Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions. In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD. In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-12T14:48:04Z No. of bitstreams: 2 DISSERTAÇÃO Danyllo Felipe de Oliveira.pdf: 2356321 bytes, checksum: 65e8ae7586ecebdf70bf8d998cba93d3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T14:48:04Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Danyllo Felipe de Oliveira.pdf: 2356321 bytes, checksum: 65e8ae7586ecebdf70bf8d998cba93d3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / CAPES / Entende-se por Deficiência Intelectual (DI), conforme a mais recente versão do Código Internacional de Doenças (CID-10), como sendo uma “parada do desenvolvimento ou desenvolvimento incompleto do funcionamento intelectual”. Essa alteração se dá essencialmente durante o desenvolvimento do indivíduo e afeta, principalmente, “funções cognitivas, de linguagem, motricidade e do comportamento social”. Ainda segundo as definições teóricas do CID-10, a DI pode ser subdividida de acordo com o grau de severidade de comprometimento intelectual, sendo este medido pelo nível do quociente de inteligência (QI) obtido dos pacientes em testes de neuropsicologia A deficiência intelectual é uma característica bastante recorrente, e tem sido extensivamente estudada do ponto de vista genético desde a identificação da trissomia do cromossomo 21 como um fator etiológico para a Síndrome de Down. Posteriores investigações usando técnicas citogenéticas de bandeamento cromossômico e hibridização in situ, contribuíram para melhor compreender a base molecular da DI, através da descrição de perdas e ganhos de grandes fragmentos cromossômicos além de microdeleções e microduplicações. Estas alterações têm sido crescentemente descritas para a Síndrome de Cri Du Chat, Smith-Magenis, Wolf- Hischhron, entre outras. Dentre as condições genéticas onde DI é encontrada, as cujo padrão de herança está ligado ao cromossomo X representam de 10 a 15 % dos casos. Em face disso, a deficiência mental de herança ligada ao cromossomo X (DMLX) apresenta-se como um complexo grupo de mais de mais de 200 entidades nosológicas, com uma considerável heterogeneidade genética, uma vez que mais de 100 genes têm sido identificados até o momento. Entretanto, a despeito da grande quantidade de genes envolvidos, mais de 50 % das famílias identificadas com deficiência mental ligada ao X possuem uma etiologia genética desconhecida. Duas famílias com um provável padrão de herança ligado ao cromossmo X, originárias do Estado de Pernambuco foram avaliadas do ponto de vista clínico e molecular. A família 1 apresentou quatro afetados, sendo três homens e uma mulher, que eram portadores de deficiência mental grave. O indivíduo probando, cuja idade é 29 anos, apresenta além da DI, um perímetro cefálico abaixo do segundo percentil. Nenhuma outra alteração dismórfica foi evidenciada nos outros indivíduos, embora entre os afetados haja o registro de crises convulsivas. A segunda família, com três afetados, sendo dois deles gêmeos univitelinos, apresenta indivíduos com deficiência intelectual grave, com nenhum outro sinal dismórfico aparente. A análise por Southern blotting de ambas famílias para a verificação das expansões do gene FMR1, associado à síndrome do cromossomo X frágil, revelou que o probando da família 1 apresentava a pré-mutação (fragmentos de 3 kb), assim como a sua genitora e a irmã desta. O outro afetado, filho desta última portadora da pré-mutação, apresentou a mutação completa (fragmentos de 6 , 6,3 e 8kb). O último indivíduo afetado e sua respectiva genitora apresentaram fragmentos correspondentes ao de indivíduos normais (2,8 e 5,2 kb). Tais dados levam ao diagnóstico da Síndrome do cromossomo X frágil para a primeira família. A segunda família foi semelhantemente testada, mas os indivíduos e sua genitora são normais para o teste do gene FMR1. O estudo do teste de desvio de inativação do cromossomo X na mãe dos afetados da família 2 revelou que este indivíduo apresenta um desvio total de inativação (100:0), um indicativo do padrão de herança ligado ao cromossomo X. A hibridização genômica comparativa (array CGH) não identificou nenhuma alteração cromossômica microestrutural que pudesse explicar o fenótipo.

