Poon Cho Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-91). / Abstract also in Chinese. / ACKNOWLEDGEMENT --- p.v / ABSTRACT --- p.vi / LIST OF ABBREVIATIONS --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.5 / Chapter 2.1 --- Epidemiology and aetiology of cervical cancer --- p.5 / Chapter 2.1.1 --- Incidence and mortality --- p.5 / Chapter 2.1.2 --- Aetiology --- p.6 / Chapter 2.1.2.1 --- Oral contraceptive pills and cervical cancer --- p.7 / Chapter 2.1.2.2 --- Human papilloma virus (HPV) and cervical cancer --- p.7 / Chapter 2.1.2.3 --- Immunity and cervical cancer --- p.8 / Chapter 2.1.2.4 --- Socio-economic differences and cervical cancer --- p.9 / Chapter 2.1.2.5 --- Smoking and cervical cancer --- p.9 / Chapter 2.1.2.6 --- Male role and cervical cancer --- p.9 / Chapter 2.1.2.7 --- Nutrition and cervical cancer --- p.10 / Chapter 2.2 --- Oncogenes and tumour suppressor genes --- p.10 / Chapter 2.2.1 --- Oncogene --- p.10 / Chapter 2.2.2 --- Tumour suppressor gene --- p.13 / Chapter 2.2.3 --- Alterations of oncogene in cervical cancer --- p.16 / Chapter 2.2.4 --- Alterations of tumour suppressor genes in cervical cancer --- p.18 / Chapter 2.3 --- Alterations of chromosome 1 in cervical cancer --- p.19 / Chapter 2.3.1 --- Cytogenetic tudy --- p.19 / Chapter 2.3.2 --- Molecular genetic study --- p.21 / Chapter 2.4 --- Loss of heterozygosity (LOH) --- p.21 / Chapter Chapter 3 --- Materials and methods --- p.24 / Chapter 3.1 --- Materials --- p.24 / Chapter 3.1.1 --- Patients --- p.24 / Chapter 3.1.2 --- Specimens --- p.24 / Chapter 3.1.2.1 --- Blood samples --- p.24 / Chapter 3.1.2.2 --- Tumour tissue specimens --- p.24 / Chapter 3.1.3 --- Chemicals and reagents --- p.25 / Chapter 3.1.3.1 --- Chemicals --- p.25 / Chapter 3.1.3.2 --- Reagents --- p.27 / Chapter 3.1.3.3 --- Markers --- p.29 / Chapter 3.1.4 --- Major equipment --- p.33 / Chapter 3.2 --- Methodology --- p.33 / Chapter 3.2.1 --- DNA extraction --- p.33 / Chapter 3.2.2 --- DNA amplification --- p.35 / Chapter 3.2.2.1 --- Validation of PCR primers and optimisation of PCR condition --- p.35 / Chapter 3.2.2.2 --- End labelling of the primer by (γ-32p)ATP --- p.35 / Chapter 3.2.2.3 --- PCR for LOH detection --- p.36 / Chapter 3.2.2.4 --- Electrophoresis --- p.37 / Chapter 3.2.2.5 --- Gel dry and radioautography --- p.38 / Chapter 3.2.2.6 --- PCR analysis of the D1S80 and D1S76 loci --- p.39 / Chapter 3.3 --- Determination of Loss of heterozygosity (LOH) --- p.39 / Chapter 3.4 --- Statistical analysis --- p.40 / Chapter Chapter 4 --- Results --- p.41 / Chapter 4.1 --- LOH analysis in cervical cancer --- p.41 / Chapter 4.2 --- LOH and age in cervical cancer --- p.60 / Chapter 4.3 --- LOH and pathological grade in cervical cancer --- p.62 / Chapter 4.4 --- LOH and clinical stage in cervical cancer --- p.64 / Chapter 4.5 --- LOH and clinical status in cervical cancer --- p.66 / Chapter Chapter 5 --- Discussion --- p.68 / Chapter 5.1 --- Microsatellite markers --- p.69 / Chapter 5.2 --- PCR condition --- p.70 / Chapter 5.3 --- LOH in cervical cancer --- p.72 / Chapter 5.4 --- Correlation of LOH with clinico-pathologic characteristics of cervical cancer --- p.76 / Chapter 5.4.1 --- LOH and age --- p.78 / Chapter 5.4.2 --- LOH and clinical stage --- p.78 / Chapter 5.4.3 --- LOH and pathologic grade --- p.79 / Chapter 5.4.4 --- LOH and clinical status --- p.79 / Chapter Chapter 6 --- Conclusion --- p.80 / Chapter Chapter 7 --- References --- p.83

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

Existe grande interesse na determinação das bases genéticas do carcinoma basocelular (CBC) que expliquem seu fenótipo pouco agressivo e comportamento metastático infreqüente. Investigamos a instabilidade de microssatélites (MSI) e perda de heterozigosidade (LOH) nos microssatélites D6S251 (6q14) e D6S252 (6q16) de CBCs esporádicos de alto e baixo risco histológico através da análise de bandas obtidas pelo gel de poliacrilamida após PCR em comparação com o tecido normal. Não houve alteração do microssatélite D6S252 nas 15 amostras estudadas. Para o microssatélite D6S251, houve alterações em 6 das 26 amostras estudadas (23,07%). MSI e LOH ocorreram em 46,15% das amostras de alto risco (respectivamente 15,38% e 30,76), o que sugere