La prise d’un supplément d’acide folique en période préconceptionnelle réduit le risque d’une anomalie du tube neural (ATN), une malformation du système nerveux. Dans le but d’en réduire la prévalence, la Société des Obstétriciens et Gynécologues du Canada a émis de nouvelles directives cliniques en 2007 qui tenaient compte de différents facteurs de risque pour les ATN et pour qui la dose recommandée variait selon le profil de risque de la femme, allant de 0,4 à 5,0 mg d’acide folique. Jusqu’à présent, peu de données sont disponibles sur les effets de la prise d’une haute dose d’acide folique. Les objectifs de cette étude étaient: 1) d’évaluer la concordance entre la supplémentation en acide folique chez les femmes enceintes et les nouvelles recommandations canadiennes; 2) d’identifier les déterminants d’une utilisation concordante et 3) d’évaluer si la prise de hautes doses d’acide folique en période périconceptionnelle réduisait le risque de malformations congénitales autre que les ATN. Pour répondre à ces objectifs, une étude transversale et une étude écologique ont été effectuées. La première incluait 361 femmes enceintes recrutées aux cliniques d’obstétriques du CHU Sainte-Justine et la deuxième utilisait le Registre Québécois des Grossesses, issu du jumelage de trois banques de données administratives au Québec (RAMQ, Med-Écho et ISQ), où 152 392 couples mère-enfant ont été identifiés. Seul 27% des femmes enceintes ayant participé à l’étude transversale avaient une supplémentation en acide folique, avec ou sans ordonnance, concordante aux lignes directrices canadiennes. La concordance variait selon leur profil de facteurs de risque pour les ATN. Notre étude écologique montre que la prévalence annuelle de l’utilisation de haute dose d’acide folique (avec ordonnance) en période périconceptionnelle a augmenté de 0,17% à 0,80% (p < 0,0001) entre 1998 et 2008 et que la prévalence des malformations congénitales majeures a augmenté de 15% au cours de la même période (3,35% à 3,87%, p<0,0001). Les résultats de nos deux études montrent que l’acide folique n’est pas largement utilisé par les femmes en âge de procréer et ce, peu importe la dose. De nouvelles campagnes de santé publique devront être mises sur pied, afin d’inciter les femmes à consommer de l’acide folique avant et pendant leur grossesse. Également, la prise de haute dose d’acide folique ne semble pas avoir diminué le risque de malformations congénitales, à l’échelle populationnelle. / The use of folic acid during the preconceptionnal period reduces the risk of neural tube defects (NTD), a malformation of the nervous system. In order to reduce it’s prevalence, the Society of Obstetricians and Gynaecologists of Canada proposed new practice clinical guidelines, in 2007, on the use of pre-conceptional vitamin/folic acid supplementation for the prevention of NTDs, with specific recommendations to prevent recurrences and occurrences among women with intermediate to high health risk factors and for whom the dose was different. The objectives of this study were to evaluate the concordance between the new guidelines and folic acid use in real life; 2) to identify predictors associated with a recommended folic acid supplementation, and 3) to evaluate if the use of folic acid could reduce the risk of congenital malformations other than NTDs. A cross-sectional study and an ecological study have been conducted. 361 women were recruited in obstetrics outpatient clinic at the CHU Ste-Justine for the first study and 152,392 pregnancies and babies were identified in the Quebec Pregnancy Registry, which results from the linkage of three administrative health care databases from Quebec (RAMQ, Med-Echo and ISQ) for the second study. Only 27% of the wowen recruited for the first study had periconceptional folic acid supplementation intake that was concordant with guideline. Concordance varied according to their health risk factors profile for NTD. Our ecological study showed that the annual prevalence of periconceptional folic acid use increased from 0.17% to 0.80% (p < 0,0001) from 1998 to 2008 and birth prevalence of major congenital malformations increased by 15% (3.35% to 3.87%, p < 0,0001) during the same period. Our findings highlight the fact that folic acid is not widely used by women of childbearing age, regardless of the dose. There is a need for new public health programs to encourange women to consume folic acid every day before and during pregnancy. Moreover, the use of high dose folic acid does not seem to be correlated with a decline in the prevalence of major congenital malformations, on a populational level.

Les anomalies du tube neural (ATN) sont des malformations congénitales très fréquentes chez l’humain en touchant 1-2 nouveau-nés sur 1000 naissances. Elles résultent d’une fermeture incomplète du tube neural lors de l’embryogenèse. L’étiologie des ATN est complexe impliquant des facteurs environnementaux et des facteurs génétiques. La souris représente un outil puissant afin de mieux comprendre la génétique des ATN. Particulièrement, la souris modèle a impliqué fortement la voie de la polarité cellulaire planaire (PCP) dans ces malformations. Dans cette étude, nous avons identifié et caractérisé une nouvelle souris mutante, Skam26Jus dans le but d’identifier un nouveau gène causant les ATN. Skam26Jus a été générée par l’agent mutagène N-Ethyl-N-Nitrosuera. Cette souris est caractérisée par une queue en forme de boucle ou de crochet, soit un phénotype associé aux ATN. La complémentation génétique de la souris Skam26Jus avec une souris mutante d’un gène de la voie PCP Vangl2 (Looptail) a montré une interaction génétique entre le gène muté chez Skam26Jus et Vangl2, suggérant que ces deux gènes fonctionnent dans des voies de signalisation semblables ou parallèles. Un total de 50% des embryons doubles hétérozygotes avec un phénotype de la queue présentent un spina bifida. La cartographie par homozygotie du génome entier suivie par un clonage positionnel a permis d’identifier Lrp6 comme le gène muté chez Skam26Jus. Une mutation homozygote, p.Ile681Arg, a été identifiée dans Lrp6 chez les souris ayant une queue en boucle/crochet. Cette mutation était absente dans 30 souches génétiques pures indiquant que cette mutation est spécifique au phénotype observé. Une étude de phénotype-génotype évalue la pénétrance à 53 % de la mutation Ile681Arg. Lrp6 est connu pour activer la voie canonique Wnt/β-caténine et inhiber la voie non canonique Wnt/PCP. Le séquençage de la région codante et de la jonction exon-intron de LRP6 chez 268 patients a mené à l’identification de quatre nouvelles rares mutations faux sens absentes chez 272 contrôles et de toutes les bases de données publiques. Ces mutations sont p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile; p.Tyr1541Cys et leur pathogénicité prédite in silico indiquent que p.Val1386Ile est bénigne, et que p.Tyr306Hiset p.Tyr373Cys et p.Tyr1541Cys sont i possiblement dommageables. Les mutations p.Tyr306His, p.Tyr373Cys et p.Tyr1541Cys ont affecté l’habilité de LRP6 d’activer la voie Wnt/β-caténine en utilisant le système rapporteur luciférase de pTOPflash. Nos résultats suggèrent que LRP6 joue un rôle dans le développement des ATN chez une petite fraction de patients ayant une ATN. Cette étude présente aussi Skam26Jus comme un nouveau modèle pour étudier les ATN chez l’humain et fournit un outil important pour comprendre les mécanismes moléculaires à l’origine des A TN. / Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating both environmental