Introduction: Les données des analyses génomiques des tumeurs de vessie ont défini des sous-groupes qui présentent une agressivité et une réponse à la chimiothérapie différents. Notre but était d’identifier des marqueurs moléculaires prédictifs de l'évolution tumorale.Matériels et méthodes: Les ADNs de 54 tumeur solides et de 50 échantillons d’ urines de patients avec une tumeur de vessie ont été hybridés sur la puce CGH (Comparative Genomic Hybridization) BCA-oligo. Des TMAs (Tissue Micro Array) de 140 tissus paraffinés de tumeurs primitives et métastatiques, ont été analysés en immunohistochimie pour les marqueurs impliqués dans: stabilité de l’ADN, prolifération cellulaire et définition de sous-groupe basal ou luminal. Des corrélations avec les données cliniques ont été recherchées à tous les niveaux d’analyse.Résultats: Le nombre d'altérations chromosomiques augmentait significativement avec le stade tumoral. La distribution de ces altérations était cohérente entre les ADNs extraits de tissu tumoral et de prélèvements urinaires. Si aucun marqueur immunohistochimique ne permettait de différencier les patients métastatiques ou non, des profils de marquage similaires ont été observés entre tumeurs primitives et métastases. La présence d'emboles tumoraux lymphatiques était prédictive du statut métastatique. Conclusion: l’utilisation dans la pratique clinique de la puce BCA-oligo pour prédire une évolution d'une tumeur de vessie vers un stade ou un grade plus élevé peut être envisagée, et réalisée sur un simple prélèvement urinaire. La recherche systématique d’emboles lymphatiques sur tumeurs primitives peut être utilisée pour prédire une évolution métastatique. / Introduction: Recent data from genomic analysis of bladder tumors have identified subgroups with different aggressiveness patterns and chemotherapy response profiles. The aim of our study was to identify molecular markers that can be used in clinical practice, to predict the evolution of these tumors in order to personalize their management.Materials and Methods: DNAs extracted from 54 solid tumors and 50 urine samples from patients with bladder cancer were hybridized on the BCA-oligo CGH (Comparative Genome Hybridization) chip. TMAs (Tissue Micro Array) from 140 paraffine-embedded tissues of primary and metastatic tumors, were analyzed in immunohistochemistry with antibodies directed against proteins involved in DNA stability, cell proliferation and the definition of basal or luminal subgroup. Correlations with clinical data were sought at all levels of analysis.Results: The number of chromosomal alterations increased significantly with the tumor stage. In addition, the distribution of these alterations was consistent between the DNAs extracted from tumor tissue and those from urinary samples. If no immunohistochemical marker differentiated between metastatic and non-metastatic patients, similar labeling patterns were observed between primary tumors and metastases. Notably, the presence of lymphatic emboli was predictive of metastatic status.Conclusion: The use in clinical practice of the BCA-oligo chip to predict a progression of a bladder tumor to a higher stage or grade may be considered. This analysis is feasible on urine sample. The systematic search for lymphatic emboli on primary tumors can be used in clinical practice to predict a metastatic evolution.

A obesidade se tornou uma das maiores preocupações de saúde pública. É um distúrbio neuroendócrino, no qual fatores ambientais e genéticos agem em conjunto, levando ao excesso de armazenamento de energia na forma de gordura corporal. A síndrome de Prader-Willi (PWS) é a mais freqüente das síndromes que possui a obesidade como uma de suas características, com incidência de 1:25.000 nascimentos. É caracterizada por hipotonia neonatal com dificuldade de sucção, atraso do desenvolvimento neuropsicomotor (DNPM), hiperfagia, obesidade, baixa estatura em adolescentes, mãos e pés pequenos, hipogonadismo, distúrbios do sono, características faciais dismórficas, deficiência intelectual leve a moderada e comportamento obsessivo-compulsivo. Pacientes com