o provável envolvimento da região 6q14 na diferenciação histológica do CBC / A lot of interest lies in determining the genetic basis of basal cell carcinoma (BCC) to explain the lack of aggressive phenotype and infrequent metastatic behavior. We have analyzed the microsatellite instability (MSI) and loss of instability (LOH) in the D6S251 (6q14) and D6S252 (6q16) microsatellites patterns of histological low- high-risk sporadic BCC tumor samples using PCR-based assay in comparison with normal tissue. We have not found any alteration in D6S252 microsatellite 15 samples studied. We have encountered D6S251 alterations in 6 of 26 BCC samples (23.07%).MSI and LOH occurred in 46.15% of high-risk samples (15.38% and 30.76%), These results probably suggests participation of 6q14 region in histological differentiation of BCC

Aproximadamente 80% dos casos com Neoplasia endócrina múltipla tipo 1 (NEM1) possuem mutações germinativas no gene supressor de tumor MEN1, que os predispõem a tumores nas glândulas paratireóides, pâncreas endócrino e hipófise, além de outros tumores não endócrinos. A tumorigênese dos mais de 20 diferentes tipos de neoplasias já descritas na NEM1 ocorre pela presença da mutação germinativa MEN1 associadas a um segundo evento mutacional nas células desses tecidos, levando à perda de heterozigose (LOH) do locus do gene MEN1 (11q13) e à inativação da proteína supressora de tumor codificada por esse gene, a proteína MENIN. Recentemente, mutações germinativas em outros genes foram descritas em casos com NEM1 sem mutações no gene MEN1. Esses novos genes (CDKN1A, CDKN1B, CDNK2B e CDKN2C) codificam proteínas envolvidas no controle do ciclo celular (p21, p27, p15 e p18), chamadas proteínas inibidoras de quinases dependentes de ciclinas. Outro gene, chamado AIP, que codifica uma proteína chaperona de mesmo nome, também foi recentemente descrito associado à NEM1. Esses trabalhos descreveram o papel desses novos genes na NEM1, em nível germinativo, entretanto não esclareceu se esses novos genes estão inativados nos tumores de pacientes com NEM1 com mutação MEN1. O presente estudo investigou, pela primeira vez, o status somático do gene p27Kip1 em pacientes com mutação MEN1 e identificou quatro possíveis perda de heterozigose (LOH) em tumores de paratireóides e pâncreas, sugerindo que além de 11q13LOH, os tumores NEM1 podem sofrer raras perdas adicionais do gene supressor tumoral p27Kip1. Essas são as primeiras evidências na literatura de um processo de tumorigênese multi-step na NEM1, envolvendo três eventos genéticos: 1- Mutação germinativa MEN1; 2- 11q13-LOH; 3- Perda somática do gene supressor de tumor p27Kip1/CDKN1B / Approximately 80% of cases with multiple endocrine neoplasia type 1 (MEN1) harbor a germline mutation in the tumor suppressor gene MEN1, which predisposes these patients to tumors comprehending the parathyroid and pituitary glands, endocrine pancreas and others non-endocrine tumors. The tumorigenesis of the more than 20 different types of tumors already described in the MEN1 syndrome occurs due to a MEN1 germline mutation associated with a second mutational event in the cells of these tissues, leading to loss of heterozygosity (LOH) of the MEN1 gene locus (11q13) and therefore inactivation of tumor suppressor protein encoded by this gene, MENIN protein. Recently, germline mutations in other genes have been described in cases with MEN1 without any detectable mutations in the MEN1 gene. These novel genes (CDKN1A, CDKN1B, CDNK2B and CDKN2C) encode proteins involved in the control of the cell cycle (p21, p27, p15 and p18), called cyclin dependent kinases inhibitors. Another gene, called AIP, which encodes a chaperon protein with the same name, was recently described associated with MEN1 phenotypes. These data described a role for these novel genes in the germline level, however whether they are inactivated in tumors of patients with MEN1 mutation is so far not clarified. The present study investigated for the first time, the somatic status of p27KIP1 gene mutation in patients with MEN1 and identified four possible loss of heterozygosity (LOH) in tumors of the parathyroid and pancreas, suggesting that in addition 11q13-LOH, MEN1 tumors may suffer rare loss additional tumor suppressor gene p27KIP1. These are the first evidence in the literature of a process of tumorigenesis in MEN1 multi-step, involving three genetic events: 1-MEN1germlinemutation; 2-11q13LOH; 3-Loss of somatic tumor suppressor gene p27Kip1/CDKN1B

