Next-generation sequencing methods now allow rapid and cost-effective sequencing of DNA on a scale not previously possible. This offers great opportunities for the research of Mendelian disorders, but also significant challenges. The sequencing of exomes, or whole genomes, has emerged as a powerful clinical research tool, with targeted gene analyses generally being preferred in the clinical diagnostic setting. These methods have been employed here with the aim of identifying novel genetic causes of inherited heart disorders and to gain insights into the utility and limitations of these techniques for clinical diagnosis in these disorders. Data produced from the introduction of a targeted multi-gene next-generation sequencing test into clinical practice has been studied. Variation within the mitochondrial genome has been analysed to assess the importance of mitochondrial DNA variants in patients with hypertrophic cardiomyopathy. The m.4300A>G mutation is identified as an important cause of this disorder, with other previously cardiomyopathy-associated and novel variants also identified. Such multi-gene tests can facilitate interpretable and phenotype-relevant results, but at the expense of limiting more extensive data acquisition. Whole-genome sequencing has been performed in five families with different autosomal dominant inherited heart disease phenotypes of unknown genetic aetiology. In two of these likely pathogenic variants were identified, one in the gene encoding titin (TTN) and the other in the calcium channel subunit gene CACNA1C. In vitro studies were undertaken to support the pathogenicity of the TTN variant and understand the functional effects of this. In the other three families either multiple candidate gene variants were identified or no clear candidate variant was identified. This highlights the difficulties in interpreting these results, even in carefully selected families. Overall, although the research benefits of exome or genome studies are evident, the interpretation and validation of genetic variant data produced remains highly challenging for clinical diagnosis.

Les dystrophies héréditaires vitelliformes de transmission autosomique dominante représentent la 2ème cause de maculopathie après la maladie de Stargardt, maladie récessive monogénique (ABCA4). BEST1 et PRPH2 sont les deux gènes connus associés aux dépôts vitellins. L'étude d'une large cohorte de 88 patients ayant une dystrophie vitelliforme juvénile ou de l'adulte avec un criblage systématique des deux gènes BEST1 et PRPH2 nous a permis d'établir des recommandations en fonction des trois critères : l'âge, l'histoire familiale et le rapport d'Arden. Nous avons ensuite recherché de larges réarrangements (délétions, insertions) dans les familles négatives par MLPA. Deux cas de délétion exonique ont été retrouvés (délétion de l'exon 4 du gène BEST1, délétion de l'exon 2 du gène PRPH2). L'étude de l'exome d'une grande famille (3 générations, 10 sujets atteints) n'ayant pas de mutations exoniques ou de réarrangements, a permis de démontrer l'implication du gène IMPG1 qui code pour une glycoprotéine de la matrice interphotoréceptrice. La même mutation faux-sens hétérozygote a été retrouvée dans deux autres familles. Nous avons ensuite testé son paralogue IMPG2 qui code également une protéine de la matrice interphotoréceptrice. Une seule famille avec une forme modérée de dystrophie vitelliforme a une mutation faux-sens hétérozygote dans ce second gène. IMPG1 et IMPG2, deux gènes de la matrice interphotoréceptrice sont désormais à ajouter à la liste des gènes des dystrophies vitelliformes après BEST1 le gène majeur et PRPH2. / Vitelliform dystrophies represent the second cause of inherited macular dystrophies after Stargardt disease (monogenic disease linked to ABCA4). To date, BEST1 and PRPH2 are the only known genes involved in vitellin deposits. Considering a large cohort of 88 unrelated patients with juvenile or adult form of vitelliform dystrophy and after a systematic screening of both genes, we propose a rational for BEST1 and PRPH2 analysis according to age of onset, positive family history and Arden ratio. The second step was to consider large deletions or insertions in these genes in patients negative for BEST1 and PRPH2. Exonic deletions are rare: one exon 4 deletion of BEST1 and one exon 2 deletion of PRPH2. Whole exome sequencing in a large family (3 generations, 10 affected patients) revealed a hetezogygous missense variation in IMPG1 an interphotoreceptor matrix gene. IMPG1 was the causal gene in two additionnal families. In the same way, its paralog IMPG2 have been tested : only one family with an heterozygous missense mutation was found. IMPG1 and IMPG2 are two new genes involved in vitelliform dystrophies after BEST1 the main gene and PRPH2.

