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Journal of Human Hypertension (2007) 21, 154–158 & 2007 Nature Publishing Group All rights reserved 0950-9240/07 $30.00 www.nature.com/jhh

ORIGINAL ARTICLE

High frequency of methylenetetrahydrofolate reductase 677TT genotype in Hungarian HELLP syndrome patients determined by quantitative real-time PCR B Nagy, P Hupuczi and Z Papp 1st Department of Obstetrics and Gynaecology, Semmelweis University, Budapest, Hungary

Our aim was to determine the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in hypertensive disorders during pregnancy. We conducted our experiments on isolated DNA samples of 73 healthy pregnant, 101 severe pre-eclamptic and 63 HELLP syndrome women in this study. The MTHFR C677T polymorphism was determined by quantitative real-time PCR method. A significantly higher number of the TT genotype (25.4%) was found in the HELLP syndrome group compared to the healthy (8.2%) and severe pre-

eclamptics group (8.9%) (P ¼ 0.03). The frequency of the mutant T allele was found to be 45.2% of HELLP syndrome, whereas it was 32.2% of the healthy pregnant (P ¼ 0.03) and 30.2% (P ¼ 0.008) of the severe preeclamptic patients. In the HELLP group a high frequency of eclampsia was observed (12.6%) and among them 75% had the MTHFR C677T mutation. Journal of Human Hypertension (2007) 21, 154–158. doi:10.1038/sj.jhh.1002122; published online 30 November 2006

Keywords: pre-eclampsia; HELLP syndrome; methylenetetrahydrofolate reductase; allele frequency

Introduction Pre-eclampsia (PE) is a pregnancy-related disorder characterized by high blood pressure (BP) and proteinuria occurring in the second or third trimester of pregnancy.1–7 It is a major cause of maternal and perinatal mortality. PE can be complicated by haemolysis, elevated liver enzymes and low platelets, called HELLP syndrome, named by Weinstein in 1982.8 Although PE causes complications approximately in about 5–8% of all pregnancies, the pathophysiology remains unclear. The same holds true for HELLP syndrome that occurs in about 24% of all PE pregnancies.7 Family studies have shown that the genetic factor plays a role in the development of PE. Dekker and Sibai9 found that placental ischaemia, oxidative stress and maternal–fetal immune maladaptation are the key elements in pathophysiology. Several other studies have observed endothelial damage and dysfunction to be a common pathological feature

Correspondence: Dr B Nagy, Baross u. 27, Budapest, H-1088 Hungary. E-mail: [email protected] Received 28 July 2006; revised 12 October 2006; accepted 16 October 2006; published online 30 November 2006

in the disease.10,11 Severe pregnancy complications such as severe PE, intrauterine growth retardation, abruption placenta and intrauterine fetal death are associated with thrombophilia. There are three mutations connected to thrombophilia that have been well studied. These three inherited thrombophilias are factor V Leiden mutation, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism. These have been studied in PE with contradictory results. MTHFR plays a role in the metabolism of homocysteine, where it effects the NADPH-linked reduction of 5,10-methylenetetrahydrofolate to 5methyltetrahydrofolate. The C to T missense mutation at nucleotide 677 results in a thermolabile format with reduced activity. TT genotypes are predisposed to increased homocystein levels. Hyperhomocysteinaemia is implicated in premature vascular disease, venous thrombosis and unexplained early pregnancy loss.12 Hyperhomocysteine is an independent risk factor for atherosclerosis, stroke, peripheral vascular disease and cardiovascular disease. Nutrition affects the concentrations too, for example low B-6 and B-12 vitamin intake causes elevation of homocysteine levels. Hyperhomocysteinemia promotes vascular damage that can be found in PE.12 The prevalence of homozygosity

High frequency of MTHFR 677TT genotype in HELLP B Nagy et al 155

for the MTHFR C677T polymorphism is reported to be 9–10% in Caucasian Europeans.13–15 We determined the MTHFR polymorphism in healthy and in well-defined groups of severe preeclamptic and HELLP syndrome patients in a Caucasian, Hungarian population.