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune com forte componente genético, caracterizada por inflamação crônica e produção de autoanticorpos. O objetivo deste trabalho foi determinar o perfil de variação no número de cópias (CNVs) e de regiões de perda de heterozigose (LOH) na patogênese do LES. A detecção de CNVs e LOH foi feita pela metodologia Cytoscan HD array em pacientes com LES (n = 23) e indivíduos saudáveis (n = 110). Devido à formação tri-híbrida da população brasileira, foi desenvolvido e validado um painel de 345 marcadores informativos de ancestralidade, a partir dados provenientes do próprio array, para estimar as proporções de ancestralidade individual e, em última instância, inseri-las nos modelos de regressão logística como variável de controle nas análises de distribuição de CNVs e LOH. O perfil de CNVs evidenciou que o número e o tamanho de duplicações são maiores nos indivíduos saudáveis do que nos pacientes com LES. Duplicações nos genes FCGR3B e ADAM3A foram descritas como fator de proteção ao LES, quando tais genes foram avaliados por PCR quantitativa em maior grupo amostral de pacientes (n = 135) e controles (n = 200). Além disso, mostrou-se o efeito sinérgico da presença da deleção em ambos os loci FCGR3B e ADAM3A no aumento do risco para desenvolver a doença. Deleções em pacientes com LES envolvendo os genes CFHR4, CFHR5 e HLA-DPB2, previamente descritos em associação com o LES na literatura, foram identificadas por array e confirmadas por PCR digital. O protocolo desenvolvido para identificação de variantes raras, resultou em um conjunto de 21 CNVs raras em pacientes com LES. Em relação às regiões de perda de heterozigose, não foram encontradas evidências de que o número médio e a extensão dos segmentos LOH seja diferente entre pacientes e indivíduos saudáveis. No entanto, os cromossomos 6 e 12 em pacientes exibem regiões de perda de heterozigose em maior quantidade e tamanho do que os de indivíduos saudáveis, além de apresentarem 17 segmentos LOH restritos ao grupo de pacientes com LES. Os resultados aqui descritos evidenciam que novos loci de susceptibilidade ao LES podem ser encontrados quando a distribuição de CNVs é analisada em todo o genoma, em que a investigação de sua relação com a patogênese pode contribuir para a compreensão da base genética da doença. / Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic background characterized by chronic inflammation and autoantibody production. The purpose of this study was to determine the copy number variation (CNV) and loss of heterozygosity (LOH) profiles in the susceptibility to SLE. The detection of CNVs and LOH was performed by the Cytoscan HD array methodology in SLE patients (n = 23) and healthy subjects (n = 110). Due to the tri-hybrid composition of the Brazilian population, a panel of 345 ancestral informative markers was developed and validated, based on data from the array itself, to estimate the proportions of individual ancestry and, ultimately, to insert them into the logistic regression models as a control variable in the analysis of CNV and LOH distribution. The CNVs profile showed that the burden and the size of duplications are higher in healthy individuals than in SLE patients. Duplications in FCGR3B and ADAM3A genes were described as a protective factor for SLE, when these genes were evaluated by quantitative PCR in a larger SLE (n = 135) and control (n = 200) groups. In addition, the synergistic effect of the presence of deletion in both FCGR3B and ADAM3A loci increase the risk of developing the disease. Deletions in SLE patients encompassing the CFHR4, CFHR5 and HLA-DPB2 genes, previously described in the literature in association to SLE, were identified by the array and confirmed by droplet digital PCR. The pipeline developed here for the identification of rare variants resulted in a set of 21 rare CNVs in SLE patients. Regarding the loss of heterozygosity regions, no evidence was found that the mean number and extent of LOH segments is different between patients and healthy individuals. However, the chromosomes 6 and 12 in SLE patients exhibit greater quantity and size of LOH than those of healthy individuals, besides showing 17 LOH segments restricted to the group of SLE. The results described here show that novel susceptibility loci to SLE can be found once the distribution of variants is analyzed throughout the genome, in which the investigation of its relation to the pathogenesis may contribute to the understanding of the genetic basis of the disease.

