The methylenetetrahydrofolate reductase C677T polymorphism and the risk of congenital heart diseases: a literature review

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1 Giorn. It. Ost. Gin. Vol. XXXVI - n. 3 Maggio-Giugno Department of Obstetrics and Gynecology, Paolo Giaccone University Hospital, Palermo, Italy 2 Department of Obstetrics and Gynecology, Cervello Hospital, Palermo, Italy 3 Department of Cardiology, Paolo Giaccone University Hospital, Palermo, Italy Corresponding Author: Alessandro Svelato, alessandrosvelato@virgilio.it clinical case The methylenetetrahydrofolate reductase C677T polymorphism and the risk of congenital heart diseases: a literature review A. SVELATO 1, W. BERTOLINO 2, G. CALAGNA 1, F. FIORINO 1, A. VASSILIADIS 1, R. VENEZIA 1, E. BERTOLINO 3, A. PERINO 1 summary: The methylenetetrahydrofolate reductase C677T polymorphism and the risk of congenital heart diseases: a literature review. A. svelato, w. BerTolIno, G. CAlAGnA, F. FIorIno, A. vassiliadis, r. venezia, e. BerTolIno, A. PerIno Congenital Heart Diseases (CHDs) are the most common and serious developmental anomaly and the leading non-infectious cause of mortality in the first year of life. Despite the advances in diagnosis and treatment, understanding of the developmental causes and aetiologies of CHDs has been limited. The hyperhomocysteinemia is one of the proved risk factors related to the occurrence of CHDs. The connection between cardiac defects, folate and hyperhomocysteinemia could be explained by a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Indeed, the C677T MTHFR mutation produces a thermolabile variant of MTHFR with reduced enzymatic action resulting in higher plasma levels of homocysteine, especially in individuals with low folate levels. Studies regarding MTHFR C677T polymorphism in relation to CHDs have yielded conflicting conclusions. Our aim is to perform a literature review about the suspected interrelation between MTHFR C677T mutation and the risk of congenital heart diseases. Furthermore, considering that exist populations with a higher prevalence of these type of mutation, we have started a multicentre case-control study in order to further elucidate this topic. riassunto: La mutazione C677T del metilenetetraidrofolasi reduttasi e il rischio di anomalie cardiache congenite: revisione della letteratura. A. svelato, w. BerTolIno, G. CAlAGnA, F. FIorIno, A. vassiliadis, r. venezia, e. BerTolIno, A. PerIno Le anomalie cardiache congenite (Congenital Heart diseases, CHDs) sono le più frequenti e gravi anomalie dello sviluppo e le principali cause di mortalità non infettiva durante il primo anno di vita. Nonostante i progressi nella diagnosi e nel trattamento, la comprensione delle cause e dell eziologia che conduce allo sviluppo delle CHDs è limitata. L iperomocisteinemia è uno dei comprovati fattori di rischio delle CHDs. La connessione tra difetti cardiaci, folati e iperomocisteinemia potrebbe essere spiegata dalla mutazione nel gene che codifica per l enzima metilenetetraidrofolato reduttasi (MTHFR). Infatti, la mutazione C677T per l enzima MTHFR produce una variante termolabile dell enzima MTHFR con ridotta attività enzimatica, con conseguente aumento dei livelli plasmatici di omocisteina, specialmente nei soggetti con bassi livelli di folati. Gli studi riguardo l associazione tra polimorfismo C677T per l enzima MTHFR e le CHDs hanno dato fino ad ora risultati contrastanti. Il nostro scopo è eseguire una revisione della letteratura riguardo alla sospetta correlazione tra mutazione C677T dell enzima MTHFR e il rischio di CHDs. Inoltre, considerando che esistono popolazioni con una prevalenza maggiore di questo tipo di mutazione, abbiamo iniziato uno studio multicentrico caso-controllo, al fine di chiarire questa relazione. Key words: Congenital heart diseases - Methylenetetrahydrofolato reductase - MTHFr - Birth defects - Folic acid - Hyperhomocysteinemia - MTHFr polymorphism - C677T MTHFr mutation. Anomalie cardiache congenite - Metilenetetraidrofolato reduttasi - MTHFr - difetti congeniti - Acido folico - Iperomocisteinemia - Polimorfismo MTHFr - Mutazione C677T per MTHFr. Copyright 2014, CIC Edizioni Internazionali, Roma 398