and genetic factors. The mouse model represents a powerful tool to investigate the genetics of NTDs. Particularly, mouse mutants at genes belonging to the planar polarity pathway (PCP) developed severe forms of NTDs strongly implicating this pathway in the pathogenesis of NTDs. In this study, we identified and characterized a novel mouse mutant, Skam26Jus, as a model for NTDs. Skam26Jus was generated by N-Ethyl-N-Nitrosuera mutagenesis and displayed a characteristic kinky or loop tail that is considered as the minimal sign if NTDs. Complementation of Skam26Jus mutant with a PCP mouse mutant called Looptail (Lp) showed a genetic interaction between Skam26Jus and Vangl2, the gene mutated in Lp. This led to spina bifida in 50% of double heterozygotes with a kinky or looptail phenotype. Homozygosity mapping followed by a positional candidate gene approach led to the identification of Lrp6 as the gene mutated in Skam26Jus. We detected a homozygous mutation, p.Ile681Arg, in Lrp6 in Skam26Jus mice having loop/kinky tail phenotype. This mutation was absent in 30 inbred strains analyzed indicating that it is disease specific. Genotype-phenotype studies indicated a 52 % penetrance of the p.Ile681Arg mutation. Lrp6 is known to activate Wnt canonical β-catenin pathway and inhibit Wnt non canonical PCP pathway. Sequencing analysis of the open reading frame and exon-intron junctions of human LRP6 in 268 NTD patients led to the identification of 4 novel rare missense mutations that were absent in 272 controls analyzed and in all public databases. These mutations were p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile ; p.Tyr1541Cys, and of these, p.Val1386Iso was predicted to be benign, and p.Tyr306His ; p.Tyr373Cys and p.Tyr1541Cys were predicted to be possibly pathogenic using bioinformatics tools. Functional validation of these mutations with the luciferase reporter system pTOPflash assay demonstrated that mutation p.Tyr306His, p.Tyr373Cys and iii p.Tyr1541Cys reduced the ability of LRP6 to activate the Wnt canonical β-catenin pathway. Our data suggest that LRP6 could play a role in the development of NTDs in a small fraction of NTD patients. Our study also presents Skam26Jus as a new mouse model for the study of human NTDs and provides an important tool for better understanding of the molecular pathogenic mechanisms underlying NTDs.

Les anomalies du tube neural (ATN) sont des malformations congénitales parmi les plus fréquentes chez l’humain en touchant 1-2 nouveau-nés par 1000 naissances. Elles résultent d’un défaut de fermeture du tube neural pendant l’embryogenèse. Les formes les plus courantes d'ATN chez l'homme sont l'anencéphalie et le spina-bifida. Leur étiologie est complexe impliquant à la fois des facteurs environnementaux et des facteurs génétiques. Un dérèglement dans la signalisation Wnt, incluant la signalisation canonique Wnt/β-caténine et non-canonique de la polarité planaire cellulaire (PCP), peut causer respectivement le cancer ou les anomalies du tube neural (ATN). Les deux voies semblent s’antagoniser mutuellement. Dans cette étude, nous investiguons les rôles de Lrp6 et deANKRD6, entant qu’interrupteurs moléculaires entre les deux voies de signalisation Wnt, et CELSR1, en tant que membre de la PCP, chez la souris mutante Skax26m1Jus, générée par l’agent mutagène N-Ethyl-N-Nitrosuera, et dans une cohorte de patients humains ATN. Pour Lrp6, nous avons démontré que Skax26m1Jus représente un allèle hypermorphe de Lrp6 avec une augmentation de l’activité de la signalisation Wnt/canonique et une diminution de l’activité JNK induite par la voie PCP. Nous avons également montré que Lrp6Skax26m1Jus interagit génétiquement avec un mutant PCP (Vangl2Lp) où les doubles hétérozygotes ont montré une fréquence élevée d’ATN et des défauts dans la polarité des cellules ciliées de la cochlée. Particulièrement, notre étude démontre l'association des nouvelles et rares mutations faux-sens dans LRP6 avec les ATN humaines. Nous montrons que trois mutations de LRP6 causent une activité canonique réduite et non-canonique élevée. Pour ANKRD6, nous avons identifié quatre nouvelles et rares mutations faux-sens chez 0,8% des patients ATN et deux chez 1,3% des contrôles. Notamment, seulement deux, des six mutations validées (p.Pro548Leu et p.Arg632His) ont démontré un effet significatif sur l’activité de ANKRD6 selon un mode hypomorphique. Pour CELSR1, nous avons identifié une mutation non-sens dans l'exon 1 qui supprime la majeure partie de la protéine et une délétionde 12 pb. Cette perte de nucléotides ne change pas le cadre de lecture et élimine un motif putatif de phosphorylation par la PKC " SSR ". Nous avons également détecté un total de 13 nouveaux et rares variants faux-sens qui avaient été prédits comme étant pathogènes in silico. Nos données confirment le rôle inhibiteur de Lrp6 dans la signalisation PCP pendant la neurulation et indiquent aussi que les mutations faux-sens identifiées chez LRP6 et ANKRD6 pourraient affecter un équilibre réciproque et un antagonisme très sensible à un dosage précis entre les deux voies Wnt. Ces variants peuvent aussi agir comme facteurs prédisposants aux ATN. En outre, nos résultats impliquent aussi CELSR1 comme un facteur de risque pour les anomalies du tube neural ou l’agénésie caudale. Nos résultats fournissent des preuves supplémentaires que la voie de signalisation PCP a un rôle pathogène dans ces malformations congénitales et un outil important pour mieux comprendre leurs mécanismes moléculaires. / Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating environmental and genetic factors. Wnt signaling has been classified as canonical Wnt/ β-catenin dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs) respectively. Both pathwaysseem to antagonize each other. In this study, we investigate the role of Lrp6andANKRD6 as molecular switches between both Wnt pathways as well as CELSR1 as PCP member, in a novel ENU mouse mutant of Lrp6 (Skax26m1Jus) and in human NTDs. For Lrp6, we demonstrate that Skax26m1Jus represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6Skax26m1Jusgenetically interacts with a PCP mutant (Vangl2Lp) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells’ polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. For ANKRD6: We identified four rare missense mutations in 0.8% of the NTD patients and 2 rare missense mutations in 1.3% of the controls. Notably, when all 6 mutations were validated, only two mutations identified in NTD patients, p.Pro548Leu, p.Arg632His, significantly altered DIVERSIN activity in Wnt signaling assays in a hypomorphic fashion. For CELSR1: We identified one nonsense mutation in exon 1 of CELSR1 that truncates the majority of the protein in one NTD patient and one in-frame 12 bp deletion that removes a putative PKC phosphorylation“SSR” motif in one caudal agenesis patient. We also detected a total of 13 novel missense variants in 12 patients (11 NTDs and 1 caudal agenesis) that were predicted to be pathogenic in silico. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate that rare missense mutations in LRP6 and ANKRD6 could affect a balanced reciprocal and a highly dosage sensitive antagonism between both Wnt pathways in neurulation and act as predisposing factors to NTDs in a subset of patients. Also, our findings implicate CELSR1 as a risk factor for NTDs or caudal agenesis. Our findings provide additional evidence for a pathogenic role of PCP signaling in thesemalformations and an important tool for better understanding their molecular mechanisms.

