gestational hypertension was defined as an absolute blood pressure of 140 or 90 mm Hg or more or an increase of 30 mm Hg systolic or 15 mm Hg diastolic blood pressure compared with values obtained before 20 weeks’ gestation. Eighty-nine percent (24) of the women in this study met the absolute increased blood pressure criteria. Proteinuria was defined as greater than 300 mg per 24-hour urine collection or greater than 2+ on a voided or greater than 1+ on a catheterized random urine sample. Hyperuricemia was defined as greater than one standard deviation above values at gestational age of sampling (5.5 mM or more at term). All patients were nulliparous, and no patient was known to have chronic hypertension, renal, or metabolic disease.

Ethylenediaminetetra-acetic acid plasma and genomic deoxyribonucleic acid samples were collected at admission to labor and delivery and stored at -80C until assayed.

The MTHFR genotype for each woman was determined by restriction fragment length polymorphism polymerase chain reaction, as previously described by Frosst et al and Powers et al.19,20

Total plasma homocysteine was measured by high-performance liquid chromatography with electrochemical detection. Briefly, 40 µL of plasma was mixed with 20 µL of 75 µM of penicillamine, and the sample was reduced by adding 7 µL of a 60-mg/mL in-water solution of tris (carboxyethyl) phosphine. The sample was incubated at room temperature for 10 minutes, and then 170 µL of 0.3 N perchloric acid was added to precipitate the protein. The sample was centrifuged at 12,000g to pellet the protein, and 20 µL of the supernatant was injected onto a prepared high-performance liquid chromatography column (HR-80 [ESA Inc., Chelmsford, MA], 80 x 4.6 mm 3 µm, C18 packing with guard column). The mobile phase for the system was 0.14 M of NaH2PO4, 1 mM of sodium dodecylsulfate, and 10% acetonitrile (pH = 2.9), with phosphoric acid, and the flow rate was 1.2 mL per minute. The electrochemical detector was a 5010 analytical cell from ESA, and the settings were E1 = + 400 mV, 5-µAmp full-scale 5-s filter, 1-V output; E2 = +750 mV, 5-µAmp full-scale 5-s filter, 1-V output, and the setting for the guard cell was Egc = +850 mV. The standard curve was prepared daily by spiking L-homocysteine into control plasma to obtain the following final homocysteine concentrations: 0, 1, 2, 5, 10, and 20 µM. The interassay variability was 10%.

The plasma folic acid concentration was determined with a radioimmunoassay from Diagnostics Products Corp. (Los Angeles, CA). The assay procedure was that described by the manufacturer. The detection limit of the assay for folic acid was 0.3 ng/mL. The interassay coefficient of variation was less than 10%.

Statistical analysis was by unpaired Student t test for the subjects’ clinical variables and two-factor (pregnancy outcome and MTHFR genotype) analysis of variance for plasma homocysteine and folic acid. Correlations were by simple regression analysis. Sample size power analysis indicated that we required 29 subjects per group to obtain a 35% difference in mean plasma homocysteine concentration with an {alpha} of 0.05 and 80% power. Significance was accepted at P < .05. Means and standard deviations are reported.


   RESULTS
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ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The subjects’ clinical characteristics are summarized in Table 1Go. Briefly, maternal age, prepregnancy body mass index, gestational age at delivery, and blood pressure were significantly different between the preeclamptic and control subjects. However, these variables were not different when analyzed by the subjects’ MTHFR genotype.


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  		Table 1. Subjects’ Clinical Characteristics Divided by Genotype or Outcome
 
Differences in maternal total plasma homocysteine were analyzed by two-way analysis of variance (pregnancy outcome and MTHFR genotype), and the results of this analysis are presented in Table 2Go. Maternal plasma homocysteine concentrations were not significantly different between the two MTHFR genotypes (8.7 ± 5.5 µM versus 9.0 ±5.7 µM, CC and TT, respectively, P = .83). However, plasma homocysteine was significantly increased in the women with preeclampsia compared with that in the normal pregnant women (10.6 ±7.3 µM versus 7.2 ± 2.6 µM, respectively, P < .03).


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  		Table 2. Maternal Plasma Homocysteine by MTHFR Genotype and Pregnancy Outcome
 
There was no significant difference in plasma folic acid between the two MTHFR genotypes (18 ± 8.2 ng/mL versus 18.3 ± 5.7, CC and TT, respectively, P = .81) (Table 3Go). However, maternal plasma folic acid was significantly higher in the preeclamptic than in normal pregnant women (21.1 ± 6.2 ng/mL versus 15.5 ± 6.8 ng/mL, respectively, P < .01) (Table 3Go), despite increased homocysteine concentrations in the women with preeclampsia. Lastly, there was one normal pregnant woman who had significantly increased plasma homocysteine (greater than 90th centile) with the 677 TT MTHFR genotype (Figure 1Go), despite increased plasma folic acid. Further investigation of her medical history and analysis of her cystathionine ß-synthase gene for a common pyrodoxine-nonresponsive mutation (G307S)21,22 failed to explain the elevated homocysteine concentrations.


