Étude De Remaniements Chromosomiques Apparemment Équilibrés Associés À Des Phénotypes Anormaux Anouck Schneider

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Étude De Remaniements Chromosomiques Apparemment Équilibrés Associés À Des Phénotypes Anormaux Anouck Schneider Étude de remaniements chromosomiques apparemment équilibrés associés à des phénotypes anormaux Anouck Schneider To cite this version: Anouck Schneider. Étude de remaniements chromosomiques apparemment équilibrés associés à des phénotypes anormaux. Médecine humaine et pathologie. Université Montpellier, 2015. Français. NNT : 2015MONTT033. tel-01344094 HAL Id: tel-01344094 https://tel.archives-ouvertes.fr/tel-01344094 Submitted on 11 Jul 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. S ! "#! S $%% Cellules Souches Mésenchymateuses. Environnement Articulaire et Immunothérapie de la Polyarthrite Rhumatoïde Spécialité : GÉNÉTIQUE & '( & '( Née le 22 Juillet 1974 &(1 &&1 "&&1 " &I(3&& 4&I(3&& 4 )* + ,*)- ./ M. D. GENEVIEVE, PUPH, CHU de Montpellier Directeur M. J.M. DUPONT, PUPH, APHP- Hôpitaux Universitaire Paris Centre Rapporteur M. B. KEREN, PH, Pitié Salpétrière de Paris XIII Examinateur M. D. SANLAVILLE, PUPH, CHU de Lyon Examinateur M. F. VIALARD, PUPH, CHI de Poissy - St Germain Rapporteur S ! "#! S $%% Cellules Souches Mésenchymateuses. Environnement Articulaire et Immunothérapie de la Polyarthrite Rhumatoïde Spécialité : GÉNÉTIQUE & '( & '( Née le 22 Juillet 1974 &(1 &&1 "&&1 " &I(3&& 4&I(3&& 4 )* + ,*)- ./ M. D. GENEVIEVE, PUPH, CHU de Montpellier Directeur M. J.M. DUPONT, PUPH, APHP- Hôpitaux Universitaire Paris Centre Rapporteur M. B. KEREN, PH, Pitié Salpétrière de Paris XIII Examinateur M. D. SANLAVILLE, PUPH, CHU de Lyon Examinateur M. F. VIALARD, PUPH, CHI de Poissy - St Germain Rapporteur Résumé (français) La déficience intellectuelle (DI) est définie par un QI < 70. La DI, répartie en formes non syndromiques et en formes syndromiques, est observée dans 3 % de la population. Des anomalies chromosomiques sont identifiées dans 15 % des DI syndromiques. Les translocations chromosomiques réciproques (TR) apparemment équilibrées sont observées chez 1 individu sur 1000 et seul 6 % des patients avec une TR de novo apparemment équilibrée ont une DI. Plusieurs mécanismes chromosomiques peuvent expliquer la DI syndromique associée à une TR : (i) un microremaniement déséquilibré identifié par l'utilisation de techniques plus résolutives, (ii) la formation d'un gène de fusion, (iii) un effet de position, (iv) la modification d’une région soumise à une empreinte parentale, (v) une interruption d'un gène au niveau d'un ou des deux points de cassure, (vi) une mutation génique sans rapport avec la TR, (vii) ou encore une cause acquise ou multifactorielle. Nous rapportons l'étude de 12 patients avec DI et porteurs d'une TR de novo apparemment équilibrée. L'analyse systématique par puces à ADN de ces individus a été réalisée avec une résolution de 25 kb. Un déséquilibre infracytogénétique au niveau des points de cassure ou ailleurs dans le génome a été observé chez 3/12 patients. Chez les 9 patients sans anomalies sur puces à ADN, nous avons étudié les points de cassure des remaniements de novo apparemment équilibrés. En dehors de la technique de marche sur le chromosome par FISH, deux autres approches ont été mises en œuvre : (i) l'Array-Painting qui correspond à l'hybridation sur puces à ADN de chacun des dérivés chromosomiques préalablement séparés par Cytométrie en Flux, (ii) et le séquençage haut débit (WGS - Whole Genome Sequencing). Grâce à l'Array-Painting, nous avons identifié (i) chez 2 patients, des interruptions de gènes pouvant expliquer leur phénotype, à savoir les gènes : KIF1A , AUTS2 et EPHA6 ; (ii) et chez 1 patiente, un point de cassure entraînant une dérégulation de la transcription du gène MEF2C . L'étude par WGS a permis (i) chez 1 patiente, de diagnostiquer un déséquilibre plus complexe que celui observé par puce à ADN ; (ii) chez 2 patients, de mettre en évidence un chromothripsis , qui pourrait avoir un impact dans les pathologies constitutionnelles par interruption de gènes et/ou par effet de position ; (iii) et chez 2 autres patients, de caractériser