Introdução: Malformações congênitas afetam 2-3% recém-nascidos e estima-se que metade desses problemas poderiam ser prevenidos. Considerando que o ácido fólico reduz o risco de DTN, que a fortificação compulsória das farinhas, de trigo e milho com ferro ácido fólico ocorre desde julho de 2004 e ainda que o Brasil é um país de grande heterogeneidade. Justifica-se, conhecer a evolução dos DTN e sua distribuição espaço-temporal, com vistas a contribuir para o aperfeiçoamento das políticas públicas que visem a prevenção e a minimização desse problema na população brasileira. Objetivo: Analisar a intervenção da fortificação de farinhas com ácido fólico na prevalência de defeitos do fechamento do tubo neural no Brasil. Método: Estudo ecológico, transversal, de desenho misto. Dados foram obtidos do Sinasc. População de estudo foi composta pelos 12.992 casos de DTN (anencefalia, encefalocele e espinha bífida) entre 32.996.065 nascidos no período de 2000-2010. A prevalência de DTN foi calculada para cada 10.000 nascidos vivos. Foi realizada uma analise descritiva exploratória e posterior analise de regressão segmentada e análise espacial com utilização do Índide Global de Moran e indicador Local de Associação espacial. Foram utilizados os softwares Epi info versão 3.4; SPSS versão 17 Terraview versão 3.2.1 e o programa R (The R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org).. Todos os aspectos éticos foram respeitados quanto ao uso das informações obtidas no banco de dados disponíveis no Datasus. Resultados: A prevalência global dos DTN foi de 3,94 casos para cada 10000nv. Ao longo dos anos, entre os anos de 2000 a 2010, verifica-se que o número de casos apresentou uma taxa de variação positiva de 17,3%. Crescimento representado em 2000, por 3,26 casos/10000nv que passou em 2010 para 4,28 casos/10000nv. Em 2005, verificou-se um declínio na prevalência, momento em que a fortificação estava de fato sendo implantada. Constata-se na análise de regressão segmentada que a tendência em praticamente todos os estados tem três segmentos: uma tendência de aumento, seguido de queda no momento da intervenção e posterior tendência de aumento. Foram mapeadas as prevalências da doença em dois triênios, o primeiro antes da fortificação, entre 2001-2003 e o segundo após entre 2008-2010. A prevalência de DTN aumentou no segundo triênio em 19 estados brasileiros. Observou-se áreas de conglomerados embora nem sempre exista a autocorrelação espacial. Conclusão: Conclui-se que embora a prevalência dos DTN tenha declinado em meados de 2005, ela volta a crescer após esse período de modo significativo em alguns estados. Deve-se buscar monitorar o teor do ácido fólico nas farinhas através da implantação de uma metodologia analítica para monitoramento dos alimentos fortificados. A distribuição espaço temporal dos agravos abordados é importante, pois permite a compreensão desses eventos complexos e dinâmicos. Estudos nessa área contribuem na elaboração de políticas públicas para reduzir a prevalência dessas doenças. / Introduction: Congenital malformations affect 2-3% of newborns and it is estimated that half of these problems could be prevented. Considering that folic acid reduces the risk of NTDs, the mandatory fortification of flour, wheat and corn with iron folic acid occurs since July 2004 in Brazil and this is a country of great diversity. These aspects justify studying the evolution of NTD and their distribution in time and space, to contribute to the improvement of public policies for the prevention and reduction of these diseases in Brazilian population. Objective: To analyze the intervention of the fortification of flour with folic acid in the prevalence of neural tube defects in Brazil. Methods: Ecological study, cross, mixed design. Data were obtained from SINASC. Study population consisted of 12 992 cases of NTDs (anencephaly, encephalocele and spina bifida) between 32,996,065 born in 2000-2010 period. The prevalence of NTDs was calculated for every 10,000 live births. An exploratory descriptive analysis and later segmented regression analysis and spatial analysis using the Global index Moran and Local. Epi Info version 3.4 software were used; SPSS version 17, Terraview version 3.2.1 and the R program (The R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org). All ethical aspects were respected in the use of information obtained the database available in Datasus. Results: The overall prevalence of NTDs was 3.94 cases per 10000nv. Over the years between 2000 and 2010, it was found that the number of cases showed a positive rate of change of 17.3%. Growth represented in 2000 by 3.26 cases / 10000nv that passed in 2010 to 4.28 cases / 10000nv. In 2005, there was a decline in the prevalence, at which time the fortification was actually being implemented. Notes on the segmented regression analysis that the trend in every state has three segments: an increasing trend, followed by a decrease at the time of intervention and subsequent increasing trend. The prevalence of the disease in two periods were mapped, the first before fortification, between 2001-2003 and between 2008-2010 after the second. The prevalence of NTDs increased in the second three years in 19 Brazilian states. It was noted areas where clustering though not always there spatial autocorrelation. Conclusion: Although the prevalence of NTDs has declined in mid-2005, it grows back after this period significantly in some states. It should seek to monitor the content of folic acid in flour through the implementation of an analytical methodology for monitoring of fortified foods. The timeline distribution of diseases covered is important because it gives an understanding of these complex and dynamic events. Studies in this area contribute to the development of public policies to reduce the prevalence of these diseases.