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  		Table 3. Plasma Folic Acid by MTHFR Genotype and Pregnancy Outcome
 


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  		Figure 1. Correlation between maternal total plasma homocysteine and plasma folic acid in the normal pregnant women. Women with the 677 CC genotype are represented by the open circles (r2 = .39, P = .01), and women with the 677 TT genotype are represented by the filled circles (r2 = .01, P = .91). Despite plasma folic acid concentrations above the mean of nonpregnant values in our population (10.3.± 4.9 ng/mL, horizontal line), one woman with the MTHFR 677 TT genotype still had an increase in plasma homocysteine (greater than 90th centile, vertical line).

Powers. MTHFR and Maternal Homocysteine. Obstet Gynecol 2003.

 

   DISCUSSION
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have addressed whether the presence of the 677 C-T missense mutation of the MTHFR gene results in increased homocysteine, among pregnant women with increased plasma folic acid. Although the 677 C-T MTHFR polymorphism has been associated with an increase in plasma homocysteine, as well as an increased risk of thrombosis,23,24 the association with increased homocysteine is extremely dependent upon folic acid concentrations. In assessing an individual’s risk of thrombosis, the presence of 677 C-T MTHFR may not result in increased homocysteine.

We report that mean plasma homocysteine is not different between pregnant women with or without the 677 C-T MTHFR polymorphism. Nonetheless, despite significantly increased plasma folic acid, one normal pregnant woman with the 677 TT genotype still had a significant increase in total plasma homocysteine (less than 90th centile). We failed to identify a cause for her increased homocysteine after studying her medical record as well as searching for possible mutations in her cystathionine ß-synthase gene. However, folic acid and the presence of the 677 C-T MTHFR polymorphism are just two of the many factors that may affect plasma homocysteine, including additional mutations in other homocysteine-regulating genes (methionine synthase, cystathionine ß-synthase, as well as other mutations in MTHFR), other nutritional mediators (vitamin B12, B6, or betaine), age, hormonal effects, and renal function. Despite this one outlier, our general conclusion remains that the presence of the 677 C-T MTHFR polymorphism does not necessarily result in increased plasma homocysteine in populations with increased folic acid.

In contrast to finding no difference in plasma homocysteine between subjects with or without the 677 C-T MTHFR polymorphism, we did find a significant increase in maternal total plasma homocysteine in women with preeclampsia compared with normal pregnant women, regardless of the presence or absence of the 677 C-T MTHFR polymorphism. This result confirms previous studies that also reported increased homocysteine in preeclampsia.20,25–28 In addition, we observed an increase in plasma folic acid among the women with preeclampsia compared with normal pregnant women. One possible explanation for this result may be that the increase in plasma folic acid is the result of compromised renal function in the women with preeclampsia as has been described previously for patients with renal dysfunction.29

In conclusion, this study suggests that the 677 C-T MTHFR polymorphism likely contributes minimally to maternal homocysteine concentrations in vitamin-supplemented women during pregnancy. The increase in plasma folic acid (from prenatal multivitamins) during pregnancy likely affects maternal homocysteine more than the 677 C-T MTHFR polymorphism. Lastly, if increased homocysteine concentrations are considered to increase the risk of thrombophilias and adverse pregnancy outcomes in pregnancy, then homocysteine concentrations should be measured instead of focusing on particular mutations.


   Footnotes
 
This study was supported by National Institutes of Health grant number RO1 HD36110-02 and National Research Service Award fellowship number 1 F32 HD08310-01.

doi:10.1016/S0029-7844(02)03120-4

Received July 8, 2002. Received in revised form September 24, 2002. Accepted November 13, 2002.


   REFERENCES
TOP
ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Verhoef P, Meleady R, Daly LE, Graham IM, Robinson K, Boers GH. Homocysteine, vitamin status and risk of vascular disease; effects of gender and menopausal status. European COMAC Group. Eur Heart J 1999;20: 1234–44.[Abstract/Free Full Text]

2. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Ueland PM, et al. Plasma homocysteine as a risk factor for vascular disease: The European Concerted Action Project. JAMA 1997;277:1775–81.[Abstract]

3. Verhoef P, Stampfer MJ. Prospective studies of homocysteine and cardiovascular disease. Nutr Rev 1995;53: 283–8.[Medline]

4. Mayer EL, Jacobsen DW, Robinson K. Homocysteine and coronary atherosclerosis. J Am Coll Cardiol 1996;27: 517–27.[Abstract]

5. Wouters MGAJ, Boers GHJ, Blom HJ. Hyperhomocysteinemia: A risk factor in women with unexplained recurrent early pregnancy loss. Fertil Steril 1993;60:820–5.[Medline]

6. Harpel PC, Zhang X, Borth W. Homocysteine and hemostasis: Pathogenetic mechanisms predisposing to thrombosis. J Nutr 1996;126:1285S–9S.