No description available.

Hintergrund: Karzinoid-Tumoren des embryonalen Mitteldarms sind seltene intestinale neuroendokrine Tumoren, bei denen zum Zeitpunkt der Diagnose häufig Metastasen vorliegen. Im Gegensatz zu Karzinoiden des Vorderdarms und Respirationstraktes sind sie nicht mit der Multiplen Endokrinen Neoplasie Typ 1 (MEN1) vergesellschaftet. Die Mechanismen ihrer Tumorigenesis sind weitgehend unbekannt. Methoden: Tumorgewebe acht sporadischer, maligner Dünndarm-Karzinoide war Objekt dieser Studie über Verlust der Heterozygotie ("Loss Of Heterozygosity" (LOH)) mit 131 fluoreszierenden Mikrosatelliten. DNA Sequenz-Analyse mit Oligonucleotid Primern, die Exon 8-11 des SMAD4/DPC4 Gens flankieren sowie immunhistochemische Färbung mit Smad4/DPC4 antikörpern wurde durchgeführt. Ergebnis: Chromosom 18 wies Deletionen in 88% der Tumoren auf. Alle außer einem Tumor hatten sowohl 18p als auch 18q verloren, in einem der Tumoren war eine kleine Region telomer zu den SMAD4/DPC4/DCC Genen auf 18q21 verloren. Andere Chromosomen waren nur in drei Tumoren betroffen. LOH auf Chromosom 11q13, dem MEN1 Lokus, wurde nicht gefunden.Sequenzierung der DNA und immunhistochemische Färbung für das SMAD4/DPC4 Gen zeigten keine Aberrationen. Diskussion: Die Funde der Chromosom 18 Deletionen weisen eindeutig auf ein entscheidendes Ereignis in der Tumorigenese von Karzinoiden des Mitteldarms hin. An der Entstehung dieser Tumoren könnte ein mutmaßliches Tumor Suppressor Gen beteiligt sein, welches auf Chromosom 18 lokalisiert ist. Dahingegen ist SMAD4/DPC4 wahrscheinlich nicht in die Tumorneogenese von Carcinois Tumoren involviert. / Background: Midgut carcinoid tumors are rare malignant tumors with origin in the neuroendocrine cells of the small intestine. Due to secretion of a variety of peptide hormones and biogenic amines they cause the carcinoid syndrome. Metastases are often present at first diagnosis. Despite this, patients have a realistic chance to survive for a prolonged period (30% (unresectable/metastatic disease) -79% (non-metastatic disease) 5-year survival rate) if treated by a combination of surgery and medication. Unlike their foregut counterparts, midgut carcinoid tumors are not or rarely associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome. The genetic back-ground to tumorigenesis of these neoplasms is unknown. In contrast, the events involved in tumorigenesis of gastroenteropancreatic adenocarcinomas are better characterized with frequent mutations e.g. of the Smad4/DPC4, Smad2/MADR2/JV18-1 and DCC genes on chromosome 18. Methods: Eight metastatic midgut carcinoids were analysed by a genome-wide screening for loss of heterozygosity using 131 PCR-amplified fluorescent-labelled microsatellite markers. DNA sequence analysis using oligonucleotide primers flanking exons 8-11 of the Smad4/DPC4 gene and immunohistochemical staining with Smad4/DPC4 antibodies was performed. Results: Chromosome 18 was deleted in seven out of eight tumors (88%). All but one of these tumors had lost both 18p and 18q, the remaining tumor had lost the long arm but retained the short arm. Several other chromosomal alleles were lost in a subset of the tumors. Loss of heterozygosity (LOH) on chromosome 11q13, the MEN 1 locus, was not found. Smad4/DPC4 wild-type sequence and normal immunohistochemical staining for Smad4/DPC4 protein was found for all analysed tumors. Conclusions: Our finding of a high frequency of chromosome 18 deletions in 88% of the tumors strongly suggests that midgut carcinoid tumorigenesis might involve inactivation of a candidate tumor suppressor gene located in that region while Smad4/DPC4 is unlikely to be involved in that process. A more detailed analysis of the genetic events in midgut carcinoid tumors is warranted to clarify their neogenetic origin.

