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Obstetrics & Gynecology 2000;96:277-280
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

677 C->T Polymorphism of the Methylenetetrahydrofolate Reductase Gene and Preeclampsia

HANNELE LAIVUORI, MD, RISTO KAAJA, MD, OLAVI YLIKORKALA, MD, TIMO HILTUNEN, MD and KIMMO KONTULA, MD

From the Departments of Obstetrics and Gynecology and Medicine, Helsinki University Central Hospital, Helsinki, Finland.

Address reprint requests to: Hannele Laivuori, MD Helsinki University Central Hospital Department of Obstetrics and Gynecology Box 140 00029 HYKS Helsinki Finland E-mail: hannele.laivuori{at}pp.fimnet.fi


   Abstract
Top
Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To evaluate C to T substitution at nucleotide 677 of N5,N10-methylenetetrahydrofolate reductase gene in women with prior preeclamptic or normotensive pregnancies.

Methods: Methylenetetrahydrofolate reductase genotypes were determined in 113 Finnish women with preeclamptic first pregnancies and 103 controls with one or more normotensive pregnancies, using polymerase chain reaction and restriction enzyme analysis. Preeclampsia was defined as severe in 100 women who fulfilled one or more of the subsequent criteria: systolic blood pressure (BP) at least 160 mmHg, diastolic BP at least 110 mmHg, or proteinuria at least 2 g per 24-hour urine collection.

Results: There were no significant differences in prevalences of the methylenetetrahydrofolate reductase genotypes (CC, CT, and TT) between groups (57%, 40%, and 3% in the preeclamptic group and 54%, 39%, and 7%, respectively, in controls). The frequency of the T677 allele was 0.23 in the preeclamptic group and 0.26 in the control group (difference 0.03; 95% confidence interval -0.08, 0.14; P = .51). Our sample had 60% power to detect a difference of the allele frequencies similar to that (0.12) reported previously. The result was similar when analysis was restricted to patients with severe preeclampsia (T677 allele frequency 0.22).

Conclusion: A carrier status for the T677 allele of the methylenetetrahydrofolate reductase gene does not predispose to preeclampsia, at least in the Finnish population.

Prospective studies have shown that a moderately elevated plasma homocyst(e)ine concentration is an independent risk factor for cardiovascular disease.1,2 A common cause of hyperhomocyst(e)inemia is the thermolabile variant of N5,N10-methylenetetrahydrofolate reductase, an enzyme involved in vitamin B12–dependent remethylation of homocyst(e)ine to methionine.3 The thermolability, caused by a C to T substitution at methylenetetrahydrofolate reductase gene nucleotide 677 and corresponding with a substitution of an alanine to a valine at position 222, leads to reduced enzyme activity and elevated plasma homocyst(e)ine concentration, especially in response to folic acid depletion.4 We and others have reported elevated homocyst(e)ine levels in preeclampsia.5–7 The known causes of hyperhomocyst(e)inemia, such as dietary factors (folate, vitamin B12, and vitamin B6 deficiencies), renal insufficiency, and genetic defects in homocyst(e)ine metabolism,8 also might be relevant to preeclampsia, as well as the fact that maternally derived homocyst(e)ine is extracted by the fetus.9

Initial reports on the possible association of the T677 allele to preeclampsia appear contradictory,10–13 which might indicate differences in diagnostic classification or ethnic background of study patients. We studied whether the T677 allele predisposes to preeclampsia, taking advantage of the genetically homogenous Finnish population and setting strict criteria for severe preeclampsia. We reasoned that any association between the methylenetetrahydrofolate reductase polymorphism and risk of preeclampsia would justify clinical studies in which effects of folic acid supplementation on plasma homocyst(e)ine levels and risk of preeclampsia would be prospectively investigated in pregnant women genotyped for that polymorphism.