was extracted from 0.2 ml samples by using the High Pure PCR Template Isolation kit (Roche, Mannheim, Germany), according to the manufacturer’s instructions.16,17

MTHFR C677T analysis

Materials and methods Patients

All patients were enrolled at the 1st Department of Obstetrics and Gynaecology at the Semmelweis University, Budapest, Hungary, during the period 1 January 2000 and 31 December 2005. We included three groups of patients, a healthy pregnant (n ¼ 73), a severe pre-eclamptic (n ¼ 101) and an HELLP syndrome group (n ¼ 63), and the mean characteristics of the groups are shown in Table 1. Control patients were consecutively selected from a group of normotensive, healthy pregnant women, who were undergoing routine blood tests, and were excluded if they developed hypertensive disorder. The severe pre-eclamptic and HELLP syndrome patients were diagnosed according to the recommendations of the ACOG.7 Briefly, patients in the severe PE group had systolic BP4160 mm Hg and diastolic BP 490 mm Hg with significant proteinuria and intrauterine growth restriction (IUGR). IUGR was diagnosed in neonates when their birth weight was below 10 percentile for gestational age by weight based on Hungarian population tables. In HELLP syndrome cases, the following criteria were defined: haemolysis, classified by increased lactic dehydrogenase activity (4600 IU/l); elevated liver enzyme activity, defined as increased aspartate aminotransferase and alanine aminotransferase levels (470 IU/l); and thrombocytopenia (p100 platelets 109/l). The Ethical Committee of the Semmelweis University has approved the study, all participants were informed and they have agreed to their involvement in the study. DNA isolation

Three millilitres of peripheral blood were drawn from each patient into an EDTA tube. Genomic DNA

The MTHFR C677T was determined according to the Aslanidis and Schmitz18 method using the primer and probe system on the LightCycler instrument (Roche GmbH, Penzberg, Germany). We used the following primers and probes, forward primer CGA AGC AGG GAG CTT TGA GGC TG, reverse primer AGG ACG GTG CGG TGA GAG TG, probes TGA CCT GAA GCA CTT GAA GGA GAA GGT GTCfluorescein and LC-Red640-CGG GAG YCG ATT TCA TCA T-phosphate. This set up produced a 233 bp PCR product. The PCR mix contained 1 ml of the genomic DNA, 5 mM of the primers and probes, 1 ml of LightCycler FastStart DNA Master HybProbe kit (Roche) and 2.5 mM MgCl2. PCR condition and melting curve reading parameters were used according to the published protocol.18 One MTHFR heterozygote, one MTHFR homozygote sample, a distillated water sample and samples in two copies were included in each PCR run. The wild-type sequence showed one peak with a Tm of 611C, whereas the mutant homozygotes had one peak with Tm of 531C, and the heterozygotes had both peaks during the melting curve analysis.

Stastistical analysis of the data

The w2 test was used for the statistical analysis of data with 95% of confidence interval (CI). Po0.05 was considered statistically significant.

Results Seventy-three healthy pregnant, 101 severe preeclamptic and 63 HELLP syndrome patients were involved in the study. There was no statistically significant difference in the average maternal age, the percentage of smokers and primiparas between the hypertensive groups and the control group (Table 1).

Table 1 Clinical characteristic of the patients involved in the study Controls n ¼ 73 Average age (years) Primipara Systolic BP (mm Hg) Diastolic BP (mm Hg) Gestational age at delivery (weeks) Foetal growth restriction Eclampsia Smokers

44 110 70 40 0 0

28.3 (60.2%) (105–120) (60–80) (39–40) (0%) (0%) 0

Pre-eclampsia n ¼ 101

59 170 104 32 22 4 6

29.5 (58.4%) (140–220) (100–115) (25–39) (21.7%) (3.9%) (6%)

HELLP n ¼ 63

38 160 102 31.5 19 8 4

27.5 (60.3) (130–220) (90–140) (23–39) (30.15%) (12.6%) (6.3%)