2 The methylenetetrahydrofolate reductase C677T polymorphism and the risk of congenital heart diseases: a literature review Introduction Congenital heart diseases (CHds) are a cluster of heterogeneous heart disease that includes abnormalities in the structure of the heart and great vessels that results from incomplete development of the heart during the first 6 weeks of pregnancy. CHds are the most common and serious developmental anomalies and the leading non-infectious cause of mortality in the first year of life (1, 2). The incidence of CHds in underdeveloped countries is not known, and in the western industrialized world has varied from a low value of about 3 to 5 per 1000 live births to about 12 per 1000 live births, but for those aborted spontaneously or stillborn the figure is much higher (5-10%) (2, 3). CHds accounts for more than 25% of all infants deaths, 10% of total admissions to neonatal intensive care units, and 26% of total neonatal intensive care mortality (4). CHds at birth occur as an isolated malformation, but are also associated with other anomalies or occurs as part of a syndrome. despite the advances in diagnosis and treatment, understanding of the developmental causes and etiologies of CHds has been limited. In < 20% of the cases, a cause can be found, including 22q11 deletion, trisomy 21, and established environmental risk factors as maternal diabetes, taking certain medications or alcohol or drug abuse during pregnancy and maternal viral infection, such as rubella in the first trimester of pregnancy; however the cause is unknown for the vast majority (2, 5, 6). The etiology of nonsyndromic CHds is considered to be multifactorial, resulting from a complex interaction between genetic, epigenetic, environmental and lifestyle factors (7-9). Formation of the fetal heart involves multiple developmental pathways. Two interdependent candidate metabolic pathways that may contribute to fetal heart defects are folic acid-dependent nucleotide synthesis and methionine/homocysteine transmethylation (8). epidemiologic studies have shown that the protective effect of maternal supplement of folic acid or multivitamins containing folic acid in the periconceptional period could significantly reduce the risk of having a child with neural tube defects (ntd) (10, 11), but also urinary tract defects (12), cleft lip and palate defects and cardiac defects (13-16). Furthermore, an inverse relationship between daily maternal folic acid intake and the offspring s incidence of cardiac outflow tract defects has also been mentioned in the literature (17). Although the underlying mechanisms by which folic acid, in isolation or in concert with other multivitamins, protects against CHds is unclear, the teratogenic process resulting from folate insufficiency may be related to hyperhomocysteinemia, which is one of the proved risk factors related to the occurrence of CHds (8, 18). Previous studies have observed an association between elevated homocysteine levels and pregnancy complications (19, 20), particularly with regard to ntd. owing to the existence of a connection in the morphogenesis of ntd and CHds several studies implicate a relation between maternal hyperhomocysteinemia and CHds, too (21, 22). This conclusion is based on the presence of a specific neural crest population, named cardiac neural crest, which originates in a site conterminous with the site of the neural tube closure and is involved in the development of the outflow tract region of the heart (2, 23, 24). The cardiac neural crest cells are a group of cells that migrate from the dorsal side of the neural tube, through pharyngeal arches, during a specific time window and contribute to the septation of the developing heart, specifically the outflow tract (24). Ablation of the cardiac neural crest in chicken embryos leaded chiefly to conotruncal heart defects (24, 25). In several experimental animal studies, a disturbed folate and homocysteine metabolism have been related to congenital anomalies, including CHds (23, 26). studies of animal models suggest that heart defects associated with low folate/high homocysteine may result from abnormal differentiation, migration, and apoptosis in neural crest cells affecting primarily the interventricular septum and the conotruncal region (23, 27). Tang et al. (23) observed that reduced availability of folate by inactivating the folate transporter, Folbp1, in mice leads to an extensive reduction of migrating cardiac neural crest cells that contribute to the formation of the truncus arteriosus and its division into the aorta and pulmonary artery, thus probably affecting the conotruncal defects in particular (24). Chicken embryos exposed to homocysteine induced several kinds of heart defects (28). In vitro, folic acid and homocysteine affected outgrowth and differentiation of neural crest cells (27, 29). The connection between cardiac defects, folate and hyperhomocysteinemia could be explained by a mutation in the methylenetetrahydrofolate reductase (MTHFr) gene (30). The MTHFr gene is located on chromosome 1p36.3, consists of 11 exons and encodes an enzyme that is essential for folate-mediated onecarbon metabolism (3, 31-35). MTHFr catalyzes the biologically irreversible reduction of dietary folate, 5,10-methylenetetrahydrofolate, into 5-methylenete- 399