7. Rees MM, Rodgers GM. Homocysteinemia: Association of a metabolic disorder with vascular disease and thrombosis. Thromb Res 1993;71:337–59.[Medline]

8. Vollset SE, Refsum H, Irgens LM, Emblem BM, Tverdal A, Gjessing HK, et al. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: The Hordaland homocysteine study. Am J Clin Nutr 2000;71:962–8.[Abstract/Free Full Text]

9. Cotter AM, Molloy JM, Scott JM, Daly SF. Elevated plasma homocysteine in early pregnancy: A risk factor for the development of severe preeclampsia. Am J Obstet Gynecol 2001;185:781–5.[Medline]

10. Powers RW, Evans RW, Majors AK, Ojimba JI, Ness RB, Crombleholme WR, et al. Plasma homocysteine is increased in preeclampsia and is associated with evidence of endothelial activation. Am J Obstet Gynecol 1998;179: 1605–11.[Medline]

11. Bailey LB, Gregory JF. Polymorphisms of methylenetetrahydrofolate reductase and other enzymes: Metabolic significance, risks and impact on folate requirement. J Nutr 1999;129:919–22.[Abstract/Free Full Text]

12. Ubbink JB. Vitamin nutrition status and homocysteine: An atherogenic risk factor. Nutr Rev 1994;52:383–93.[Medline]

13. Goyette P, Christensen B, Rosenblatt DS, Rozen R. Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene, and description of five novel mutations in MTHFR. Am J Hum Genet 1996;59: 1268–75.[Medline]

14. Daly S, Molloy A, Mills J, Kirke AS, Whitehead AS, Conley D, et al. The effect of 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T genotype on plasma folate and red cell folate concentrations in pregnant and non pregnant women. J Gynecol Investig 1997;4:113A.

15. Brenner B, Sarig G, Weiner Z, Younis J, Blumenfeld Z, Lanir N. Thrombophilic polymorphisms are common in women with fetal loss without apparent cause. Thromb Haemost 1999;82:6–9.[Medline]

16. van der Molen EF, Verbruggen B, Novakova I, Eskes TK, Monnens LA, Blom HJ. Hyperhomocysteinemia and other thrombotic risk factors in women with placental vasculopathy. Br J Obstet Gynaecol 2000;107:785–91.

17. Stern LL, Bagley PJ, Rosenberg IH, Selhub J. Conversion of 5-formyltetrahydrofolic acid to 5-methyltetrahydrofolic acid is unimpaired in folate-adequate persons homozygous for the C677T mutation in the methylenetetrahydrofolate reductase gene. J Nutr 2000;130:2238–42.[Abstract/Free Full Text]

18. Guenther BD, Sheppard CA, Tran P, Rozen R, Matthews RG, Ludwig ML. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat Struct Biol 1999;6:359–65.[Medline]

19. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3.[Medline]

20. Powers R, Minich L, Lykins D, Ness R, Crombleholme W, Roberts J. Methylenetetrahydrofolate reductase polymorphism, folate, and susceptibility to preeclampsia. J Soc Gynecol Investig 1999;6:74–9.[Medline]

21. Kraus JP, Janosik M, Kozich V, Mandell R, Shih V, Sperandeo MP, et al. Cystathionine beta-synthase mutations in homocystinuria. Hum Mutat 1999;13:362–75.[Medline]

22. Gallagher PM, Ward P, Tan S, Naughten E, Kraus JP, Sellar GC, et al. High frequency (71%) of cystathionine beta-synthase mutation G307S in Irish homocystinuria patients. Hum Mutat 1995;6:177–80.[Medline]

23. Christensen B, Frosst P, LussierCacan S, Selhub J, Goyette P, Rosenblatt DS, et al. Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. Arterioscler Thromb Vasc Biol 1997;17: 569–73.[Abstract/Free Full Text]

24. Kluijtmans LA, Whitehead AS. Methylenetetrahydrofolate reductase genotypes and predisposition to atherothrombotic disease; evidence that all three MTHFRC677T genotypes confer different levels of risk. Eur Heart J 2001;22:294–9.[Abstract/Free Full Text]

25. Raijmakers MT, Zusterzeel PL, Steegers EA, Hectors MP, Demacker PN, Peters WH. Plasma thiol status in preeclampsia. Obstet Gynecol 2000;95:180–4.[Abstract/Free Full Text]

26. De Falco M, Pollio F, Scaramellino AD, Pontillo AD, Lieto AD. Homocysteinaemia during pregnancy and placental disease. Clin Exp Obstet Gynecol 2000;27:188–90.[Medline]

27. Rajkovic A, Catalano PM, Malinow MR. Elevated homocyst(e)ine levels with preeclampsia. Obstet Gynecol 1997; 90:168–71.[Abstract]

28. Dekker GA, deVries JIP, Doelitzsch PM, Huijgens PC, von Blomberg BM, Jakobs C, et al. Underlying disorders associated with severe early-onset preeclampsia. Am J Obstet Gynecol 1995;173:1042–8.[Medline]

29. Litwin M, Abuauba M, Wawer ZT, Grenda R, Kuryt T, Pietraszek E. Folate, vitamin B12, and sulfur amino acid levels in patients with renal failure. Pediatr Nephrol 2001; 16:127–32.[Medline]




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