Le médulloblastome est une des tumeurs les plus fréquentes du système nerveux central chez l’enfant. Son impact clinique, ainsi que les effets secondaires engendrés par les traitements actuels, sont significatifs en matière de morbidité et de mortalité. La caractérisation moléculaire des tumeurs du système nerveux central a grandement évolué, et ce, particulièrement en ce qui concerne le médulloblastome. Des travaux antérieurs ont permis d’établir qu’un des sous-groupes de médulloblastome est caractérisé par l’activation de la voie sonic hedgehog. La mutation la plus fréquente menant à ce sous-type de médulloblastome est la mutation du gène suppresseur de tumeur PTCH1. Grâce au modèle de souris Ptch1+/-, des données issues de notre laboratoire ont permis de caractériser le développement de cette tumeur comme étant en deux étapes. Ce travail porte sur la caractérisation du mécanisme par lequel cette première étape, soit la perte d’hétérozygotie de Ptch1, survient. Tout d’abord, nous revisitons le rôle in vivo du corécepteur Boc dans la tumorigenèse. Selon nos résultats, la modulation de Boc ne semble pas avoir un impact significatif sur le développement tumoral dans des expériences de transplantation orthotopiques. Ensuite, nous démontrons que le ligand Shh augmente le dommage à l’ADN, ce qui mène à une hausse des évènements de recombinaisons qui peuvent causer une perte d’hétérozygotie. Nous tentons de moduler l’activité de Rad51 en observant une tendance non statistiquement significative des évènements de recombinaison avec des inhibiteurs de Rad51. Nous démontrons ensuite qu’un inhibiteur de Cdc7 permet la diminution des évènements de recombinaisons ainsi qu’une diminution du stress réplicatif de l’ADN. En intervenant sur le gène Mcm2 grâce à un modèle de souris transgénique, nous parvenons à prouver qu’une diminution de l’action de Mcm2 permet une diminution du stress réplicatif de l’ADN. En somme, la première étape du développement du médulloblastome sonic hedgehog-activé est la perte d’hétérozygotie de Ptch1. Celle-ci est caractérisée par une augmentation du dommage à l’ADN engendrant une hausse des évènements de recombinaison. Plusieurs cibles potentielles de modulation s’avèrent prometteuses pour un éventuel traitement ciblé. / Medulloblastoma is one of the most common central nervous system tumors of the child. Its clinical impact, as well as the adverse effects caused by current treatments, are significant in terms of morbidity and mortality. The molecular characterization of tumors of the central nervous system has greatly evolved, particularly in the case of medulloblastoma. Previous work has established that one of the medulloblastoma sub-groups is characterized by the activation of the sonic hedgehog (Shh) pathway. The most common mutation leading to this medulloblastoma subtype is the PTCH1 tumor suppressor gene mutation. Working with the Ptch1+/- mouse model, data from our la-boratory characterized the medulloblastoma tumorigenesis as a two-step process. This work focuses on the characterization of the mechanism by which this first step, the loss of heterozygosity of Ptch1, occurs. First, we revisit the in vivo role of the Boc coreceptor in the medulloblastoma tumor-igenesis. According to our results, Boc modulation does not seem to have a significant impact on tumor development. Next, we show that the Shh ligand increases DNA dam-age. This leads to an increase in recombination events which predispose to loss of het-erozygosity. We attempt to modulate Rad51 activity and observe a non-statistically sig-nificant trend to decrease recombination events with Rad51 inhibitors. We then demonstrate that Cdc7 inhibition reduces recombination events as well as DNA replica-tive stress. Using an Mcm2 transgenic mouse model, we demonstrate that a reduction in the action of Mcm2 reduces DNA replicative stress. To conclude, the first step in the development of Shh-activated medulloblastoma is the loss of heterozygosity of Ptch1. This is characterized by an increase in DNA damage leading to an increase in recombination events. Several potential modulation targets hold promise for possible targeted therapy.