   Materials and Methods
Top
Abstract
 Materials and Methods
Results
 Discussion
 References
 
We studied 113 women who had had preeclampsia in their first pregnancy (104 with one preeclamptic pregnancy, seven with two, and two with three) and 103 controls who had one or more normotensive pregnancies (Table 1Go). Our sample had 60% power to detect the difference of 0.12 in the allele frequencies reported in an earlier study.10 Subjects and controls were of Finnish origin and lived in Southern Finland. All had been healthy before their first pregnancy, without evidence of renal or autoimmune disease. Using discharge records of the Helsinki University Central Hospital, we sought women who had had severe preeclampsia between January 1988 and April 1998. Blood samples were collected between January 1997 and April 1998 after index pregnancy. During the same period, we also collected blood samples from controls who had given birth in the same hospital after uncomplicated pregnancies.


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  		Table 1. Clinical Characteristics
 
Preeclampsia was defined as blood pressure (BP) level at least 140/90 mmHg confirmed by two measurements at least 6 hours apart and proteinuria at least 0.3 g per 24-hour urine collection (or dipstick positive) after 20 weeks’ gestation. Preeclampsia was defined as severe14 in 100 women who also fulfilled one or more of the following criteria: systolic BP at least 160 mmHg, diastolic BP at least 110 mmHg, or proteinuria at least 2 g during any 24-hour urine collection. Data on a number of laboratory examinations were available for a portion of them: thrombocytopenia (thrombocyte value lower than 140 · 109/L) was present in 37 of 83 (44.6%), hyperuricemia (serum uric acid at least 340 µmol/L) in 34 of 42 (81.0%), and fetal growth restriction (FGR) (birth weight less than -2 standard deviation) in 44 of 100 (44.0%) of pregnancies with severe preeclampsia. At 12 weeks postpartum, all women were normotensive and proteinuria had disappeared. Each subject provided written informed consent, and the study was approved by the local ethical review committee.

Genomic DNA was extracted from peripheral blood leukocytes by standard methods.15 DNA samples were first amplified by polymerase chain reaction (PCR) using the primers 5'-TGAAGGAGAAGGTGTCT-GCGGGA-3' and 5'-AGGACGGTGCGGTGAGAGTG- 3'.4 The PCRs were done in a 25-µL volume, containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 200 µmol/L of deoxynucleotide triphosphate mixture, 1 U of Taq polymerase (Ampli-Taq Gold; Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, NJ), 1 µmol/L of each of the primers, and 100 ng of genomic DNA (20 ng/µL). The PCR conditions were 95C for 9 minutes in the first cycle followed by 95C for 1 minute, 60C for 1 minute, and 72C for 1 minute for 33 cycles, and final extension for 10 minutes at 72C. Eight microliters of the PCR product was digested by using 10 U of the restriction enzyme HinfI (10,000 U/mL; New England BioLabs, Inc., Beverly, MA). The C to T substitution at nucleotide 677 creates an extra HinfI restriction site that cleaves the original 198–base pair PCR fragment into 175– and 23–base pair fragments. Size fractionation of the PCR products was accomplished by electrophoresis (2.5% SeaKem agarose; FMC BioProducts, Rockland, ME).

Continuous variables are presented as mean (95% confidence intervals [CIs]) and median (interquartile range) values. Distribution of the data was skewed, so we used the nonparametric Mann–Whitney test for statistical analysis. The differences in allele and genotype frequencies were tested with {chi}2 test.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
Women with and without preeclampsia were of similar age, and their mean body mass indices (BMIs) did not differ from each other (Table 1Go). The mean highest BP values attained were 171/107 and 119/73 mmHg for preeclamptic women and controls, respectively (P < .001). On average, deliveries were 5 weeks earlier in preeclamptic women than controls, and preeclamptic women had significantly smaller babies than controls.

The methylenetetrahydrofolate reductase genotype and allele frequencies in preeclamptic and control women did not differ significantly from each other (Table 2Go). The frequency of the T677 allele was 0.23 in the preeclamptic group and 0.26 in the control group (difference 0.03, 95% CI -0.08, 0.14; P = .51), and TT homozygosity was found in four preeclamptic women (3%) and seven controls (7%) (Table 2Go). The methylenetetrahydrofolate reductase genotype and allele frequencies were similar when analysis was restricted to patients with severe preeclampsia (T677 allele frequency 0.22; prevalence of genotypes CC 59%, CT 39%, TT 2%).