Abbreviation: BP, blood pressure. Journal of Human Hypertension


Table 2 MTHFR genotype distribution in healthy pregnant, severe pre-eclamptic and HELLP syndrome Hungarian women Patient group Healthy pregnant (n ¼ 73) Severe pre-eclamptic (n ¼ 101) HELLP syndrome (n ¼ 63)

Wild type (CC)

Heterozygote (CT)

Homozygote (TT)

32 (43.8%) 49 (48.5%) 22 (34.9%)

35 (47.9%) 43 (42.5%) 25 (39.6%)

6 (8.2%) 9 (8.9%) 16 (25.4%)*

Table 3 MTHFR C677T allele frequencies in healthy pregnant, severe pre-eclamptic and HELLP syndrome Hungarian women Patient group Healthy pregnant (n ¼ 73) Severe pre-eclamptic (n ¼ 101) HELLP syndrome (n ¼ 63)

C allele

T allele

99 (67.8%) 141 (69.8%) 69 (54.8%)

47 (32.2%) 61 (30.2%) 57 (45.2%)a,b

a

P ¼ 0.03; odds Ratio ¼ 1.74.; 95% Confidence Interval: 1.062–2.851. HELLP syndrome patients compared to healthy pregnant women. P ¼ 0.008; Odds Ratio 1.909; 95% Confidence Interval: 1.203–3.031. HELLP syndrome patients compared to severe pre-eclamptic women.

Fluorescence -d(F3)/dT

Abbreviation: MTHFR, methylenetetrahydrofolate. *P ¼ 0.03; Odds Ratio ¼ 3.058; 95% Confidence Interval: 1.178–7.939.

0.070 0.065 0.060 0.055 0.050 0.045 0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 0.000 46.0 48.0 50.0 52.0

b

Table 2 shows the MTHFR C677T genotype distribution in the study groups. There was a significantly higher number of TT genotype (25.4%) in the HELLP syndrome group compared to the healthy (8.2%) and severe pre-eclamptic (8.9%) groups. This difference was proven significant by using w2 test (P ¼ 0.03). We found almost the same genotype distribution of the wild types, heterozygotes and homozygotes in the healthy pregnant and PE groups. Table 3 shows the distribution of the C and T alleles in the three groups. In the HELLP syndrome group, the frequency of the mutant T allele was 45.2%, whereas it was 32.2% in the healthy pregnant (P ¼ 0.03) and 30.2% (P ¼ 0.008) in the severe pre-eclamptic patients. There was no difference between the healthy and severe pre-eclamptics. The Hardy–Weinberg equilibrium was calculated using the Hardy–Weinberg Equilibrium calculator (www.changbioscience.com/genetics/hardy.html), and the rule seems to be valid for the studied population involved in this study. Figure 1 shows the melting curves of a wild type, a homozygote and a heterozygote sample. The melting point (Tm) of the wild-type sample was 611C, whereas it was 531C in the homozygous sample and both are detectable in the heterozygous samples. Reliable and safe classification of the samples is made possible using the melting curve analysis programme. From the studied clinical parameters, an interesting finding was the high number of mutations in eclamptic cases. We observed eclampsia in eight (12.6%) HELLP cases, and from those three were homo- and three were heterozygotes for MTHFR C677T. Meanwhile, the number of eclamptic cases Journal of Human Hypertension

54.0 56.0

58.0 60.0

62.0

64.0 66.0

68.0

70.0 72.0 73.0

Temperature (°C)

Figure 1 Melting curve analysis of the MTHFR C677T detection. The figure shows the melting curves converted to melting peaks by plotting the negative derivative of the fluorescence against the temperature. The melting point (Tm) is 611C in the case of wildtype sample and 531C in the case of homozygous sample, and both are detectable in the case of heterozygous samples.

was lower in PE, only four cases (3.96%), including two MTHFR heterozygote (50%).