3 trahydrofolate, the predominant circulatory form of folate and the methyl donor for the vitamin B12 dependent remethylation of homocyseine to methionine (3, 32, 33, 36). Methionine is ultimately converted to s-adenosylmethionine, which acts as a methyl donor for dna, rna, proteins and phospholipids methylation (34). In addition, methylenetetrahydrofolate participates in dna synthesis by converting uracil to thyamine (34). A common missense mutation, more precisely a cytosine (C) to thymine (T) substitution at base 677 that cause a replacement of valine for alanine at position 222 of the protein, produces a thermolabile variant of MTHFr with reduced enzymatic action resulting in higher plasma levels of homocysteine, especially in individuals with low-folate levels (37, 38). Individuals who are homozygous for the 677T allele have only 30% of the MTHFR enzyme activity measured in individuals who are homozygous for the wildtype C allele, whereas heterozygotes retain 65% of wild-type MTHFR enzyme activity (39). About 10-12% of Caucasians and 1-2% of those of African origin carry this C677T polymorphism (5, 40). Variation in the genes involved in the folate pathway plays an important role in the etiology of birth defects, but studies regarding MTHFr C677T polymorphism in relation to CHds have yielded conflicting conclusions. our aim is to perform a literature review about the suspected interrelation between C677T methylenetetrahydrofolate reductase mutation and the risk of CHds. Furthermore, considering that exist populations with a higher prevalence of these mutations, we have started a multicentre case-control study in order to further elucidate this topic. Data sources and study selection A. Svelato et al. we conducted a research on PubMed and Google scholar to retrieve relevant published reports, from 1999 to 2013, on linking the C677T polymorphism in the MTHFr gene and CHds risk. The searches were carried out using the search terms: MTHFr or methylenetetrahydrofolate reductase, polymorphism and congenital heart diseases or CHd or congenital abnormalities or congenital anomalies or birth defects. reference lists of the retrieved articles were also scanned for more eligible studies. In relation to the fact that this article is not a meta-analysis, but a review of the literature, we decided to include only articles with the greatest number of citations and to exclude all articles not exclusively focused on the review s subject. only studies in the english language were included. Description of published studies In 1999 Kapusta et al. (21) were first to describe a significant association between higher median fasting homocysteine levels in 27 mothers and the occurrence of CHds in their offspring compared to the control subjects. A report by li et al. (41) on the incidence of heart malformations in murine embryos showed that mild MTHFr deficiency, low dietary folate, or both increase the incidence of fetal loss and heart defects; the majority of observed defects were ventricular septal defects. Both Junker et al. (42) and wenstrom et al. (22) have reported, in 2001, that the C677T MTHFr mutation was linked to multifactorial cardiac defects. Junker et al. (42), through the use of a case-control design, observed a higher frequency of the 677TT genotype versus the combined group of CT and CC genotypes among 114 Caucasian patients children with a CHds, compared with 228 healthy controls. They estimated that children with CHds were approximately 2.2 times more likely to have the MTHFr 677TT genotype than children without CHds. In particular, the frequency of the 677TT genotype in patients with pulmonary valve stenosis, hypoplastic left heart syndrome, coarctation of the aorta, aortic valve stenosis or subaortic stenosis was significantly elevated. However, the absolute numbers of cases investigated in the subgroups of CHds were rather small, so that no definitive statement on the benefit of multivitamin use for a specific genotype and particular CHds was given. wenstrom et al. (22) analyzed amniotic fluid from a small number of pregnancies complicated by any type of isolated fetal cardiac defect and investigated the interrelation between homocysteine levels and the MTHFr gene polymorphism. They did not provide the full genotype distribution, but compared the combined group of 677CT and TT genotypes with CC individuals in samples of amniotic fluid of 26 pregnancies complicated by CHds, and with 116 controls. They found the 677CT and TT genotypes in 35% of the samples versus 13% in controls. After these reports, a string of studies has subsequently explored the association between this variant and risk of CHds, however the results were controversial. 400