La forêt n’est pas un espace naturel comme nous l’avons longtemps pensé. En utilisant l’ensemble des services que lui offre la forêt, l’homme influence la dynamique des espaces forestiers. L’objectif de cette thèse, s’inscrivant dans le champ de l’histoire de l’environnement, est d’analyser, dans le temps long, les interrelations entre paysages et sociétés riveraines, d’identifier les ruptures et continuités paysagères qui ont jalonné l’histoire forestière de l’Avesnois pour aboutir à ce que nous connaissons aujourd’hui. Cette recherche a été menée dans le cadre d’un contrat Cifre participant au Plan Forêt Régional - dont l’objectif est de doubler la superficie boisée sur l’ensemble du territoire d’ici une vingtaine d’années- et au Schéma Régional de Cohérence Ecologique Trames Vertes et Bleues. Ce dispositif en Sciences humaines et plus particulièrement en Histoire étant rare, il a fallu construire une démarche au carrefour de la démarche fondamentale et la démarche appliquée. Car non seulement il s’agissait d’analyser les modalités des actions humaines et leurs impacts sur les espaces forestiers mais il fallait plus particulièrement répondre à une demande des acteurs du monde forestier actuel conditionnant ainsi certaines problématiques scientifiques. Essentiel à la compréhension des interactions entre l’homme et son milieu, l’emboîtement des échelles spatio-temporelles constitue le cœur de cette recherche. La prise en considération de l’importance des emboîtements des échelles d’analyses, impliquant un croisement de sources de nature variée, ont conduit à une réflexion sur les outils et les méthodes à employer pour répondre aux questionnements initiaux. Tout en composant avec les limites des sources qu’il étudie, l’historien offre un recul sur les processus spatio-temporels qui ont fabriqué le paysage forestier d’aujourd’hui. Cette distanciation est nécessaire pour mener à bien les politiques environnementales actuelles : préserver un paysage, sa biodiversité doit nécessairement interroger le temps. / The forest is not a space natural as we thought of it for a long time. By using all the services which offers him the forest, the man influences the dynamics of the forest spaces. Our objective is to analyse, in the long time, the relations between landscapes and societies, to reveal the weight of the silvicultural inheritances in the contemporary sylvo-systems, to identify the breaks and the continuities of the landscape which marked out the forest history of the Avesnois, to end what we know today. This research was led within the framework of a Cifre contract participating in the Plan Forest Regional – the objectif of which is to double the surface afforested on the whole territory- and in the Regional Plan of Ecological Coherence Green and blue Wefts. This Cifre contract in Human sciences and more particularly in History being rare, it was necessary to build an approach in the crossroads of the fundamental approach and the applied approach. Because not only it was a question of analyzing the modalities of the human actions and their impacts on the forest spaces but it was more particularly necessary to answer a demand of the actors of the current forest world, conditioning certain scientific problems. The main part in the understanding of the interactions between the man and its environnement, the crossing of the spatiotemporal scales establishes the heart of this research. The taking into consideration of the importance of the crossings of the scales of analyse, implying a crossing of sources of varied nature, led to a reflection on tools and methods to use to answer the initial questionings. While composing with the limits of the sources which he studies, the historian offers a backward movement on the spatiotemporal processes which made the forest landscape of today. This distance is necessary to bring to a successful conclusion the current environmental policies : to protect a landscape, its biodiversity, it is inevitable to resort to the past.