atraso do DNPM e/ou dificuldade de aprendizado, distúrbios de comportamento, obesidade e/ou hiperfagia, com teste negativo para PWS, foram estudados com plataformas de SNP array, “The GeneChip® Mapping 500K Set” da Affymetrix, ou array-CGH, CytoSure ISCA 4x180k da OGT, para identificar genes relacionados a obesidade e hiperfagia, assim como, novas regiões genômicas implicadas na etiologia de síndromes genéticas associadas à obesidade. Dentre os 31 pacientes estudados, oito apresentaram variações de número de cópias (CNVs) em seu genoma: deleção em 1p22.1p21.2; deleção em 3q25.33q26.1 e deleção em 13q31.2q32.1; duplicação em 7q36.2; deleção em 8p23.3p23.1 e duplicação em 12p13.33p13.31; duplicação 16p13.11p12.3; duplicação em 17q11.2; deleção em 20p12.1; duplicação em 21q22.13. Duas dessas alterações foram herdadas de pais fenotipicamente normais. Algumas dessas CNVs sobrepõem regiões genômicas previamente relacionadas com obesidade, incluindo a microdeleção de 1p21.3 e as duplicações dos cromossomos 12 e 21. Identificamos genes anteriormente descritos como associados à obesidade (PTBP2, DPYD, MIR137, GNB3 e PPM1L), ou possivelmente envolvidos com este fenótipo (HTR5A e KCNJ6), mapeados em várias dessas CNVs. Além disso, os genes RNF135, NF1, DPP6, GPC5, DYRK1A e MACROD2 são os prováveis causadores da deficiência intelectual, atraso do desenvolvimento neuropsicomotor, dificuldades de aprendizagem, distúrbios de comportamento e outras características clínicas encontrados nos pacientes. O diagnóstico e prognóstico dos pacientes e o Aconselhamento Genético aos pais e familiares é fornecido / Obesity has become a major concern for public health. It is a neuroendocrine disorder, in which genetic and environmental factors act together, leading to excessive storage of energy as fat. Prader-Willi syndrome (PWS) is the main obesity-related syndrome with a birth incidence of 1:25,000. It is characterized by neonatal hypotonia, poor sucking, developmental delay, hyperphagia, obesity, short stature in adolescents, small hands and feet, hypogonadism, sleep disturbance, dysmorphic facial features, mild to moderate intellectual disability and obsessive-compulsive behavior. Patients with psychomotor developmental delay and/or learning disabilities, behavior disorders, obesity and/or hyperphagia, who tested negative for PWS, were studied by chromosomal microarray analysis, including the SNP-based platform “The GeneChip® Mapping 500K Set” (Affymetrix), and the array-CGH platform “CytoSure ISCA 4x180k (OGT)”, to identify genes related to hyperphagia and obesity, as well as new genomic regions implicated in the etiology of genetic syndromes associated with obesity. Of 31 patients studied, eight had copy number variants (CNVs) in the genome: 1p22.1p21.2 deletion; 3q25.33q26.1 deletion and 13q31.2q32.1 deletion; 7q36.2 duplication; 8p23.3p23.1 deletion and 12p13.33p13.31 duplication; 16p13.11p12.3 duplication; 17q11.2 duplicaton; 20p12.1 deletion; 21q22.13 duplication. Two of these CNVs were inherited from an unaffected father. Some of these CNVs overlap genomic regions that have previously been related to obesity, including the 1p21.3 microdeletion and the duplications of chromosomes 12 and 21. Furthermore, we identified genes previously described as associated with obesity (PTBP2, DPYD, MIR137, GNB3 and PPM1L), or possibly involved with this phenotype (HTR5A and KCNJ6), mapped to several of these CNVs. In addition, the genes RNF135, NF1, DPP6, GPC5, DYRK1A and MACROD2 are likely implicated in intellectual disability, developmental delay, learning disabilities, behavioral disorders and other clinical features found in patients. The diagnosis and prognosis of patients and genetic counseling to parents and families is provided