4 The methylenetetrahydrofolate reductase C677T polymorphism and the risk of congenital heart diseases: a literature review In 2002 Klerk et al. (43) observed that the individuals with MTHFr 677TT genotype had a significantly higher risk of CHds. In 2003 storti et al. (44) genotyped 103 Italian mothers with conotruncal defects offspring, 200 control mothers, 103 affected children and their fathers in the only study to date that has used family control data to assort a possible association between this genetic marker and CHds. They did not observe any association between the fetal MTHFr genotypes and the risk of conotruncal heart defects. The CHds-affected children were included consecutively, but methods of case ascertainment and definition of conotruncal heart defects were not described (5). Beside, data on periconceptional folic acid supplementation or intake were lacking, which might explain the absence of the possible detrimental action of C677T MTHFr polymorphism (5). In the Hordaland homocysteine study (2004), nurk et al. (45) did not find an association between the MTHFr C677T polymorphism and CHds. In that study, the incidence of CHds was very low, probably because the birth registry covered only until the first 8 days of life, and consequently missed many heart defects that were diagnosed after this period. Furthermore, information concerning periconceptional folic acid use was not available (5). Mc Bride et al. (2004) didn t find significant relation between MTHFr C677T polymorphism and CHds (46). The study only included children with left ventricular outflow tract malformations and was performed after folate food fortification. Hobbs et al. (2005) studied mothers with CHds in their offspring and identified homocysteine, s-adenosylhomocysteine and methionine as the most important biomarkers predictive of CHds (47). lee et al. (2005) observed that the frequencies of overall MTHFr C677T genotypes were not significantly different between the CHds patients and non- CHds controls (48). They observed a significantly increased proportion of homozygous TT genotypes in specific subgroups of CHds patients (valvular pulmonary stenosis or pulmonary atresia with an intact ventricular septum). whether the CHds was isolated, or part of a syndrome or genetic defect was unknown (5). shaw et al. (2005) performed a population-based case-control study and did not find an increased risk of conotruncal heart defects for the MTHFr 677CT or TT genotype, even in the absence of maternal use of multivitamin supplements with folic acid (49). Pereira et al. (2005) reported that there were no sufficient evidence for an association between the MTHFr C677T genotypes and CHds (50). very limited data on study design, case ascertainment and CHds specification were given (5). In 2006, Hobbs et al. did not find an effect of the MTHFr C677T polymorphism on the estimated risk of having a CHds affected pregnancy (51). This study was done after the introduction of food fortification with folic acid (5). van Beynum et al. (2006) performed a study in the netherlands and reported that the maternal MTHFr 677CT and TT genotypes in combination with no use of periconceptional folate supplements were associated with a three-fold and six-fold increased risk of CHds in offspring, especially for a conotruncal heart defects (24). The maternal MTHFr 677CT and TT genotypes were no genetic risk factors for CHds in women using folate supplements during the entire advised period (2, 24). zhu et al. (2006) genotyped 56 cases with an atrial septal defect or patent ductus arteriosus and found that the fetal MTHFr TT genotype was significantly associated with increased risk of CHds, especially for these two specific lesions (52). The effect of periconceptional folic acid supplements was not investigated (5). van Beynum et al. (2007) published a meta-analysis and found no substantial evidences of increased CHds risk in individuals with MTHFr 677CT and TT genotypes (5). In another meta-analysis, verkleij-hagoort et al. (2007) found that MTHFr polymorphisms C677T did not show to be significantly associated with CHds, but that maternal hyperhomocysteinemia was associated with a greater than four-fold increase in risk for CHds (53). since the publication of these meta-analysis in 2007, many more new studies have appeared. wintner et al. (2007) found a relationship between C677T polymorphism and aortic arch anomalies in a small sample, but could not confirm these anomalies in an Austrian population (18). Marinho et al. (2009) found that MTHFr C677T polymorphism was just partially associated with a specific phenotype of CHds, such as Tetralogy of Fallot (30). shaw et al. (2009), in a population-basedcontrol study involving conotruncal heart defects and spina bifida patients, did not find an association between MTHFr C677T polymorphism and conotruncal heart defects (54). A more recent human study, by van Beynum (2010), suggests that periconceptional folate supple- 401