Chondrodysplasia of Texel sheep is a newly described recessively inherited disorder distinct from other chondrodysplasias described in sheep. Phenotypically normal at birth, affected lambs develop microscopic lesions as early as 9 days of age, and usually demonstrate gross deformities and markedly reduced rates of bone growth by 2 to 3 weeks. Individual bone growth rates are most severely affected in the proximal bones of the forelimbs. Chondrodysplastic lambs typically have short stature, angular limb deformities, a barrel-shaped chest and a wide-based stance. Gross lesions include tracheal narrowing and contortion, enlarged costochondral junctions, and erosion of articular cartilage in major limb joints. Microscopic lesions are confined to hyaline cartilage, and are characterised by degeneration of the interterritorial matrix and dense perichondrocytic rings consisting predominantly of type VI collagen. These lesions are identical in appearance to those in achondrogenesis 1b and diastrophic dysplasia, two diseases caused by defects of the diastrophic dysplasia sulphate transporter (DTDST) in human beings. An investigation to measure the uptake of radiolabelled sulphate by dermal fibroblasts in vitro did not provide evidence of a defect in the DTDST in chondrodysplastic Texel sheep. A linkage disequilibrium study of ovine chromosomes 1, 5, 6, 13 and 22 using microsatellite DNA markers was unable to identify evidence of a mutation causing this form of chondrodysplasia. Capillary electrophoresis of unsaturated chondroitin sulphate disaccharides demonstrated a relative reduction in the ratio of chondroitin 4-sulphate to chondroitin 6-sulphate in affected animals of all ages. This biochemical feature enables the potential determination of the phenotype of newborn lambs prior to the emergence of gross or microscopic lesions. The pathology of the disease, combined with the findings of the genetic, biochemical and in vitro studies, suggest that a mutation may be present in the CHST11 gene. This gene is a good candidate for future studies aimed at discovering the genetic defect in chondrodysplasia of Texel sheep and developing a test to identify heterozygous animals.

Chondrodysplasia of Texel sheep is a newly described recessively inherited disorder distinct from other chondrodysplasias described in sheep. Phenotypically normal at birth, affected lambs develop microscopic lesions as early as 9 days of age, and usually demonstrate gross deformities and markedly reduced rates of bone growth by 2 to 3 weeks. Individual bone growth rates are most severely affected in the proximal bones of the forelimbs. Chondrodysplastic lambs typically have short stature, angular limb deformities, a barrel-shaped chest and a wide-based stance. Gross lesions include tracheal narrowing and contortion, enlarged costochondral junctions, and erosion of articular cartilage in major limb joints. Microscopic lesions are confined to hyaline cartilage, and are characterised by degeneration of the interterritorial matrix and dense perichondrocytic rings consisting predominantly of type VI collagen. These lesions are identical in appearance to those in achondrogenesis 1b and diastrophic dysplasia, two diseases caused by defects of the diastrophic dysplasia sulphate transporter (DTDST) in human beings. An investigation to measure the uptake of radiolabelled sulphate by dermal fibroblasts in vitro did not provide evidence of a defect in the DTDST in chondrodysplastic Texel sheep. A linkage disequilibrium study of ovine chromosomes 1, 5, 6, 13 and 22 using microsatellite DNA markers was unable to identify evidence of a mutation causing this form of chondrodysplasia. Capillary electrophoresis of unsaturated chondroitin sulphate disaccharides demonstrated a relative reduction in the ratio of chondroitin 4-sulphate to chondroitin 6-sulphate in affected animals of all ages. This biochemical feature enables the potential determination of the phenotype of newborn lambs prior to the emergence of gross or microscopic lesions. The pathology of the disease, combined with the findings of the genetic, biochemical and in vitro studies, suggest that a mutation may be present in the CHST11 gene. This gene is a good candidate for future studies aimed at discovering the genetic defect in chondrodysplasia of Texel sheep and developing a test to identify heterozygous animals.

Chondrodysplasia of Texel sheep is a newly described recessively inherited disorder distinct from other chondrodysplasias described in sheep. Phenotypically normal at birth, affected lambs develop microscopic lesions as early as 9 days of age, and usually demonstrate gross deformities and markedly reduced rates of bone growth by 2 to 3 weeks. Individual bone growth rates are most severely affected in the proximal bones of the forelimbs. Chondrodysplastic lambs typically have short stature, angular limb deformities, a barrel-shaped chest and a wide-based stance. Gross lesions include tracheal narrowing and contortion, enlarged costochondral junctions, and erosion of articular cartilage in major limb joints. Microscopic lesions are confined to hyaline cartilage, and are characterised by degeneration of the interterritorial matrix and dense perichondrocytic rings consisting predominantly of type VI collagen. These lesions are identical in appearance to those in achondrogenesis 1b and diastrophic dysplasia, two diseases caused by defects of the diastrophic dysplasia sulphate transporter (DTDST) in human beings. An investigation to measure the uptake of radiolabelled sulphate by dermal fibroblasts in vitro did not provide evidence of a defect in the DTDST in chondrodysplastic Texel sheep. A linkage disequilibrium study of ovine chromosomes 1, 5, 6, 13 and 22 using microsatellite DNA markers was unable to identify evidence of a mutation causing this form of chondrodysplasia. Capillary electrophoresis of unsaturated chondroitin sulphate disaccharides demonstrated a relative reduction in the ratio of chondroitin 4-sulphate to chondroitin 6-sulphate in affected animals of all ages. This biochemical feature enables the potential determination of the phenotype of newborn lambs prior to the emergence of gross or microscopic lesions. The pathology of the disease, combined with the findings of the genetic, biochemical and in vitro studies, suggest that a mutation may be present in the CHST11 gene. This gene is a good candidate for future studies aimed at discovering the genetic defect in chondrodysplasia of Texel sheep and developing a test to identify heterozygous animals.

