A long standing axiom in the field of teratology states that the teratogenic period, when most birth defects are produced, occurs during the third to eighth weeks of development post-fertilization. Any insults prior to this time are thought to result in a slowing of embryonic growth from which the conceptus recovers or death of the embryo followed by spontaneous abortion. However, new insights into embryonic development during the first two weeks, including formation of the anterior-posterior, dorsal-ventral, and left-right axes, suggests that signaling pathways regulating these processes are prime targets for genetic and toxic insults. Establishment of the left-right (laterality) axis is particularly sensitive to disruption at very early stages of development and these perturbations result in a wide variety of congenital malformations, especially heart defects. Thus, the time for teratogenic insults resulting in birth defects should be reset to include the first two weeks of development.

The prevalence of congenital heart defects is approximately 1% of all births, yet the causative factors remain largely uncharacterized. For the majority, the physiopathogenesis is believed to be multifactorial, hindering the identification of causative factors. However, several genes have been identified for septation defects that are part of a syndrome. Yet, in non-syndromic septal defects it has been difficult to identify predisposing genetic factors. When I started this thesis project, only one gene had been identified to be responsible for non-syndromic septal defects. We collected families in which two or more individuals were affected with non-syndromic cardiac septal defects. In five families, arrhythmia was associated with ASD/VSD. We screened the CSX/NKX2-5 gene, previously identified to be responsible for ASD and PR prolongation, and identified 3 novel missense mutations. In parallel, we screened the CSX/NKX2-5 gene in sporadic and familial cases of other cardiopathies, but additional mutations were not found. Substitutions in this gene seem to be a rare cause of cardiopathies without conduction defect. The absence of CSX/NKX2-5 mutations in two families suggest locus heterogeneity. We also examined whether the VEGF gene, which modifies the cardiac phenotype in del22q11 patients, could be responsible for the phenotypic variability observed in the three CSX/NKX2-5 mutated families. No statistically significant association was observed. We also evaluated the role of five candidate genes (GATA4, FOG2, CRELD1, HEY2 and BMP4) in a series of 66 patients affected with structural cardiac malformations, especially septal defects. Twenty nine nucleotide changes were identified. Based on their presence on dbSNP, dbEST and their position in multiple alignements, they were not considered to be mutations. However, we cannot exclude that some of them have an effect on RNA stability or abnormal splicing. We conclude that none of these five genes is a major cause of structural cardiac defects in man. Finally, we studied the genetic basis of heterotaxy. Among the collected families, there was one which was consanguineous, composed of two unaffected parents and three children, two of which presented situs inversus with or without Kartagener syndrome. We hypothezised an autosomal recessive mode of inheritance. Genotype analysis with polymorphic markers did not show linkage to known candidate genes or to loci causing laterality disorders. Array CGH did not detect duplication or microdeletion. Genome wide screening using 10K Affymetrix SNP chips allowed the identification of two autozygous regions, one in chromosome 1 and the other in chromosome 7. Interestingly, the kinesin associated protein 3 (KIF3AP) gene is located in chromosome 1q23.1-1q32.1. We screened this gene for mutations in all members of the family and excluded that mutations in this gene caused the situs inversus/Kartagener syndrome. In conclusion, we identified three new mutations in the CSX/NKX2-5 gene, evaluated the role of five candidate genes in structural cardiac malformations and identified two new candidate loci in situs inversus/Kartagener syndrome. / Les anomalies cardiaques ont une fréquence de 1% chez les nouveaux-nés. Leurs causes peuvent être multiples, mais pour la plupart l'étiologie n'a pas encore été élucidée. Cependant plusieurs gènes ont déjà été identifiés dans les défauts de septation non-syndromiques. En ce qui concerne les anomalies touchant la septation et ne faisant pas partie d'un syndrome, très peu est encore connu. Lorsque j'ai commencé mon travail de thèse, un seul gène était impliqué dans les défauts de septation non-syndromiques. Parmi les familles collectées, cinq familles présentant des défauts de septation associés à des troubles de conduction nous ont particulièrement intéressés. Nous avons criblé le gène CSX/NKX2-5, dont des mutations avaient été décrites auparavant comme étant responsables de CIA associées à des prolongements de l'intervalle PR. Ceci nous a permis d'identifier trois nouvelles mutations dans le gène CSX/NKX2-5. En parallèle, nous avons étudié la fréquence des mutations dans ce gène parmi des patients sporadiques et des cas familiaux atteints de diverses cardiopathies. Comme aucune nouvelle mutation n'a été identifiée, ce gène ne semble donc pas être fréquemment impliqué dans les cardiopathies non associées à des troubles de conduction. L'absence de mutations dans les deux autres familles suggère une hétérogénéité de locus. Nous avons de plus étudié si le gène VEGF, qui modifie le phénotype cardiaque chez les patients atteints du syndrome de DiGeorge, pouvait être responsable de la variabilité phénotypique observée dans les trois familles ayant des mutations dans le gène CSX/NKX2-5. Aucune association significative n'a été observée. Nous avons aussi évalué l'implication de cinq gènes candidats (GATA4, FOG2, CRELD1, HEY2 et BMP4) dans une série de 66 patients atteints de malformations cardiaques structurelles, constituées en grande partie par des défauts de septation. Vingt neuf changements nucléotidiques ont été identifiés. Aucun de ces changements nucléotidiques n'a été considéré comme étant une mutation sur base de leur présence dans les banques de données dbSNP et/ou dbEST. De même l'exclusion s'est basée sur le fait que les changements en acide aminé étaient localisés dans un domaine conservé. Nous ne pouvons cependant pas exclure que les variations des régions non codantes ou exons non transcrits, n'induisent pas une instabilité au niveau de l'ARN ou un épissage anormal. Cette étude nous a permis de conclure que les cinq gènes candidats n'ont aucune implication majeure dans les malformations cardiaques structurelles chez l'homme. La dernière partie du travail a été consacrée à l'étude de l'hétérotaxie. Parmi toutes les familles contactées, une famille consanguine présentait un intérêt particulier. Les parents n'étaient pas atteints et deux de leur trois enfants présentaient un situs inversus ou un syndrome de Kartagener. Sur base de ces données, nous avons émis l'hypothèse d'un mode de transmission autosmique récessif. Tous les gènes et loci connus, de même que des nouveaux candidats ont été exclus à l'aide du génotypage de marqueurs polymorphiques. L'analyse par array CGH n'a pas démontré la présence de microdélétion ou d'amplification. Nous avons donc entrepris un criblage du génome entier sur puce à SNP d'Affymetrix. Ceci nous a permis d'identifier deux régions homozygotes candidates, l'une dans le chromosome 1 et l'autre dans le chromosome 7. Un gène candidat, KIF3AP, situé dans le chromosome 1q23.1-1q32.1 et codant une protéine qui s'associe à la kinésine a été criblé pour tous les membres de la famille. Nous avons exclu l'implication de ce gène dans le situs inversus et le syndrome de Kartagener de la famille étudiée. En conclusion, nous avons identifié trois nouvelles mutations dans le gène CSX/NKX2-5, étudié l'implication de cinq gènes candidats dans des malformations cardiaques structurelles et finalement identifié deux nouveaux loci associés au situs inversus et au syndrome de Kartagener.

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Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.