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  		Table 2. Methylenetetrahydrofolate Reductase Gene Polymorphism (677 C->T) Related Genotypes and Alleles
 
To study a possible relationship to clinical variables, CT and TT genotypes were pooled. Body mass index, systolic and diastolic BP, weeks of gestation at delivery, proteinuria, and infant birth weights did not differ between women with CC or CT/TT genotypes in the preeclampsia (Table 3Go) or the control group (data not shown). Among preeclamptic women severe proteinuria (2 g per 24 hours) occurred in 48 of 64 (75%) of women with CC genotype and 31 of 49 (64%) of women with CT/TT genotypes (P = .17).


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  		Table 3. Clinical Characteristics Related to Methylenetetrahydrofolate Reductase Gene Polymorphism (677 C->T) Genotype in Preeclamptic Women
 

   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study was done in the genetically homogeneous Finnish population,16 which should be ideally suited for genetic association studies. Most women had severe preeclampsia with high proteinuria, and controls were well matched for residence, age, and BMI. Our results showed that frequency of methylenetetrahydrofolate reductase T677 allele and the genotype homozygous for the T677 allele did not increase in women with preeclamptic first pregnancies. The 677 C->T polymorphism was not associated with variation of clinical phenotype as assessed within the group of preeclamptic women alone.

Our results disagree with two earlier studies that reported increased methylenetetrahydrofolate reductase T677 allele frequency in preeclampsia compared with controls.10,11 One possible reason is the different severity of preeclampsia in those studies. In the study of Sohda et al, parity and severity of preeclampsia were not reported,10 and in the study of Grandone et al, 24% of preeclamptic women were multiparous and 53% had no significant proteinuria (at least 0.3 g per 24 hours).11 Our results agree with two recent studies from the United Kingdom12 and United States13 that used clinical criteria (obligate proteinuria and nulliparity) similar to ours, and found that the T677 allele was not a genetic risk factor for preeclampsia.

A possible explanation for those discrepant results is the different ethnic backgrounds of the study subjects. The 677 C->T allele frequencies of the methylenetetrahydrofolate reductase gene seem to vary between populations. Frosst et al found that the frequency of the T677 allele was 0.38 in the French Canadian population,4 whereas Sohda et al reported the following T677 allele frequencies among Japanese: 0.37 in nonpregnant controls, 0.36 in pregnant controls, and 0.48 in preeclamptic women.10 In the European Atherosclerosis Research Study II, 785 individuals from 12 countries across Europe were genotyped for this polymorphism, with an overall frequency of 0.32 for the T677. That frequency was lower in the Baltic countries (0.23) than in other regions of Europe (0.37).17 Compared with those figures, Grandone et al reported a somewhat higher T677 allele frequency (0.43 in pregnant controls and 0.52 in preeclampsia) in an Italian study population.11 Those frequencies of T677 allele in our Finnish healthy population (0.26) were relatively low and comparable with those in the Baltic countries.17 Whether the lack of association between T677 allele and preeclampsia in Finns is caused by low allele frequency predisposing to hyperhomocyst(e)inemia remains to be investigated. The only prospective study in which a raised plasma homocyst(e)ine was not found to be a risk factor for the development of vascular disease was done in Finland.18 Unfortunately, we did not measure plasma homocyst(e)ine in the present study. The available collected information emphasizes the need for prospective studies in which plasma homocyst(e)ine levels are followed during pregnancies of normotensive and preeclamptic women and related to polymorphism of the methylenetetrahydrofolate reductase gene.

Although we found no association between the methylenetetrahydrofolate reductase gene 677 C->T polymorphism and risk of severe preeclampsia, we cannot exclude the possibility that a linkage disequilibrium between the polymorphism and a yet unknown functional alteration of the same gene, predisposing to preeclampsia, might exist in another population. Other genetic variants of the methylenetetrahydrofolate reductase gene or other factors that affect plasma homocyst(e)ine levels could influence the risk or severity of preeclampsia.