Le caryotype moléculaire permet d’identifier un CNV chez 10-14% des individus atteints de déficience intellectuelle et/ou de malformations congénitales. C’est pourquoi il s’agit maintenant de l’analyse de première intention chez ces patients. Toutefois, le rendement diagnostique n’est pas aussi bien défini en contexte prénatal et l’identification de CNVs de signification clinique incertaine y est particulièrement problématique à cause du risque d’interruption de grossesse. Nous avons donc testé 49 fœtus avec malformations majeures et un caryotype conventionnel normal avec une micropuce CGH pangénomique, et obtenu un diagnostic dans 8,2% des cas. Par ailleurs, des micropuces à très haute résolution combinant le caryotype moléculaire et le génotypage de SNPs ont récemment été introduites sur le marché. En plus d’identifier les CNVs, ces plateformes détectent les LOHs, qui peuvent indiquer la présence d’une mutation homozygote ou de disomie uniparentale. Ces anomalies pouvant être associées à la déficience intellectuelle ou à des malformations, leur détection est particulièrement intéressante pour les patients dont le phénotype reste inexpliqué. Cependant, le rendement diagnostique de ces plateformes n’est pas confirmé, et l’utilité clinique réelle des LOHs n’est toujours pas établie. Nous avons donc testé 21 enfants atteints de déficience intellectuelle pour qui les méthodes standards d’analyse génétique n’avaient pas résulté en un diagnostic, et avons pu faire passer le rendement diagnostique de 14,3% à 28,6% grâce à l’information fournie par les LOHs. Cette étude démontre l’utilité clinique d’une micropuce CGH pangénomique chez des fœtus avec malformations, de même que celle d’une micropuce SNP chez des enfants avec déficience intellectuelle. / Molecular karyotyping identifies a CNV in 10-14% of individuals affected with intellectual disability and/or congenital abnormalities. Therefore, it is now the first-tier analysis for these patients. However, the diagnostic yield is not as clear in the prenatal context, and the risk of pregnancy termination makes the detection of variants of uncertain clinical significance particularly problematic. We tested 49 fetuses with major malformations and a normal karyotype, using a pangenomic CGH array, and obtained a diagnosis in 8.2% of cases. Furthermore, high-resolution microarrays combining molecular karyotyping and SNP genotyping were recently introduced on the market. In addition to identifying CNVs, these platforms detect LOHs, which can indicate the presence of a homozygous mutation or of uniparental disomy. Since these abnormalities can be associated with intellectual disability or congenital abnormalities, their detection is of particular interest for patients whose phenotype remains unexplained. However, the diagnostic yield obtained with these platforms is not confirmed, and the real clinical value of LOH detection is not yet established. We tested 21 children affected with intellectual disability for whom standard genetic analyses failed to provide a diagnosis, and were able to increase the diagnostic yield from 14.3% to 28.6% as a result of the information provided by LOHs. This study shows the clinical usefulness of pangenomic CGH arrays in fetuses with malformation(s), as well as that of SNP arrays in children with intellectual disability.