<p>Traditionally in human genetic linkage analysis, extended families were only used in the analysis of dichotomous traits, such as Disease/No Disease. For quantitative traits, analyses initially focused on data from family trios (for example, mother, father, and child) or sib-pairs. Recently however, there have been two very important developments in genetics: It became clear that if the disease status of several generations of a family is known and their genetic information is obtained, researchers can pinpoint which pieces of genetic material are linked to the disease or trait. It also became evident that if a trait is quantitative (numerical), as blood pressure or viral loads are, rather than dichotomous, one has much more power for the same sample size. This led to the&nbsp / development of statistical mixed models which could incorporate all the features of the data, including the degree of relationship between each pair of family members. This is necessary because a parent-child pair definitely shares half their genetic material, whereas a pair of cousins share, on average, only an eighth. The statistical methods involved here have however been developed by geneticists, for their specific studies, so there does not seem to be a unified and general description of the theory underlying the methods. The aim of this dissertation is to explain in a unified and statistically comprehensive manner, the theory involved in the analysis of quantitative trait genetic data from extended families. The focus is on linkage analysis: what it is and what it aims to do.&nbsp / There is a step-by-step build up to it, starting with an introduction to genetic epidemiology. This includes an explanation of the relevant genetic terminology. There is also an application section where an appropriate human genetic family dataset is analysed, illustrating the methods explained in the theory sections.</p>

<p>Traditionally in human genetic linkage analysis, extended families were only used in the analysis of dichotomous traits, such as Disease/No Disease. For quantitative traits, analyses initially focused on data from family trios (for example, mother, father, and child) or sib-pairs. Recently however, there have been two very important developments in genetics: It became clear that if the disease status of several generations of a family is known and their genetic information is obtained, researchers can pinpoint which pieces of genetic material are linked to the disease or trait. It also became evident that if a trait is quantitative (numerical), as blood pressure or viral loads are, rather than dichotomous, one has much more power for the same sample size. This led to the&nbsp / development of statistical mixed models which could incorporate all the features of the data, including the degree of relationship between each pair of family members. This is necessary because a parent-child pair definitely shares half their genetic material, whereas a pair of cousins share, on average, only an eighth. The statistical methods involved here have however been developed by geneticists, for their specific studies, so there does not seem to be a unified and general description of the theory underlying the methods. The aim of this dissertation is to explain in a unified and statistically comprehensive manner, the theory involved in the analysis of quantitative trait genetic data from extended families. The focus is on linkage analysis: what it is and what it aims to do.&nbsp / There is a step-by-step build up to it, starting with an introduction to genetic epidemiology. This includes an explanation of the relevant genetic terminology. There is also an application section where an appropriate human genetic family dataset is analysed, illustrating the methods explained in the theory sections.</p>