Introdução: Defeitos do tubo neural (DTN) são as malformações mais freqüentes do sistema nervoso. Decorrem de falha no fechamento do tubo neural embrionário entre 21-28 dias após a concepção e representam importante causa de morbimortalidade infantil passível de prevenção. Os defeitos mais freqüentes são anencefalia e espinha bífida. Considerando que o ácido fólico reduz o risco de DTN, a fortificação compulsória das farinhas de trigo e milho com ferro e ácido fólico passou a ser obrigatória no Brasil desde junho de 2004. Assim, delineou-se este estudo com vistas a proporcionar uma base de referência sobre a evolução do problema no Estado de São Paulo e contribuir para o aperfeiçoamento das políticas públicas que visam a prevenção e a minimização desse problema de saúde em nível populacional. Objetivo: Comparar a prevalência de DTN no Estado de São Paulo, antes e após a fortificação das farinhas com ácido fólico. Método: Estudo transversal analítico que utilizou dados do Sistema de Informações sobre Nascidos Vivos (Sinasc) nos períodos antes (2001-2003) e após (2006-2008) a fortificação obrigatória das farinhas com ácido fólico. A variável dependente foi a presença de DTN, identificado pelos códigos Q00 (anencefalia), Q01 (encefalocele) e Q05 (espinha bífida, que inclui meningocele e mielomeningocele) da 10ª Classificação Internacional de Doenças (CID-10). Avaliou-se a prevalência de DTN segundo período (antes/após-fortificação), características maternas e do recém nascido. Odds Ratio (OR) e respectivos intervalos de confiança (IC95%) foram utilizados para análise dos dados, conduzida no software R. Utilizou-se o teste de qui-quadrado com nível de confiança de 5%. Resultados: A prevalência total de DTN diminuiu significativamente no período estudado, passando de 0,57 por mil nascidos vivos antes da fortificação para 0,37 por mil nascidos vivos após a fortificação (OR:0,65; IC95%:0,59-0,72). Tanto a espinha bífida (OR:0,52; IC95%:0,45-0,59) quanto a anencefalia (OR:0,79; IC95%:0,67-0,92) foram menos prevalentes no período após a fortificação. Encefalocele foi a menos freqüente e não mostrou diferença na prevalência entre os períodos. Análise estratificada segundo características maternas e infantis mostrou associação estatisticamente significativa de DTN com idade materna no período antes da fortificação e com escolaridade materna, número de consultas de pré-natal e duração da gestação em ambos os períodos. As variáveis do recém-nascido que se associaram estatisticamente com DTN foram sexo no período antes da fortificação e peso ao nascer em ambos os períodos. A análise estratificada da prevalência de DTN mostrou redução significativa após a fortificação para mulheres de todas as faixas etárias (exceto para <15 anos), para aquelas com mais de três anos de estudo, com ou sem companheiro, com sete consultas de pré-natal ou mais e menos de 42 semanas de gestação. Em relação às características do recém-nascido, a análise apontou redução significativa para ambos os sexos, para nascidos com menos de 4000g e todas as raça/cor (exceto preta e outros). Conclusões: O estudo mostrou redução significativa na prevalência total de DTN no Estado de São Paulo após a fortificação das farinhas com ácido fólico e também nas prevalências de anencefalia e espinha bífida. Embora tenha que se considerar que outros fatores possam ter contribuído para esse declínio, os resultados reiteram a importância da fortificação das farinhas como medida de prevenção primária na redução da ocorrência de DTN. / Introduction: Neural tube defects (NTDs) are the most frequent malformations of the nervous system. Result of failure in the embryonic neural tube between 21-28 days after conception and are an important cause of preventable child mortality. The most frequent defects are anencephaly and spina bifida. Considering that folic acid reduces the risk of NTDs, Considering that folic acid reduces the risk of NTD, the compulsory fortification of wheat and corn flour with iron and folic acid became mandatory in Brazil since June 2004. Thus, this study was outlined in order to provide a baseline on the evolution of the problem in the State of Sao Paulo and contribute to the improvement of public policies aimed at prevention and minimization of this health problem at the population level. Objective: To compare the prevalence of NTDs in the State of Sao Paulo, before and after fortification of flour with folic acid. Methods: Analytical transversal study used data from the Information System on Live Births (Sinasc) in the periods before (2001-2003) and after (2006-2008) the mandatory fortification of flour with folic acid. The dependent variable was the presence of NTDs, identified by the codes Q00 (anencephaly), Q01 (encephalocele) and Q05 (spina bifida, meningocele and myelomeningocele including) the 10th International Classification of Diseases (ICD-10). Evaluated the prevalence of NTDs second period (before / after-fortification), and maternal characteristics of the newborn. Odds Ratio (OR) and confidence intervals (95%) were used for data analysis, conducted in the software R. Was used the chi-square test with a confidence level of 5%. Results: The total prevalence of NTDs decreased significantly during the study period, from 0,57 per thousand live births before fortification to 0,37 for a thousand live births after fortification (OR:0,65; IC95%: 0,59-0,72). Both spina bifida (OR:0,52; IC95%: 0,45-0,59) and anencephaly (OR:0,79; IC95%: 0,67-0,92) were less prevalent in the period after fortification. Encephalocele was less frequent and showed no difference in prevalence between periods. Analysis stratified by maternal characteristics and infant showed a statistically significant association of NTDs with maternal age in the period before fortification and maternal education, number of prenatal visits and duration of pregnancy in both periods. The variables of the newborn that is statistically associated with NTDs were sex in the period before fortification and birth weight in both periods. The stratified analysis of the prevalence of NTDs showed a significant decrease after fortification for women of all ages (except for <15 years) for those with more than three years of study, with or without a partner, with seven prenatal consultations or more and less than 42 weeks of gestation. In relation to the characteristics of the newborn, the analysis showed a significant reduction for both sexes, born to less than 4000g and all race/color (except black and others). Conclusions: The study showed a significant reduction in the overall prevalence of NTDs in the State of Sao Paulo after fortification of flour with folic acid and also in the prevalence of anencephaly and spina bifida. Although it is found that other factors may have contributed to this decline, the results reiterate the importance of fortification of flour as a measure of primary prevention in reducing the incidence of NTDs.

