© 2001 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Infant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart disease
aInstitute of Clinical Chemistry and Laboratory Medicine and Institute of Atherosclerosis Research, University Hospital Münster, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany
bDepartment of Pediatric Cardiology, University Hospital Münster, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany
cChildrens Hospital Lachnerstrasse, Munich, Germany
dDepartment of Pediatrics, University Hospital Münster, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany
eDepartment of Pediatric Hematology/Oncology, University Hospital Münster, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany
* Corresponding author. Tel./fax: +49-251-834-7227 junkerr{at}uni-muenster.de
Received 18 January 2001; accepted 7 March 2001
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Recently, an association between the homozygous C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in infants with congenital neural tube defects or congenital oral clefts has been shown. However, no data are available so far with respect to the MTHFR 677TT genotype in children with underlying structural congenital heart disease (CHD). Methods: We investigated the MTHFR genotype in 114 Caucasian CHD patients aged newborn to 16 years (median 0.6 years; 53% male) and in 228 age- and sex-matched healthy controls. Results: In childhood patients with CHD the homozygous MTHFR 677TT genotype was found in 21 out of 114 subjects (18.4%) compared with 21 out of 228 controls (9.2%; odds ratio (OR) 2.2, 95%-confidence interval (CI) 1.2–4.3; P=0.027). In patients with pulmonary valve stenosis, hypoplastic left heart syndrome, coarctation of the aorta, aortic valve stenosis or subaortic stenosis the frequency of the TT genotype varied between 38 and 67% with corresponding ORs from 6.1 (CI, 1.4–27.5; P=0.034) to 20.4 (CI, 1.8–235.0; P=0.025), whereas in other structural CHD the frequency of this genotype was not significantly different from the controls. Conclusions: With the present study we can show for the first time that the embryonal MTHFR 677TT genotype is significantly associated with the development of structural congenital heart malformations during early pregnancy. It remains to be clarified, whether this genotype is at least a risk marker or a risk factor for structural congenital heart malformations.
KEYWORDS Congenital defects; Developmental biology; Coronary disease; Gene expression
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Congenital heart disease (CHD) refers to structural or functional heart disease and occurs in approximately 1% of liveborn children, but in a much higher percentage of those aborted spontaneously or stillborn. Approximately 5–8% of CHD is due to chromosomal abnormalities, and 3% to classical genetic defects with a high recurrence risk in first-degree family members [1–5].
Subjects carrying the common C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene have a reduced MTHFR enzyme activity of approximately 50% of normal with subsequently increased plasma homocysteine concentrations [6,7]. In 1995 an association between the homozygous C677T mutation in infants and congenital neural tube defects was described for the first time [8,9]. This observation was confirmed by other authors [10–14], and the same TT genotype was later discussed in etiological terms in children with congenital oral clefts [15,16] or neural tube defects [12].
Up to now, no data have been available with respect to the MTHFR 677TT genotype in children with underlying CHD. We therefore performed this study focusing on patients with CHD and the MTHFR genotype.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Patients and controls
Since 1995, all patients younger than 16 years of age with CHD diagnosed in the study center were enrolled in the study. After exclusion of patients with Down syndrome or further recognized chromosomal anomalies, functional changes, and patent foramen ovale, the study population remaining for further data analysis consisted of 114 Caucasian patients aged newborn to 16 years (median 0.6 years; 53% male). Underlying CHD are shown in Table 1. All patients had isolated anomalies. No cross-over lesions were found. In all cases, diagnoses were confirmed by standard imaging procedures (color-coded echocardiography, heart catheterization).
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In both groups, patients and controls, information on maternal use of folate or multivitamins was obtained by means of a questionnaire. There was no case identified with maternal use of multivitamins or folate immediately before and during early pregnancy.
Samples from 228 age- and sex-matched healthy children (potential bone marrow donors or admitted to hospital for elective surgery) from the same geographic area were investigated as controls.
2.2 Laboratory methods
Venous blood in EDTA-treated 1.4-ml sample tubes (Sarstedt, Nümbrecht, Germany), was obtained for routine measurements of hematology. From the remaining blood, cells for genetic analysis were separated by centrifugation at 3000xg for 15 min. The buffy coat layer was then removed and stored at –70°C pending DNA extraction by a spin column procedure (Quiagen, Hilden, Germany). The C677T genotype in the MTHFR gene was determined as described by Frosst et al. [7]. Controls for each genotype and a negative control were included in each assay run. In all cases, genotyping revealed clear results.