In dieser Studie wurden erstmals die Auswirkungen von Zucht auf Kurzköpfigkeit auf ein breites Spektrum von Lebensbereichen brachyzephaler Hunde strukturiert untersucht. Hierzu wurde ein gegliederter Fragebogen für Tierbesitzer entwickelt. Erstmalig werden Validität und Reliabilität eines solchen Instruments zur Untersuchung von Symptomen einbezogen. Das Ziel der Arbeit war (1) die Konstruktion und Validierung eines internetbasierten Fragebogens für Tierbesitzer, der alle wichtigen Bereiche berücksichtigt, in denen brachyzephale Hunde Probleme zeigen; sowie die Bestimmung der Reliabilität des Fragebogens, (2) die Durchführung der Besitzerbefragung mit dem validierten Fragebogen und (3) die Analyse der Ergebnisse der Besitzerbefragung und Bestimmung von Häufigkeit und Schwere der Symptome bei den betroffenen Hunden. An der Befragung nahmen 100 Tierbesitzer teil, deren Hunde zur chirurgischen Behandlung des Brachyzephalen Syndroms an die Klinik für Kleintiere der Universität Leipzig überwiesen worden waren (61 Besitzer von Möpsen, 39 Besitzer von Französischen Bulldoggen). Die wichtigsten Ergebnisse dieser Studie sind: 100 % der Hunde haben bei Belastung laute Atemgeräusche, zwei Drittel sogar in Ruhe. Fast die Hälfte der Hunde hat in Ruhe Anzeichen für angestrengte Atmung. 41 % haben mindestens einmal wöchentlich Erstickungsanfälle und über ein Drittel ist schon mindestens einmal in ihrem Leben aufgrund von Atemnot umgefallen. Betroffene Tiere leiden an hochgradiger Belastungs- und Hitzeintoleranz. Die Belastbarkeit bei warmen Umgebungstemperaturen nimmt ab und die nach Belastung zur Erholung erforderliche Zeit steigt an. 88 % der Hunde sind nur eingeschränkt belastbar, 87 % beim Spielen; im Sommer können über ein Drittel der Hunde maximal 10 Minuten am Stück spazieren gehen, im Winter können über zwei Drittel der Hunde mindesten 30 Minuten spazieren gehen; bei 94 % der Hunde verschlimmern sich die Atembeschwerden bei warmen Temperaturen und zwar ab einer mittleren Temperatur von 19°C. Da diese Ergebnisse die hohe Temperaturabhängigkeit der Belastbarkeit zeigen, sollten für die Zuchtzulassung vorgeschriebene Belastungstests zukünftig bei eindeutig definierten Umgebungstemperaturen durchgeführt werden. Die Befragung der Tierbesitzer identifizierte ausgeprägte Atemnot während des Schla-fes als eine häufige schwerwiegende Beeinträchtigung. Ein Teil dieser Hunde entwickelt spezielle Strategien, um eine Verschlimmerung der obstruktiven Atemprobleme im Schlaf zu vermeiden. 31 % aller Hunde können nur schlafen, wenn sie ihr Kinn erhöht ablegen können; 24 % aller Hunde versuchen zeitweise im Sitzen zu schlafen und ver-meiden das Hinlegen, 6 % können nur mit geöffnetem Maul schlafen, da sie durch die pathologisch verkürzte Nase beim Schlafen keine Luft bekommen. Französische Bulldoggen haben signifikant häufiger Probleme beim Fressen als Möpse. 46 % aller Hunde zeigen Probleme im Zusammenhang mit der Futteraufnahme (Französische Bulldoggen zu 59 %, Möpse zu 38 %). Verschlucken und Erbrechen bzw. Regurgitieren sind sehr häufig genannte Probleme. Rückwärtsniesen (reverse sneezing) scheint ein weiteres durch Brachyzephalie ausge-löstes Problem zu sein. 73% der brachyzephalen Hunde zeigen Rückwärtsniesen, 25% davon täglich. Bisher ist vermehrtes Rückwärtsniesen als Symptom des Brachyzepha-len Syndroms in der Literatur nicht beschrieben. Alle brachyzephalen Hunde dieser Studie litten an tierschutzrelevanten Einschränkungen elementarer Grundbedürfnisse. Die teilweise hochgradigen Störungen in den Be-reichen Atmung, Bewegung, soziale Interaktion, Futteraufnahme, Schlafen und Thermo-regulation reduzieren die Lebensqualität erheblich. Alle Tiere hatten in mindestens einem der untersuchten Bereiche deutliche Einschränkungen. Diese Untersuchung wirft die Frage auf, ob die Zucht brachyzephaler Hunde mit den Ansprüchen eines modernen Tierschutzes überhaupt vereinbar ist.