Introdução: Malformações congênitas afetam 2-3% recém-nascidos e estima-se que metade desses problemas poderiam ser prevenidos. Considerando que o ácido fólico reduz o risco de DTN, que a fortificação compulsória das farinhas, de trigo e milho com ferro ácido fólico ocorre desde julho de 2004 e ainda que o Brasil é um país de grande heterogeneidade. Justifica-se, conhecer a evolução dos DTN e sua distribuição espaço-temporal, com vistas a contribuir para o aperfeiçoamento das políticas públicas que visem a prevenção e a minimização desse problema na população brasileira. Objetivo: Analisar a intervenção da fortificação de farinhas com ácido fólico na prevalência de defeitos do fechamento do tubo neural no Brasil. Método: Estudo ecológico, transversal, de desenho misto. Dados foram obtidos do Sinasc. População de estudo foi composta pelos 12.992 casos de DTN (anencefalia, encefalocele e espinha bífida) entre 32.996.065 nascidos no período de 2000-2010. A prevalência de DTN foi calculada para cada 10.000 nascidos vivos. Foi realizada uma analise descritiva exploratória e posterior analise de regressão segmentada e análise espacial com utilização do Índide Global de Moran e indicador Local de Associação espacial. Foram utilizados os softwares Epi info versão 3.4; SPSS versão 17 Terraview versão 3.2.1 e o programa R (The R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org).. Todos os aspectos éticos foram respeitados quanto ao uso das informações obtidas no banco de dados disponíveis no Datasus. Resultados: A prevalência global dos DTN foi de 3,94 casos para cada 10000nv. Ao longo dos anos, entre os anos de 2000 a 2010, verifica-se que o número de casos apresentou uma taxa de variação positiva de 17,3%. Crescimento representado em 2000, por 3,26 casos/10000nv que passou em 2010 para 4,28 casos/10000nv. Em 2005, verificou-se um declínio na prevalência, momento em que a fortificação estava de fato sendo implantada. Constata-se na análise de regressão segmentada que a tendência em praticamente todos os estados tem três segmentos: uma tendência de aumento, seguido de queda no momento da intervenção e posterior tendência de aumento. Foram mapeadas as prevalências da doença em dois triênios, o primeiro antes da fortificação, entre 2001-2003 e o segundo após entre 2008-2010. A prevalência de DTN aumentou no segundo triênio em 19 estados brasileiros. Observou-se áreas de conglomerados embora nem sempre exista a autocorrelação espacial. Conclusão: Conclui-se que embora a prevalência dos DTN tenha declinado em meados de 2005, ela volta a crescer após esse período de modo significativo em alguns estados. Deve-se buscar monitorar o teor do ácido fólico nas farinhas através da implantação de uma metodologia analítica para monitoramento dos alimentos fortificados. A distribuição espaço temporal dos agravos abordados é importante, pois permite a compreensão desses eventos complexos e dinâmicos. Estudos nessa área contribuem na elaboração de políticas públicas para reduzir a prevalência dessas doenças. / Introduction: Congenital malformations affect 2-3% of newborns and it is estimated that half of these problems could be prevented. Considering that folic acid reduces the risk of NTDs, the mandatory fortification of flour, wheat and corn with iron folic acid occurs since July 2004 in Brazil and this is a country of great diversity. These aspects justify studying the evolution of NTD and their distribution in time and space, to contribute to the improvement of public policies for the prevention and reduction of these diseases in Brazilian population. Objective: To analyze the intervention of the fortification of flour with folic acid in the prevalence of neural tube defects in Brazil. Methods: Ecological study, cross, mixed design. Data were obtained from SINASC. Study population consisted of 12 992 cases of NTDs (anencephaly, encephalocele and spina bifida) between 32,996,065 born in 2000-2010 period. The prevalence of NTDs was calculated for every 10,000 live births. An exploratory descriptive analysis and later segmented regression analysis and spatial analysis using the Global index Moran and Local. Epi Info version 3.4 software were used; SPSS version 17, Terraview version 3.2.1 and the R program (The R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org). All ethical aspects were respected in the use of information obtained the database available in Datasus. Results: The overall prevalence of NTDs was 3.94 cases per 10000nv. Over the years between 2000 and 2010, it was found that the number of cases showed a positive rate of change of 17.3%. Growth represented in 2000 by 3.26 cases / 10000nv that passed in 2010 to 4.28 cases / 10000nv. In 2005, there was a decline in the prevalence, at which time the fortification was actually being implemented. Notes on the segmented regression analysis that the trend in every state has three segments: an increasing trend, followed by a decrease at the time of intervention and subsequent increasing trend. The prevalence of the disease in two periods were mapped, the first before fortification, between 2001-2003 and between 2008-2010 after the second. The prevalence of NTDs increased in the second three years in 19 Brazilian states. It was noted areas where clustering though not always there spatial autocorrelation. Conclusion: Although the prevalence of NTDs has declined in mid-2005, it grows back after this period significantly in some states. It should seek to monitor the content of folic acid in flour through the implementation of an analytical methodology for monitoring of fortified foods. The timeline distribution of diseases covered is important because it gives an understanding of these complex and dynamic events. Studies in this area contribute to the development of public policies to reduce the prevalence of these diseases.