Discussion We determined the MTHFR C677T polymorphism in the DNA samples of genetically homogeneous Hungarian Caucasian women (n ¼ 237) using quantitative real-time PCR method combined with melting curve analysis. From these females, 101 had severe PE and 63 were diagnosed with HELLP syndrome. Controls were matched for ethnicity, residence and age. Our results showed no difference in the distribution of the MTHFR C677T genotypes between the healthy controls and PE women. We had similar findings in our previous study of 120 pre-eclamptic women in 2000.7 In the present study, however, there was a substantial difference between the incidence of MTHFR 677TT genotype among HELLP syndrome patients compared to the other two study groups. A comparison of the clinical parameters and clinical findings led us to an interesting observation that the occurrence of eclampsia in the HELLP group was high (12.65%), and that among them the ratio of MTHFR C677T homo- and heterozygotes (75%) was extremely high. In the pre-eclamptic group, we observed only four cases (3.9%) of eclamptic patients, and among them 50% were heterozygote. Although the number of the


cases is low, it seems that the MTHFR C677T has an effect on the development of eclampsia. Studies with a higher number of cases are needed to obtain statistically useful information for the confirmation of our observation. Measurement of homocysteine levels seems to be necessary too. For some reason we could not find any association with the occurrence of thrombophilia. The Hardy–Weinberg equilibrium was calculated and it is valid in the case of our MTHFR C677T study. The observed allele frequencies are in concordance with previously published data from the neighbouring countries.19 Our results confirm Laivuori et al.20 results, which did not find differences among the 113 PE and 103 control samples in the frequency of TT genotype. It was the same in O’Saughenessy et al.’s21 publication where 283 samples were studied. A Mexican, an Italian and a Korean study concluded with similar findings,21,22,24 even though these findings are in contradiction with Grandone et al.25 and Sohda et al.’s26 results, where significant differences among these two groups were found. It is interesting, however, that even publications from the same country have contradictions in prevailing. Maybe it is caused by population differences inside the country.23,25 It seems to be clear from the previous studies and our studies that the prevalence of MTHFR C677T variant in pre-eclamptic patients does not differ from the healthy pregnant women. In these studies, the patient selection is well defined and the population is homogeneous. We did not find any literature that compares the MTHFR C677T polymorphism of controls, PE and HELLP syndrome patients, where a high number of cases is involved. Tranquilli et al.27 published a higher prevalence of this mutation in 31 patients in a combined group of patients with HELLP syndrome, placental abruption, fetal growth restriction or unexplained stillbirth. Our results call for the attention to the high prevalence of MTHFR TT genotype in HELLP syndrome and the importance of the testing. Another interesting discovery is the high prevalence of mutation among eclamptic patients. There are three important genetic causes of inherited thrombophilias: factor V Leiden, factor IIprothrombin G20210A and MTHFR C677T mutations. High number of Leiden mutation was observed among the PE women in several studies, but there is contradiction in the results of different research groups. In the case of a factor II-prothrombin mutation, most of the studies did not find a difference in the occurrence among PE and healthy subjects. We believe the contradiction in the results in the factor V Leiden and MTHFR mutation in the different studies comes from the ethnical differences among the studied populations. The prevalence of inherited thrombophilic mutations is different in

ethnic groups and there could be unknown interaction between the genetic and acquired factors. Determination of the MTHFR genotype could give helpful information on the treatment of patients diagnosed previously with PE and HELLP syndrome. Appropriate folic-acid supplementation during the upcoming pregnancy in homo- and heterozygotes might be beneficial for the patients.

What is known about the topic: K Previous studies found that the frequency of MTHFR C677T did not differ from healthy controls and in PE. K The MTHFR C677T polymorphism was not studied in large groups of patients having HELLP syndrome. What this study adds: The frequency of MTHFR 677TT genotype is high in HELLP syndrome patients. K The incidence of MTHFR C677T is high among the patients who developed eclampsia during HELLP syndrome and PE. K

Abbreviations: PE, pre-eclampsia; MTHFR, methylenetetrahydrofolate.

Acknowledgements We are particularly grateful to Dr C Aslanidis (University of Regensburg, Germany) who provided the MTHFR C677T homo- and heterozygote control samples.

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