Orientador: Egle Cristina Couto de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T14:08:16Z (GMT). No. of bitstreams: 1 Simoni_RenataZaccaria_D.pdf: 2332845 bytes, checksum: fc29ed8709fbdf31408d1968e76a01a0 (MD5) Previous issue date: 2012 / Resumo: Contexto e objetivo: Algumas malformações congênitas têm origem vascular, e a trombose durante a organogênese já foi aventada como possível mecanismo para esta ocorrência. O objetivo deste estudo foi avaliar a associação entre trombofilia fetal e malformações de origem vascular. Tipo de estudo e local: Foi realizado um estudo caso-controle, desenvolvido no ambulatório de Medicina Fetal do CAISM UNICAMP, de 2005 a 2010. Métodos: Foram incluídos no estudo 100 fetos com malformações de sistema nervoso central (SNC), gastrosquise, limb body wall e redução de membros (casos), submetidos a cordocentese como rotina do serviço, cujos resultados de cariótipo foram normais. Como controles, foram incluídos 100 fetos sem malformações cujo sangue de cordão fora previamente doado para o Banco de Sangue de Cordão do HEMOCENTRO UNICAMP. A pesquisa das mutações Fator V de Leiden, G20210A-FII e C677T-MTHFR foi realizada no sangue fetal dos dois grupos, e os resultados foram comparados. A análise descritiva foi feita utilizando Qui-quadrado e Teste Exato de Fisher. Para avaliar a associaçãoo entre as variáveis, foram utilizados o teste de Wilcoxon e a regressão logística. Resultados: Foram incluídos 78 fetos com malformações de SNC, 14 com gastrosquise, 3 com redução de membros e 2 com limb body wall. As mutações fator V de Leiden e G20210A-FII não foram encontradas nos casos e nos controles. A mutação C677T-MTHFR foi encontrada na forma heterozigota (CT) em 24 casos (24,8%) e em 6 controles. A mutação homozigota (TT) foi encontrada em 7 casos (7,2%) e em 1 controle. Estas diferenças foram estatisticamente significativas (p<0,0001). Quando avaliados os fetos com malformações de SNC (Artigo 1), a mutação CT foi encontrada com frequência significativamente maior nos casos do que nos controles (OR 10.309 IC95% 3.344-32.258), e a mutação TT também mostrou diferença significativa (OR 12.346 IC95% 1.388-111.11). A avaliação dos 14 fetos com gastrosquise (Artigo 2) não mostrou diferenças significativas quanto à presença da mutação CT ou TT entre os casos e os controles. Conclusão: A presença da mutação C677T-MTHFR no sangue fetal mostrou associação com malformações de SNC, tanto na forma homozigota quanto heterozigota / Abstract: Context and objective: Some congenital malformations have vascular origin, and a thrombosis during organogenesis is a possible mechanism for them. The aim of this study was to evaluate the association between fetal thrombophilia and malformations of vascular origin. Study type and location: A case-control study was performed at the Fetal Medicine Outpatient Clinic of CAISM UNICAMP, from 2005 to 2010. Methods: Ninety-seven fetuses with central nervous system malformations, gastroschisis, limb body wall and limb reduction were included in the study (cases), after routine cordocentesis showed normal karyotype results. A hundred fetuses without malformations were included as controls. These fetuses' cord blood had been donated to the Cord Blood Bank of HEMOCENTRO UNICAMP. DNA was extracted from fetal cord blood to study the mutations Factor V Leiden, G20210A-FII and MTHFR-C677T in both groups. Descriptive analysis was realized using Chi-square and Fisher's Exact Test. Wilcoxon test and logistic regression were used to analise the associations among variables. Results: We found 78 fetuses with central nervous system malformations, 14 with gastroschisis, 3 with member reduction and 2 with limb body wall. The mutations Factor V Leiden and G20210A-FII were not detected in cases nor in controls. The mutation MTHFR-C677T was encountered in 24 cases (24.8%) and in 6 controls its heterozygous form (CT). The homozygous mutation (TT) was found in 7 cases (7.2%) and in one control. These differences were statistically significant (p <0.0001). When the fetuses with central nervous system malformations were evaluated separately (Article 1), the frequency of the CT mutation was significantly higher in cases than in controls (OR 10.309 95% CI 3.344-32.258), as did the TT mutation (OR 12.346 95% CI 1.388-111.11). The 14 fetuses with gastroschisis were also evaluated separately (Article 2), and the results showed no statistically significant differences between cases and controls when concerning to the presence of the mutation MTHFR-C677T. Conclusion: The presence of the mutation MTHFR-C677T in fetal blood was associated with central nervous system malformations, both in homozygous and heterozygous form / Doutorado / Saúde Materna e Perinatal / Doutora em Ciências da Saúde