Les anomalies du tube neural (ATN) sont des malformations congénitales très fréquentes chez l’humain en touchant 1-2 nouveau-nés sur 1000 naissances. Elles résultent d’une fermeture incomplète du tube neural lors de l’embryogenèse. L’étiologie des ATN est complexe impliquant des facteurs environnementaux et des facteurs génétiques. La souris représente un outil puissant afin de mieux comprendre la génétique des ATN. Particulièrement, la souris modèle a impliqué fortement la voie de la polarité cellulaire planaire (PCP) dans ces malformations. Dans cette étude, nous avons identifié et caractérisé une nouvelle souris mutante, Skam26Jus dans le but d’identifier un nouveau gène causant les ATN. Skam26Jus a été générée par l’agent mutagène N-Ethyl-N-Nitrosuera. Cette souris est caractérisée par une queue en forme de boucle ou de crochet, soit un phénotype associé aux ATN. La complémentation génétique de la souris Skam26Jus avec une souris mutante d’un gène de la voie PCP Vangl2 (Looptail) a montré une interaction génétique entre le gène muté chez Skam26Jus et Vangl2, suggérant que ces deux gènes fonctionnent dans des voies de signalisation semblables ou parallèles. Un total de 50% des embryons doubles hétérozygotes avec un phénotype de la queue présentent un spina bifida. La cartographie par homozygotie du génome entier suivie par un clonage positionnel a permis d’identifier Lrp6 comme le gène muté chez Skam26Jus. Une mutation homozygote, p.Ile681Arg, a été identifiée dans Lrp6 chez les souris ayant une queue en boucle/crochet. Cette mutation était absente dans 30 souches génétiques pures indiquant que cette mutation est spécifique au phénotype observé. Une étude de phénotype-génotype évalue la pénétrance à 53 % de la mutation Ile681Arg. Lrp6 est connu pour activer la voie canonique Wnt/β-caténine et inhiber la voie non canonique Wnt/PCP. Le séquençage de la région codante et de la jonction exon-intron de LRP6 chez 268 patients a mené à l’identification de quatre nouvelles rares mutations faux sens absentes chez 272 contrôles et de toutes les bases de données publiques. Ces mutations sont p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile; p.Tyr1541Cys et leur pathogénicité prédite in silico indiquent que p.Val1386Ile est bénigne, et que p.Tyr306Hiset p.Tyr373Cys et p.Tyr1541Cys sont i possiblement dommageables. Les mutations p.Tyr306His, p.Tyr373Cys et p.Tyr1541Cys ont affecté l’habilité de LRP6 d’activer la voie Wnt/β-caténine en utilisant le système rapporteur luciférase de pTOPflash. Nos résultats suggèrent que LRP6 joue un rôle dans le développement des ATN chez une petite fraction de patients ayant une ATN. Cette étude présente aussi Skam26Jus comme un nouveau modèle pour étudier les ATN chez l’humain et fournit un outil important pour comprendre les mécanismes moléculaires à l’origine des A TN. / Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating both environmental and genetic factors. The mouse model represents a powerful tool to investigate the genetics of NTDs. Particularly, mouse mutants at genes belonging to the planar polarity pathway (PCP) developed severe forms of NTDs strongly implicating this pathway in the pathogenesis of NTDs. In this study, we identified and characterized a novel mouse mutant, Skam26Jus, as a model for NTDs. Skam26Jus was generated by N-Ethyl-N-Nitrosuera mutagenesis and displayed a characteristic kinky or loop tail that is considered as the minimal sign if NTDs. Complementation of Skam26Jus mutant with a PCP mouse mutant called Looptail (Lp) showed a genetic interaction between Skam26Jus and Vangl2, the gene mutated in Lp. This led to spina bifida in 50% of double heterozygotes with a kinky or looptail phenotype. Homozygosity mapping followed by a positional candidate gene approach led to the identification of Lrp6 as the gene mutated in Skam26Jus. We detected a homozygous mutation, p.Ile681Arg, in Lrp6 in Skam26Jus mice having loop/kinky tail phenotype. This mutation was absent in 30 inbred strains analyzed indicating that it is disease specific. Genotype-phenotype studies indicated a 52 % penetrance of the p.Ile681Arg mutation. Lrp6 is known to activate Wnt canonical β-catenin pathway and inhibit Wnt non canonical PCP pathway. Sequencing analysis of the open reading frame and exon-intron junctions of human LRP6 in 268 NTD patients led to the identification of 4 novel rare missense mutations that were absent in 272 controls analyzed and in all public databases. These mutations were p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile ; p.Tyr1541Cys, and of these, p.Val1386Iso was predicted to be benign, and p.Tyr306His ; p.Tyr373Cys and p.Tyr1541Cys were predicted to be possibly pathogenic using bioinformatics tools. Functional validation of these mutations with the luciferase reporter system pTOPflash assay demonstrated that mutation p.Tyr306His, p.Tyr373Cys and iii p.Tyr1541Cys reduced the ability of LRP6 to activate the Wnt canonical β-catenin pathway. Our data suggest that LRP6 could play a role in the development of NTDs in a small fraction of NTD patients. Our study also presents Skam26Jus as a new mouse model for the study of human NTDs and provides an important tool for better understanding of the molecular pathogenic mechanisms underlying NTDs.