Das vom endonasalen Neuroepithel der Rima olfactoria entstandenen Aesthesioneuroblastom gehört zu den seltenen malignen Tumoren der Rhinobasis. Eine generelle Therapieempfehlung für die Behandlung dieses Tumors gibt es nicht, da bis heute etablierte, durch umfassende onkologische Studien untermauerte diagnostische und therapeutische Standards fehlen und der klinische Verlauf oft unberechenbar ist. Die Aufgabe der vorliegenden Arbeit bestand in der Überprüfung des chirurgischen Konzeptes bei der Therapie von Aesthesioneuroblastomen und in der erstmaligen molekularzytogenetischen Charakterisierung von Aesthesioneuroblastomen. Dazu wurden 18 Patienten mit Aesthesioneuroblastomen, die im Zeitraum zwischen 1988 und 2001 in der HNO-Klinik (17 Patienten) sowie in der Neurochirurgischen Klinik des Klinikums Fulda operiert wurden, untersucht. Die daraus resultierenden 22 Aesthesioneuroblastome wurden alle mit Hilfe der Vergleichenden Genomischen Hybridisierung (CGH) analysiert. Nach derzeitigem Kenntnisstand besteht die optimale Therapie der Aesthesioneuroblastome in der chirurgischen Resektion des Tumors mit nachfolgender stereotaktischer Bestrahlung. Für die operative Sanierung der Aesthesioneuroblastome und auch anderer Malignome der vorderen Schädelbasis ist das nachfolgende neue Fuldaer Konzept empfehlenswert: 1) Endonasale Resektion, wenn keine intrakranielle bzw. orbitale Tumorinfiltration vorliegt; 2) Subfrontaler Zugang, bei Infiltration des Gehirns; 3) Midfacial degloving, bei weit lateraler Tumorausbreitung; 4) Laterale Rhinotomie nur bei der Notwendigkeit der simultanen Exenteratio orbitae (bei orbitaler Tumorinfiltration). Aesthesioneuroblastomen sind durch ein typisches genetisches Muster charakterisiert, das Deletionen im Bereich der chromosomalen Arme 1p, 2q, 3p/q, 4p/q, 5p/q, 6q, 8p/q, 9p, 10p/q, 11p, 12q, 13q, 18q und 21q sowie Amplifikationen der Chromosomen 1p, 7q, 9q, 11q, 14q, 16p/q, 17p/q, 19p/q, 20p/q und 22p/q umfasst. Die beim Aesthesioneuroblastom häufigen DNA-Verluste im Bereich der chromosomalen Banden 1p21-p31 scheinen mit der Prognose dieser Tumoren assoziiert zu sein. Die Tumoren aller in der vorliegenden Studie am Malignom verstorbenen Patienten zeigten eine Kombination aus 1p21-p31-Deletion, dem Vorliegen des klinischen Stadiums C oder D sowie gleichzeitig einer schlechten Differenzierung (Grad III oder IV). Vermittels der CGH ist es möglich, eine klonale Zuordnung von Metastasen bzw. auch Rezidiven zu ihren primären Aesthesioneuroblastomen vorzunehmen. Die vorliegende Arbeit zeigt nicht nur neue Ansätze in der chirurgischen Therapie von Aesthesioneuroblastomen sondern auch die erste umfassende molekularzytogenetische Analyse dieser Tumorentität, auf dem Weg, das biologische Verhalten dieser Malignome genauer charakterisieren zu können. / Esthesioneuroblastoma (ENB) is a very rare malignant neoplasm arising from the olfactory epithelium which is recognized for its propensity for local recurrence and distant dissemination. Therapeutic management approaches for this neoplasm lack uniformity. The present study describes therapeutic management in ENB: Complete surgical resection combined with adjuvant stereotactic radiation therapy. Thereby, a new surgical concept is recommended: 1) Endonasal approach in cases without tumor infiltration of the orbit and/or the brain; 2) Subfrontal approach in cases with extended tumor infiltration of the intradural space or of the brain; 3) Midfacial degloving in cases with far lateral tumor spread, particularly fossa pterygoidea or pterygopalatina; 3) Lateral rhinotomy in all cases where an exenteratio orbitae is needed. Secondarily, the study characterizes the specific chromosomal alterations of ENB analyzed using Comparative Genomic Hybridization (CGH). ENB show frequently deletions of chromosoms 1p, 2q, 3p/q, 4p/q, 5p/q, 6q, 8p/q, 9p, 10p/q, 11p, 12q, 13q, 18q and 21q as well as DNA gains of chromosoms 1p, 7q, 9q, 11q, 14q, 16p/q, 17p/q, 19p/q, 20p/q and 22p/q. Deletions of the chromosomal region 1p21-p31 could be associated with bad prognosis since the tumors of all patients who died were of stage C or D and grade III or IV, and showed 1p21-p31 deletions. The analysis of primary ENB and their corresponding metastases shows clonality by a high concordance of alterations between the tumor pairs. For the first time, this study presents the specific chromosomal alterations of ENB pathogenesis and progression.

No description available.

No description available.