5 mentation reduce the incidence of heart defects, in particular conotruncal defects and ventricular septal defect (55). In a meta-analysis, nie et al. (2011) reported that the genotypes of MTHFr C677T genetic polymorphism might be associated with the risk of CHds especially among Caucasians (3). In another meta-analysis, yin et al. (2012) found that the fetal and paternal MTHFr C677T gene may be associated with an increased occurrence of CHds (56). sanchez-urbina et al. (2012) suggested that no significant differences in allele or genotype frequencies of MTHFr C677T genetic polymorphism were found between the CHds patients and non-chds controls (P > 0.05) (57). Balderrábano-saucedo et al. (2013) found an association between the Mexican maternal MTHFr 677TT and the presence of complex CHds in their offspring (34). zidan et al. (2013) found that the MTHFr 677TT genotype and T allele were associated with and 2.0-fold increased risk of CHds in egyptian children, respectively (58). Chen et al. (2013) found a significant association of the maternal C677T polymorphism with CHds susceptibility in the Chinese Han population (2). Instead, Mamasoula et al. (2013) indicated that the MTHFr C677T polymorphism was not associated with the risk of CHds (40). In a meta-analysis, wang et al. (2013) found that both infant and maternal MTHFr C677T polymorphism may contribute to the risk of CHds (33). Conclusions After this brief survey of the main articles on the topic, it is clear that the results are very conflicting and not comparable. one possible explanation for discordant findings on the association between the MTHFr gene polymorphism and CHds could be due to different ways of classifying the particular congenital heart defects. Misclassification of outcomes might even further A. Svelato et al. weaken the association with the genotype. There is a need for use of uniform definitions and classifications of varies types of CHds. Another problem reducing possible comparison of previous publications is the difference in the study designs. Furthermore, some studies do not comment on the inclusion or exclusion criteria exerted, or include cases affected by structural malformations involving another organ system besides the heart, conditions complicating the chance of comparability between the various studies published. The sample sizes need to be large to estimate accurately the causal relationship for finer phenotype categories of CHds. several studies investigated only the genotype of the infants; others are based on genotyping trios of children and parents. A further complicating issue is the geographical and demographical variability. Information regarding dietary folic acid intake and the use of periconceptional folic acid supplement was often unknown. Ideally, the studies should be performed in conditions without food fortification with folic acid, even if owing to evidence of a positive effect of folate intake on the development of ntd in terms of a risk reduction, these types of study seems impossible for ethical reasons. In a study (59), carried out on 7130 newborns of different ethnicities from 16 different areas of the world, the authors found an interesting variability in the geographical and ethnic distribution of MTHFr C677T polymorphism. In particularly the homozygous TT genotype was common in northern China (20%), southern Italy (26%) and Mexico (32%). There was also some evidence for geographical gradients in europe (north to south increase) and China (north to south decrease). Furthermore the sicilian registry of Congenital Malformations (IsMAC) report an increase of CHds in Italy, and in particularly in the south (60). In light of this, the sicily could be defined a privileged observatory where carry out studies on this topic. For this reason we decided to perform a multicenter case-control study, with the aim to elucidate the relationship, if exist, between MTHFr C677T polymorphism and CHds. we are confident to reach promising results. 402

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