La prise d’un supplément d’acide folique en période préconceptionnelle réduit le risque d’une anomalie du tube neural (ATN), une malformation du système nerveux. Dans le but d’en réduire la prévalence, la Société des Obstétriciens et Gynécologues du Canada a émis de nouvelles directives cliniques en 2007 qui tenaient compte de différents facteurs de risque pour les ATN et pour qui la dose recommandée variait selon le profil de risque de la femme, allant de 0,4 à 5,0 mg d’acide folique. Jusqu’à présent, peu de données sont disponibles sur les effets de la prise d’une haute dose d’acide folique. Les objectifs de cette étude étaient: 1) d’évaluer la concordance entre la supplémentation en acide folique chez les femmes enceintes et les nouvelles recommandations canadiennes; 2) d’identifier les déterminants d’une utilisation concordante et 3) d’évaluer si la prise de hautes doses d’acide folique en période périconceptionnelle réduisait le risque de malformations congénitales autre que les ATN. Pour répondre à ces objectifs, une étude transversale et une étude écologique ont été effectuées. La première incluait 361 femmes enceintes recrutées aux cliniques d’obstétriques du CHU Sainte-Justine et la deuxième utilisait le Registre Québécois des Grossesses, issu du jumelage de trois banques de données administratives au Québec (RAMQ, Med-Écho et ISQ), où 152 392 couples mère-enfant ont été identifiés. Seul 27% des femmes enceintes ayant participé à l’étude transversale avaient une supplémentation en acide folique, avec ou sans ordonnance, concordante aux lignes directrices canadiennes. La concordance variait selon leur profil de facteurs de risque pour les ATN. Notre étude écologique montre que la prévalence annuelle de l’utilisation de haute dose d’acide folique (avec ordonnance) en période périconceptionnelle a augmenté de 0,17% à 0,80% (p < 0,0001) entre 1998 et 2008 et que la prévalence des malformations congénitales majeures a augmenté de 15% au cours de la même période (3,35% à 3,87%, p<0,0001). Les résultats de nos deux études montrent que l’acide folique n’est pas largement utilisé par les femmes en âge de procréer et ce, peu importe la dose. De nouvelles campagnes de santé publique devront être mises sur pied, afin d’inciter les femmes à consommer de l’acide folique avant et pendant leur grossesse. Également, la prise de haute dose d’acide folique ne semble pas avoir diminué le risque de malformations congénitales, à l’échelle populationnelle. / The use of folic acid during the preconceptionnal period reduces the risk of neural tube defects (NTD), a malformation of the nervous system. In order to reduce it’s prevalence, the Society of Obstetricians and Gynaecologists of Canada proposed new practice clinical guidelines, in 2007, on the use of pre-conceptional vitamin/folic acid supplementation for the prevention of NTDs, with specific recommendations to prevent recurrences and occurrences among women with intermediate to high health risk factors and for whom the dose was different. The objectives of this study were to evaluate the concordance between the new guidelines and folic acid use in real life; 2) to identify predictors associated with a recommended folic acid supplementation, and 3) to evaluate if the use of folic acid could reduce the risk of congenital malformations other than NTDs. A cross-sectional study and an ecological study have been conducted. 361 women were recruited in obstetrics outpatient clinic at the CHU Ste-Justine for the first study and 152,392 pregnancies and babies were identified in the Quebec Pregnancy Registry, which results from the linkage of three administrative health care databases from Quebec (RAMQ, Med-Echo and ISQ) for the second study. Only 27% of the wowen recruited for the first study had periconceptional folic acid supplementation intake that was concordant with guideline. Concordance varied according to their health risk factors profile for NTD. Our ecological study showed that the annual prevalence of periconceptional folic acid use increased from 0.17% to 0.80% (p < 0,0001) from 1998 to 2008 and birth prevalence of major congenital malformations increased by 15% (3.35% to 3.87%, p < 0,0001) during the same period. Our findings highlight the fact that folic acid is not widely used by women of childbearing age, regardless of the dose. There is a need for new public health programs to encourange women to consume folic acid every day before and during pregnancy. Moreover, the use of high dose folic acid does not seem to be correlated with a decline in the prevalence of major congenital malformations, on a populational level.

Os defeitos de fechamento de tubo neural constituem uma das malformações mais freqüentes na espécie humana, apresentando alta morbi-mortalidade. Sua etiologia é considerada multifatorial, estando envolvidos fatores genéticos e ambientais. Estes fatores estão relacionados principalmente com o metabolismo da homocisteína. Realizamos um estudo de caso-controle com o objetivo de estudar os fatores bioquímicos e genéticos relacionados ao DTN na nossa população. Em pares de afetados com DTN e suas mães e pares de pacientes normais e suas mães foram avaliados dosagem de folato, vitamina B12, homocisteína e polimorfismos da enzima metileno tetraidrofolato redutase (MTHFR), C677T e A1298C. A dosagem de folato nos casos foi 11,37 ng/mL(±6,72) e nos controles 5,64 ng/mL(±4,16) (p<0,001). O folato sérico das mães foi 7,27 ng/mL (±4,48) e 3,90 ng/mL (±1,77) nas mães controles (p<0,001). A média de dosagem de vitamina B12 foi de 641,88 pg/mL ((±262,21) nos casos e 743,27 pg/mL (±433,52) nos controles (p= 0,205). A média de dosagem de vitamina B12 nas mães dos casos foi 354,75 pg/mL (±142,06) e 465,25 pg/mL (±194,91) nas mães controles (p=0,004). O nível de homocisteína plasmático médio foi 6,89 μmol/L(±4,48) para os casos e 5,41 μmol/L (±2,55) para os controles (p=0,099). Nas mães dos casos a dosagem média de homocisteína foi 7,23 μmol/L (±2,64) e 7,00 μmol/L (±2,24) nas mães controles (p=0,666). Não houve diferença entre a freqüência dos genótipos C677T e A1298C da MTHFR nos casos e controles e suas mães. Para o polimorfismo C677T as freqüências dos alelo C e T foram respectivamente 0,6585 e 0,3414 nos pacientes com DTN; 0,6590 e 0,3410 nos controles; 0,6460 e 0,3540 nas mães dos casos e 0,6136 e 0,3860 nas mães controles. Para o polimorfismo A1298C as freqüências dos alelos A e C foram respectivamente 0,7436 e 0,2564 nos pacientes com DTN; 0,7610 e 0,2390 nos controles; 0,8055 e 0,1945 nas mães dos casos e 0,8065 e 0,1935 nas mães controles. Identificamos que indivíduos homozigotos 677TT apresentam um maior nível de homocisteína e este é inversamente relacionado com os níveis de vitamina B12. Estes achados sugerem que uma alteração metabólica relacionada ao metabolismo da homocisteína e principalmente devido à diminuição da vitamina B12 seja um fator de risco para DTN na nossa população. / Neural tube defects (NTD) are among the most common birth defect leading to great disabilities. The etiology is multifactorial, involving the combined action of both genetic and environmental factors. Those factors have been related to homocysteine metabolism. We performed a case control study in order to evaluate the biochemical and genetic factors related to NTD in the South of Brazil. A mother- NTD children pair and mother normal children were evaluated for folate, vitamin B12, homocysteine and two polymorphism of the methylene tetrahydrofolate reductase (MTHFR), C677T and A1298C. The folate level was 11,37 ng/mL (±6,72) in the NTD patients and 5,64 ng/mL(±4,16) in the controls (p<0,001). The folate was 7,27 ng/mL (±4,48) in the NTD mothers and 3,90 ng/mL (±1,77) in the control mothers (p<0,001). The level of vitamin B12 was 641,88 pg/mL ((±262,21) in the NTD case and 743,27 pg/mL (±433,52) in the controls (p= 0,205). The levels of vitamin B12 in the NTD mothers was 354,75 pg/mL (±142,06) and 465,25 pg/mL (±194,91) in the control mothers (p=0,004). The pasmatic homocysteine level was 6,89 μmol/L(±4,48) for the NTD cases and 5,41 μmol/L (±2,55) for the controls (p=0,099). The NTD mothers showed homocysteine level of 7,23 μmol/L (±2,64) and the controls mothers demonstrated 7,00 μmol/L (±2,24) (p=0,666). We could not observed a difference between the frequency of the genotypes C677T and A1298C in case and controls. The frequency of the alele C and T for the polymorphism C677T were respectively 0,6585 and 0,3414 for the NTD patients; 0,6590 and 0,3410 for the controls; 0,6460 and 0,3540 for NTD mothers; 0,6136 and 0,3860 for the control mothers. The frequencies of the allele A and C for the polymorphism A1298C were respectively 0,7436 and 0,2564 for NTD patients, 0,7610 and 0,2390 for controls; 0,8055 and 0,1945 for NTD mothers; 0,8065 and 0,1935 for controls mothers. We could demonstrated that the homozygous 677TT had a higher level of homocysteine and this was related to low level of vitamin B12. Those findings suggest that biochemical and genetic factors related to homocysteine metabolism and vitamin B12 deficiency are a risk factor to NTD in our population.