A obesidade se tornou uma das maiores preocupações de saúde pública. É um distúrbio neuroendócrino, no qual fatores ambientais e genéticos agem em conjunto, levando ao excesso de armazenamento de energia na forma de gordura corporal. A síndrome de Prader-Willi (PWS) é a mais freqüente das síndromes que possui a obesidade como uma de suas características, com incidência de 1:25.000 nascimentos. É caracterizada por hipotonia neonatal com dificuldade de sucção, atraso do desenvolvimento neuropsicomotor (DNPM), hiperfagia, obesidade, baixa estatura em adolescentes, mãos e pés pequenos, hipogonadismo, distúrbios do sono, características faciais dismórficas, deficiência intelectual leve a moderada e comportamento obsessivo-compulsivo. Pacientes com atraso do DNPM e/ou dificuldade de aprendizado, distúrbios de comportamento, obesidade e/ou hiperfagia, com teste negativo para PWS, foram estudados com plataformas de SNP array, “The GeneChip® Mapping 500K Set” da Affymetrix, ou array-CGH, CytoSure ISCA 4x180k da OGT, para identificar genes relacionados a obesidade e hiperfagia, assim como, novas regiões genômicas implicadas na etiologia de síndromes genéticas associadas à obesidade. Dentre os 31 pacientes estudados, oito apresentaram variações de número de cópias (CNVs) em seu genoma: deleção em 1p22.1p21.2; deleção em 3q25.33q26.1 e deleção em 13q31.2q32.1; duplicação em 7q36.2; deleção em 8p23.3p23.1 e duplicação em 12p13.33p13.31; duplicação 16p13.11p12.3; duplicação em 17q11.2; deleção em 20p12.1; duplicação em 21q22.13. Duas dessas alterações foram herdadas de pais fenotipicamente normais. Algumas dessas CNVs sobrepõem regiões genômicas previamente relacionadas com obesidade, incluindo a microdeleção de 1p21.3 e as duplicações dos cromossomos 12 e 21. Identificamos genes anteriormente descritos como associados à obesidade (PTBP2, DPYD, MIR137, GNB3 e PPM1L), ou possivelmente envolvidos com este fenótipo (HTR5A e KCNJ6), mapeados em várias dessas CNVs. Além disso, os genes RNF135, NF1, DPP6, GPC5, DYRK1A e MACROD2 são os prováveis causadores da deficiência intelectual, atraso do desenvolvimento neuropsicomotor, dificuldades de aprendizagem, distúrbios de comportamento e outras características clínicas encontrados nos pacientes. O diagnóstico e prognóstico dos pacientes e o Aconselhamento Genético aos pais e familiares é fornecido / Obesity has become a major concern for public health. It is a neuroendocrine disorder, in which genetic and environmental factors act together, leading to excessive storage of energy as fat. Prader-Willi syndrome (PWS) is the main obesity-related syndrome with a birth incidence of 1:25,000. It is characterized by neonatal hypotonia, poor sucking, developmental delay, hyperphagia, obesity, short stature in adolescents, small hands and feet, hypogonadism, sleep disturbance, dysmorphic facial features, mild to moderate intellectual disability and obsessive-compulsive behavior. Patients with psychomotor developmental delay and/or learning disabilities, behavior disorders, obesity and/or hyperphagia, who tested negative for PWS, were studied by chromosomal microarray analysis, including the SNP-based platform “The GeneChip® Mapping 500K Set” (Affymetrix), and the array-CGH platform “CytoSure ISCA 4x180k (OGT)”, to identify genes related to hyperphagia and obesity, as well as new genomic regions implicated in the etiology of genetic syndromes associated with obesity. Of 31 patients studied, eight had copy number variants (CNVs) in the genome: 1p22.1p21.2 deletion; 3q25.33q26.1 deletion and 13q31.2q32.1 deletion; 7q36.2 duplication; 8p23.3p23.1 deletion and 12p13.33p13.31 duplication; 16p13.11p12.3 duplication; 17q11.2 duplicaton; 20p12.1 deletion; 21q22.13 duplication. Two of these CNVs were inherited from an unaffected father. Some of these CNVs overlap genomic regions that have previously been related to obesity, including the 1p21.3 microdeletion and the duplications of chromosomes 12 and 21. Furthermore, we identified genes previously described as associated with obesity (PTBP2, DPYD, MIR137, GNB3 and PPM1L), or possibly involved with this phenotype (HTR5A and KCNJ6), mapped to several of these CNVs. In addition, the genes RNF135, NF1, DPP6, GPC5, DYRK1A and MACROD2 are likely implicated in intellectual disability, developmental delay, learning disabilities, behavioral disorders and other clinical features found in patients. The diagnosis and prognosis of patients and genetic counseling to parents and families is provided