2.3 Statistics
On an explorative basis, genotype frequencies in patients and controls were compared by 
2-analysis or Fisher's exact test. The significance level was set at 0.05. In addition, odds ratios (OR) and 95%-confidence intervals (CI) were calculated. All statistical analyses were performed using the MedCalc software package (MedCalc, Mariakerke, Belgium).
2.4 Ethics
The present study was carried out in accordance with the ethical standards laid down in a relevant version of the 1964 Declaration of Helsinki and approved by the medical ethics committee at the Westfälische Wilhelms-University, Münster, Germany. In all cases, patients and controls, written informed parental consent was given.
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3 Results |
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In childhood patients with structural CHD the genotype frequencies of the MTHFR C677T polymorphism were as follows: TT, n=21 (18.4%); CT, n=42 (36.8%); CC, n=51 (44.7%). In the control group, the homozygous MTHFR 677TT genotype was present in 21 (9.2%), the CT genotype in 78 (34.2%) and the CC genotype in 129 (56.6%) individuals. The resulting OR for patients carrying the homozygous TT genotype compared to the controls was 2.2 (CI, 1.2–4.3; P=0.027). The results of subgroup analyses are shown in the Table 1.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Fasting levels of plasma homocysteine were shown to be significantly higher in mothers of children with CHDas compared with healthy controls [17].
Periconceptional supplementation with folic acid is known to reduce the risk of occurrence and recurrence of neural tube defects [18,19], and may also reduce congenital urinary tract anomalies [20] and cardiac defects [21,22]. This preventive effect of folic acid has led investigators to search for defects or variants in candidate genes involved in the metabolic pathways of folic acid which might be overcome by vitamin supplementation. The enzyme MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which is a critical precursor in the cascade of reactions leading to methylation. A thermolabile variant of the MTHFR due to the common C677T mutation has recently been identified [7]. The resultant mild hyperhomocysteinemia observed in homozygotes and heterozygotes for the MTHFR 677T allele is accentuated when affected patients have reduced plasma folate levels, possibly due to environmental or nutritional interactions [23,24].
The present study in children with structural CHD was conducted on the basis of the hypothesis that the 677TT genotype in the MTHFR gene has an impact on the embryonal development of commonly observed congenital malformations such as neural tube defects or oral clefts. Consistent with this hypothesis, the frequency of the TT variant was significantly higher in children with CHD (18.4%) than in healthy controls (9.2%). The frequency of the homozygous TT genotype in healthy controls was no different from data recently published and represents the prevalence in the German population [25,26]. Subgroup analysis of our data revealed the highest prevalence of the TT genotype in patients with pulmonary valve stenosis, hypoplastic left heart syndrome, coarctation of the aorta, and aortic valve stenosis/subaortic stenosis (38–67%). It has been shown by other authors that periconceptional multivitamin use could reduce especially the risk of conotruncal heart defects such as transposition of the great arteries or tetralogy of Fallot [21,22]. However, the absolute number of patients investigated in subgroups of specific cardiac lesions was rather small, so that a definitive statement concerning the benefit of multivitamin use for a specific genotype and particular CHD cannot be given at the moment.
In conclusion, data presented here suggest that on an explorative basis the MTHFR 677TT genotype in the embryo is significantly associated with the development of structural congenital heart malformations during early pregnancy. It remains to be clarified, whether this genotype is at least a risk marker or a risk factor for structural congenital heart malformations. Moreover, in order to clarify whether genotype-specific periconceptional use of folate supplementation reduces the rate of specific structural CHD, further studies are required.
Time for primary review 28 days.
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Acknowledgements |
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Professor Dr Nowak-Göttl was supported by a grant from the Medical Faculty of the Westphalian Wilhelms-University. This study was supported by the Landesversicherungsanstalt Rheinprovinz and the Landesversicherungsanstalt Westfalen. The authors thank the technicians from the participating laboratories, in particular Doris Böckelmann and Anke Reinkemeier for excellent technical assistance. We also gratefully acknowledge Susan Griesbach's help in editing the manuscript.
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