Os defeitos de fechamento de tubo neural constituem uma das malformações mais freqüentes na espécie humana, apresentando alta morbi-mortalidade. Sua etiologia é considerada multifatorial, estando envolvidos fatores genéticos e ambientais. Estes fatores estão relacionados principalmente com o metabolismo da homocisteína. Realizamos um estudo de caso-controle com o objetivo de estudar os fatores bioquímicos e genéticos relacionados ao DTN na nossa população. Em pares de afetados com DTN e suas mães e pares de pacientes normais e suas mães foram avaliados dosagem de folato, vitamina B12, homocisteína e polimorfismos da enzima metileno tetraidrofolato redutase (MTHFR), C677T e A1298C. A dosagem de folato nos casos foi 11,37 ng/mL(±6,72) e nos controles 5,64 ng/mL(±4,16) (p<0,001). O folato sérico das mães foi 7,27 ng/mL (±4,48) e 3,90 ng/mL (±1,77) nas mães controles (p<0,001). A média de dosagem de vitamina B12 foi de 641,88 pg/mL ((±262,21) nos casos e 743,27 pg/mL (±433,52) nos controles (p= 0,205). A média de dosagem de vitamina B12 nas mães dos casos foi 354,75 pg/mL (±142,06) e 465,25 pg/mL (±194,91) nas mães controles (p=0,004). O nível de homocisteína plasmático médio foi 6,89 μmol/L(±4,48) para os casos e 5,41 μmol/L (±2,55) para os controles (p=0,099). Nas mães dos casos a dosagem média de homocisteína foi 7,23 μmol/L (±2,64) e 7,00 μmol/L (±2,24) nas mães controles (p=0,666). Não houve diferença entre a freqüência dos genótipos C677T e A1298C da MTHFR nos casos e controles e suas mães. Para o polimorfismo C677T as freqüências dos alelo C e T foram respectivamente 0,6585 e 0,3414 nos pacientes com DTN; 0,6590 e 0,3410 nos controles; 0,6460 e 0,3540 nas mães dos casos e 0,6136 e 0,3860 nas mães controles. Para o polimorfismo A1298C as freqüências dos alelos A e C foram respectivamente 0,7436 e 0,2564 nos pacientes com DTN; 0,7610 e 0,2390 nos controles; 0,8055 e 0,1945 nas mães dos casos e 0,8065 e 0,1935 nas mães controles. Identificamos que indivíduos homozigotos 677TT apresentam um maior nível de homocisteína e este é inversamente relacionado com os níveis de vitamina B12. Estes achados sugerem que uma alteração metabólica relacionada ao metabolismo da homocisteína e principalmente devido à diminuição da vitamina B12 seja um fator de risco para DTN na nossa população. / Neural tube defects (NTD) are among the most common birth defect leading to great disabilities. The etiology is multifactorial, involving the combined action of both genetic and environmental factors. Those factors have been related to homocysteine metabolism. We performed a case control study in order to evaluate the biochemical and genetic factors related to NTD in the South of Brazil. A mother- NTD children pair and mother normal children were evaluated for folate, vitamin B12, homocysteine and two polymorphism of the methylene tetrahydrofolate reductase (MTHFR), C677T and A1298C. The folate level was 11,37 ng/mL (±6,72) in the NTD patients and 5,64 ng/mL(±4,16) in the controls (p<0,001). The folate was 7,27 ng/mL (±4,48) in the NTD mothers and 3,90 ng/mL (±1,77) in the control mothers (p<0,001). The level of vitamin B12 was 641,88 pg/mL ((±262,21) in the NTD case and 743,27 pg/mL (±433,52) in the controls (p= 0,205). The levels of vitamin B12 in the NTD mothers was 354,75 pg/mL (±142,06) and 465,25 pg/mL (±194,91) in the control mothers (p=0,004). The pasmatic homocysteine level was 6,89 μmol/L(±4,48) for the NTD cases and 5,41 μmol/L (±2,55) for the controls (p=0,099). The NTD mothers showed homocysteine level of 7,23 μmol/L (±2,64) and the controls mothers demonstrated 7,00 μmol/L (±2,24) (p=0,666). We could not observed a difference between the frequency of the genotypes C677T and A1298C in case and controls. The frequency of the alele C and T for the polymorphism C677T were respectively 0,6585 and 0,3414 for the NTD patients; 0,6590 and 0,3410 for the controls; 0,6460 and 0,3540 for NTD mothers; 0,6136 and 0,3860 for the control mothers. The frequencies of the allele A and C for the polymorphism A1298C were respectively 0,7436 and 0,2564 for NTD patients, 0,7610 and 0,2390 for controls; 0,8055 and 0,1945 for NTD mothers; 0,8065 and 0,1935 for controls mothers. We could demonstrated that the homozygous 677TT had a higher level of homocysteine and this was related to low level of vitamin B12. Those findings suggest that biochemical and genetic factors related to homocysteine metabolism and vitamin B12 deficiency are a risk factor to NTD in our population.