ObjectifLe diagnostic prénatal (DPN) a pour but de détecter des pathologies foetales in utero. L’objectif de ce travail était de mettre au point et d’appliquer les techniques d’exploration chromosomique en prénatal. Nous avons, tout d’abord, validé et évalué une plateforme de séquençage basée sur la technologie des semi-conducteurs, Ion Proton®, pour le dépistage prénatal non-invasif (DPNI) des principales aneuploïdies en routine clinique, puis évalué l’intérêt de l’Analyse Chromosomique sur Puces à ADN (ACPA) dans le diagnostic prénatal des retards de croissance intra-utérin (RCIU) foetaux. Matériel et Méthodes Nous avons inclus prospectivement 2505 patientes enceintes analysées par huit laboratoires hospitalo-universitaires de génétique : 695 grossesses à haut risque pour la trisomie 21 (risque ≥1/250 ou avec anomalie échographique) dans une étude de validation de la technique du test ADN libre circulant (ADNlc), et 1810 grossesses à risque, sans anomalie échographique, en routine clinique. Les issues de grossesses étaient toutes disponibles dans l’étude de validation et pour 521 grossesses dans l’étude en routine clinique. L’ADNlc extrait d’échantillons plasmatiques était séquencé, puis les données étaient analysées à l’aide du logiciel WISECONDOR. Les résultats des tests ADNlc étaient comparés aux caryotypes foetaux ou 7 aux données à la naissance. Nous avons aussi évalué le taux d’échec et comparé trois méthodes d’évaluation de la fraction foetale (FF) (RASSF1A, DEFRAG et SANEFALCON). Nous avons rétrospectivement inclus tous les foetus référés pour un prélèvement invasif pour RCIU et étudié les résultats de technique d’hybridation in situ en fluorescence (FISH), caryotypes et ACPA. Résultats Les résultats des deux cohortes de l’étude sur l’ADNlc étaient cohérents et les âges gestationnels n’étaient pas significativement différents ; les données ont été combinées afin d’étoffer la cohorte à analyser. Respectivement, la sensibilité et la spécificité étaient de : 98.3% (95% IC, 93.5–99.7%) et 99.9% (95% IC, 99.4–100%) pour la trisomie 21; 96.7% (95% IC, 80.9–99.8%) et 100% (95% IC, 99.6–100%) pour la trisomie 18 ; et 94.1% (95% IC, 69.2–99.7%) et 100% (95% IC, 99.6–100%) pour la trisomie 13. Le taux de non rendus était de 1.2% initialement puis après réanalyse de 0.6%. L’estimation de la FF avec les méthodes RASSF1A et DEFRAG étaient comparables, toutes deux compatibles avec une utilisation en routine clinique. Parmi les 162 foetus RCIU (78 associés et 84 isolés) inclus dans l’étude ACPA, 15 avaient une FISH pathologique : 10 RCIU associés et cinq RCIU isolés. Parmi 143 foetus étudiés par ACPA, 10 (7%) présentaient un variant du nombre de copies (CNV), tous étaient des RCIUs associés (10/65 soit 15.4%; 95 IC: 8.4%‐26.2%), versus 0/78 dans le groupe RCIUs isolés (95% IC: 0%‐5.6%). Six foetus (4.2%) ont présenté des variants de signification inconnue (VSI) (trois RCIU associés et trois RCIU isolés). Conclusion : Notre étude évaluant le test ADNlc utilisant la technologie des semi-conducteurs est la première étude clinique à rapporter les issues de grossesses dans une population aussi large. La plateforme est performante pour le DPNI des principales aneuploïdies. Notre protocole robuste est facilement applicable en routine clinique. Notre étude souligne une augmentation de rendement diagnostique de l’ACPA de 6.1% (4/65) par rapport au caryotype pour le DPN des foetus présentant un RCIU associé. Aucun CNV pathogène n’a été mis en évidence dans le groupe RCIU isolé. L’ADNlc pourrait-il supplanter l’ACPA dans cette population de RCIU isolé ? Le développement du test ADNlc a permis de limiter le nombre de prélèvements invasifs et donc leurs complications [...]. / ObjectivePrenatal diagnosis allows to detect fetal pathologies in-utero. The goal of this work was both technical development and application of the chromosomal exploration technics in prenatal diagnosis. First, we aimed to validate and evaluate the performance metrics of the highthroughput semiconductor sequencing platform, Ion Proton®, in non-invasive prenatal genetic screening (NIPS) for common fetal aneuploidies in a clinical setting and, then to evaluate the diagnostic utility of prenatal diagnosis using the chromosomal microarray analysis (CMA) for fetuses presenting with isolated or associated intrauterine growth restriction (IUGR). Methods : First, regarding NIPS, a prospective cohort study including 2505 pregnant women from eight academic genetics laboratories (695 high risk pregnancies for trisomy 21 (risk ≥1/250 or with ultrasound anomalies) in a validation study, and 1810 such pregnancies, without ultrasound anomalies, in a real-life NIPS clinical setting) was conducted. An outcome was available for all cases in the validation cohort and for 521 in