   Footnotes
 
This study was supported by grants from the Clinical Research Institute and research funds of the Helsinki University Central Hospital, the Medical Council of the Finnish Academy, the Finnish Cardiovascular Foundation, and the Sigrid Juselius Foundation.

PII S0029-7844(00)00896-6

Received November 22, 1999. Received in revised form February 24, 2000. Accepted March 16, 2000.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Stampfer M, Malinow M, Willett W. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 1992;268:877–81.[Abstract]

2. Arnesen E, Refsum H, Bonaa K, Ueland P, Forde O, Nordrehaug J. Serum total homocysteine and coronary heart disease. Int J Epidemiol 1995;24:704–9.[Abstract/Free Full Text]

3. Kang S, Zhou J, Wong P, Kowalisyn J, Stroksch G. Intermediate homocysteinemia: A thermolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988;43:414–21.[Medline]

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

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

6. Powers R, Evans R, Majors A, Ojimba J, Ness R, Crombleholme W, et al. Plasma homocysteine concentration is increased in preeclampsia and is associated with endothelial activation. Am J Obstet Gynecol 1998;179:1605–11.[Medline]

7. Laivuori H, Kaaja R, Turpeinen U, Viinikka L, Ylikorkala O. Plasma homocysteine levels elevated and inversely related to insulin sensitivity in preeclampsia. Obstet Gynecol 1999;93:489–93.[Abstract/Free Full Text]

8. Welch G, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042–50.[Free Full Text]

9. Malinow M, Rajkovic A, Duell P, Hess D, Upson B. The relationship between maternal and neonatal umbilical cord plasma homocyst(e)ine suggests a potential role for maternal homocyst(e)ine in fetal metabolism. Am J Obstet Gynecol 1998;178:228–33.[Medline]

10. Sohda S, Arinami T, Hamada H, Yamada N, Hamaguchi H, Kubo T. Methylenetetrahydrofolate reductase polymorphism and preeclampsia. J Med Genet 1997;34:525–6.[Abstract]

11. Grandone E, Margaglione M, Colaizzo D, Cappucci G, Paladini D, Martinelli P, et al. Factor V Leiden, C>T MTHFR polymorphism and genetic susceptibility to preeclampsia. Thromb Haemost 1997; 77:1052–4.[Medline]

12. O’Shaughnessy K, Fu B, Ferraro F, Lewis I, Downing S, Morris N. Factor V Leiden and thermolabile methylenetetrahydrofolate reductase gene variants in an East Anglian preeclampsia cohort. Hypertension 1999;33:1338–41.[Abstract/Free Full Text]

13. 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]

14. Gifford R, August P, Chesley L, Cunningham G, Ferris T, Lindheimer M, et al. National High Blood Pressure Education Program Working Group report on high blood pressure in pregnancy. Am J Obstet Gynecol 1990;163:1691–712.[Medline]

15. Vandenplas S, Wiid I, Globler-Rabie A, Brebner K, Ricketts M, Wallis G, et al. Blot hybridisation of genomic DNA. J Med Genet 1984;21:164–72.[Abstract]

16. Norio R, Nevanlinna H, Perheentupa J. Hereditary diseases in Finland; rare flora in rare soil. Ann Clin Res 1973;5:109–41.[Medline]

17. Gudnason V, Stansbie D, Scott J, Bowron A, Nicaud V, Humphries S. C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): Its frequency and impact on plasma homocysteine concentration in different European populations. Atherosclerosis 1998;136:347–54.[Medline]

18. Alfthan G, Pekkanen J, Jauhiainen M, Pitkäniemi J, Karvonen M, Tuomilehto J, et al. Relation of serum homocysteine and lipoprotein (a) concentrations to atherosclerotic disease in a prospective Finnish population based study. Atherosclerosis 1994;106:9–19.[Medline]

19. Pihkala J, Hakala T, Voutilainen P, Raivio K. Uudet suomalaiset sikiön kasvukäyrät [in Finnish]. Duodecim 1989;105:1540–6.[Medline]




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