précisément les points de cassure. Ainsi, grâce aux résultats obtenus par ces différentes techniques, plusieurs mécanismes physiopathologiques responsables de DI sont mis en évidence permettant un conseil génétique adéquat. Cependant, aucun mécanisme chromosomique commun ne peut être identifié hormis le chromothripsis observé chez patients. Finalement, ce travail nous permet principalement de comparer les techniques mises en œuvre qui se sont avérées complémentaires. En conclusion, nous proposons une démarche diagnostique pour explorer un remaniement chromosomique apparemment équilibré chez des patients à phénotype anormal. Mots clefs (français) : Phénotype anormaux , Remaniements chromosomiques apparemment équilibrés, Points de cassure, Translocations, Array-Painting, Séquençage haut débit Title : Study of apparently balanced chromosomal rearrangements associated with abnormal phenotypes Abstract : Intellectual disability (ID) is defined by an IQ <70. ID, observed in 3% of the population, and displays heterogeneous origins, including acquired etiology (toxicologic, pathologic, traumatic) or genetic disorders with non-syndromic and syndromic forms. Numerical or structural chromosomal abnormalities are observed in 15% of patients with ID. Reciprocal balanced chromosomal translocations (RT) are observed in one individual in 1000. However, only 6% of patients carrying a de novo apparently balanced RT present ID. The relation between these balanced rearrangements and ID could be explained by different mechanisms namely (i) subtle rearrangement, (ii) gene fusion, (iii) position effect, (iv) disturbance of parental imprinting, (v) gene disruption at the breakpoints, (vi) mutation in gene unrelated to the translocation, (vii) or acquired or multifactorial cause. We report a chromosomal study of 12 patients with DI and carrying a de novo apparently balanced reciprocal translocation. A systematic analysis by microarrays was performed in all individuals (using a resolution of 25 kb). For three patients, a microdeletion was observed at the breakpoints or elsewhere in the genome. For the 9 remaining cases, we hypothesize that the phenotype is due to a disruption of gene(s) located at the breakpoint(s). In this context, we studied the breakpoints of the apparently balanced de novo rearrangements in these patients. Outside FISH walking, two approaches have been implemented namely Array-Painting, which combines flow chromosome sorting in an attempt to isolate derivative chromosomes from each other and DNA microarrays as well as Whole Genome Sequencing (WGS). Using Array-Painting, we identified (i) in 2 patients, a gene disruptions: in the KIF1A , AUTS2 and EphA6 genes; (ii) and in 1 patient, a breakpoint resulting in deregulation of transcription of the gene MEF2C . The WGS technology has permitted (i) in 1 patient, to diagnose more complex imbalance than that observed by micro-array; (ii) in 2 patients, to show a chromothripsis , (iii) and 2 other patients, to characterize precisely breakpoints. In conclusion, taking together, these results highlight different physiopathological mechanisms responsible for DI allowing adequate genetic counseling. However, no common chromosomal mechanism can be identified except for chromothripsis observed in 2 patients. In addition, this work allows us especially to compare the used techniques which seem to be complementary. Finally, we propose a pipeline to elucidate the etiology of the abnormal phenotype in patients carrying an apparently balanced rearrangement. Keywords : Abnormal phenotypes, Apparently balanced chromosomal rearrangements, Breackpoint, Translocations, Array-Painting, Whole genome sequencing ! # # # # # ! # # # ! % && '() # # " # $ # % ' # & " " () * + , - . / . 0# 1 + , + , . !( ) / + , # 2 - 1 # " " +# )$2 " ( ) / 0 () 03# / 0 ( # / & ! E F 2 & 56 # , # ! # # 7 ! # 7 8 ! # # 2 9 - # '# 8 # : Abréviations ............................................................................................................................... 1 Nomenclature ............................................................................................................................
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