the clinical cohort. Cell-free DNA from plasma samples was sequenced using the Ion Proton sequencer, and sequencing data were analyzed using the open-access software, WISECONDOR. Performance metrics for detection 10 of trisomies 21, 18 and 13 were calculated based on either fetal karyotype result or clinical data collected at birth. We also evaluated the failure rate and compared three methods of fetal fraction quantification (RASSF1A assay, and DEFRAG and SANEFALCON software). Then, regarding the CMA study, we retrospectively included all fetuses with IUGR referred for prenatal testing and studied by rapid fluorescence in situ hybridization (FISH), karyotype, and CMA. Results :In the NIPS study, results from both cohorts were consistent and their gestational age was not significantly different, so their data were combined to increase the sample size for analysis. Sensitivities and specificities, respectively, were as follows: for trisomy 21, 98.3% (95% CI, 93.5–99.7%) and 99.9% (95% CI, 99.4–100%); for trisomy 18, 96.7% (95% CI, 80.9–99.8%) and 100% (95% CI, 99.6–100%); and for trisomy 13, 94.1% (95% CI, 69.2–99.7%) and 100% (95% CI, 99.6–100%). Our failure rate was 1.2% initially and as low as 0.6% after retesting some of the failed samples. Fetal fraction estimation by the RASSF1A assay was consistent with DEFRAG results, and both were adequate for routine diagnosis. Among the 162 IUGR fetuses (78 associated and 84 isolated IUGR) included in the CMA study, 15 had an abnormal FISH result: 10 associated and five isolated fetal IUGRs. Among the 143 fetuses studied by CMA, 10 (7%) presented pathogenic copy number variations (CNVs). All 10 were in the associated fetal IUGR group (10/65 or 15.4%; 95% confidence interval [CI]: 8.4%‐26.2%) versus 0/78 in the isolated fetal IUGR group (95% CI: 0%‐5.6%). Six fetuses (4.2%) carried variants of unknown significance (VOUS) (three associated and three isolated fetal IUGRs). Conclusion: We described one of the largest studies evaluating Ion Proton-based NIPS and the first clinical study reporting pregnancy outcome in a large series of patients. This platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be implemented easily in any medical genetics’ laboratory. Our second study highlighted the added value of CMA in the case of associated fetal IUGR with an incremental yield of 6.1% (4/65) over karyotyping. No pathogenic CNVs were reported in the isolated fetal IUGR group. Could NIPS supplant CMA in isolated fetal IUGR? The development of the NIPS test has reduced prenatal invasive testing and therefore its complications [...].

Les fentes labio-palatines (FLP) sont la malformation cranio-faciale congénitale la plus fréquente. D'origine multifactorielle, elles sont la conséquence d'un défaut de fusion des bourgeons faciaux.Objectif et MéthodologieL'objectif de ce travail a été d'étudier la place des gènes de l'angiogenèse dans le cadre de la piste étiologique des FL/P. La méthodologie de ce travail comportait 3 étapes :- Une analyse systématique et exhaustive des gènes impliqués dans les FL/P comprenant les gènes identifiés mais aussi les gènes potentiellement impliqués.- Une analyse rétrospective des explorations génétiques des FL/P opérées au CHU de Reims entre 2003 et 2009.- La mise en place d'une analyse prospective (2009-2012, AOL) :- Génomique constitutionnelle par CGH Array- In situ au niveau des berges des fentes issues de déchets opératoires des chirurgies primaires des FL/P comprenant :Le développement d'un protocole de culture cellulaire de fibroblastesUne analyse anatomopathologiqueEt surtout le développement d'un modèle d'étude in vivo chez l'enfant d'analyse d'expression des gènes de l'angiogenèse.RésultatsL'analyse systématique des gènes impliqués dans les FL/P a mis en évidence 95 gènes dont plus d'une dizaine sont connus comme liés aux mécanismes d'angiogenèse (facteurs de croissance et protéases). Ces derniers sont en interaction entre eux mais aussi avec 18 autres gènes impliqués eux aussi dans les FL/P. Ainsi au total 1/3 des gènes d'intérêt sont soit des gènes de l'angiogenèse soit en lien avec eux.L'étude rétrospective nous a permis de mettre en exergue certaines formes cliniques originales qui ont été étudiées et publiées sous un angle « étiologique ».L'étude prospective nous a permis, après obtention des consentements, d'inclure 72 patients (30 FLP, 24FL, 18FP) opérés au CHU de Reims entre 2009 et 2012 d'une chirurgie primaire.Nous présentons :- nos résultats anatomopathologiques, et génétiques (CGH Array)- notre protocole de culture cellulaire- nos réflexions, notre cheminement aboutissant à la création du modèle d'étude d'analyse d'expression des gènes de l'angiogenèseDiscussionLa littérature a bien mis en avant une implication des phénomènes angiogéniques dans la constitution des FL/P par le biais des facteurs de croissance (TGFβ, PDGF C et Ra, FGF, TGFA, FGFR1, FGFR2 ; VEGF) et des protéases (MMP/TIMP2). L'ensemble de nos manipulations in situ nous permet aujourd'hui de disposer du matériel nécessaire pour l'étude de l'expression des facteurs impliqués dans l'angiogenèse sur les berges des fentes.Parallèlement, l'étude génomique constitutionnelle en CGH Array a permis de retrouver des variations non-connues comme polymorphiques chez 62% de nos patients. Des études familiales vont compléter notre travail. Elles permettront de savoir si ces CNV sont héritées ou De Novo et ainsi de préciser leur caractère bénin ou pathologique. L'identification chez un de nos patients d'une amplification, même de petite taille, du gène SKI (gène lié à la voie des TGFβ) nous encourage dans la poursuite de nos recherches d'anomalies constitutionnelles des gènes de l'angiogenèse dans les FL/PLa CGH array est une technique qui nous a paru particulièrement utile et fiable en terme de « scanning » et de dépistage.En conclusion, en pratique clinique, la découverte des anomalies préalablement certainement sous estimées par les cliniciens doit nous mener à une nouvelle réflexion sur le conseil génétique et sur l'utilité dans l'avenir d'un dépistage plus systématique. / Cleft lip and palate (CLP) are the most common congenital craniofacial malformation. They have a multifactorial etiology and are the consequence of incomplete fusion of the facial buds.Objective and MethodologyThe objective of this work was to study the role of the genes of angiogenesis in the framework of studying the etiology of CL/P. Our methodological approach included 3 steps:- Systematic and thorough analysis of the genes involved in CL/P including identified genes but also genes that could be potentially involved.- A retrospective analysis of the operated clefts at the University Hospital of Reims between 2003 and 2009.- Implementation of a prospective analysis (2009-2012, AOL):- Constitutional genomic study by CGH Array- In situ with tissue specimens extracted from surgically excised cleft edges including:The development of a protocol for fibroblast cell culturesHistopathological analysisAnd above all the development of an in vivo study model in children for analyzing the expression of genes of angiogenesis.ResultsThe systemic analysis of genes involved in cleft lip palate unveiled 95 genes including about ten that are known to be related to angiogenesis mechanisms (growth factor and proteases). These genes interact between themselves but also with 18 other genes also involved in CL/P. In all, 1/3 of relevant genes are either angiogenesis-related genes or in direct relation with them.The retrospective study permitted to underline the some original clinical forms that were studied and published under an « etiological » angle.The prospective study included 72 patients (30 CLP, 24CL, 18CP), for whom we obtained informed signed consents, operated at the University Hospital of Reims between 2009 and 2012 for primary cleft surgery.We present:- Our histopathological and genetic results (CGH Array)- Our cell culture protocol (submitted for publication)- Our approaches and thought process behind the design of a study model for analyzing expression profiling of angiogenic genesDiscussionThe literature has highlighted the role of angiogenesis in the formation of cleft lip/palate via growth factors (TGFβ, PDGF C and Ra, FGF, TGFA, FGFR1, FGFR2, VEGF) and proteases (MMP/TIMP2). All our manipulations in situ have yielded the necessary material, i.e. edges of resected clefts, to study the expression of factors involved in cleft angiogenesis.In parallel, the constitutional genomic study in CGH Array enabled to uncover abnormalities in 62% of our patients. Family studies will complete our work. They will help to refine if these CNV are inherited or de novo and thus indicate their benign or pathological nature. In one of our patients, the identification of the SKI gene (related to the TGFβ pathway) encourages us to continue our research of genetic abnormalities of angiogenic genes involved in cleft lip/palate. CGH array appeared to be a very useful and reliable method in terms of scanning and screening.In conclusion, in clinical practice, the discovery of abnormalities which were probably underestimated by clinicians before, leads us to rethink the issue of genetic counseling and the relevance of a more systematic screening for these abnormalities in the future.