Aberrationen in der DNA-Kopienzahl sind häufige genetische Veränderungen bei malignen Lymphomerkrankungen. Zugewinne sowie Deletionen stellen dabei Mechanismen zur Onkogen-Aktivierung sowie Tumorsuppressorgen-Inaktivierung dar und tragen somit zur Pathogenese der Erkrankung bei. Array-CGH und SNP-Array sind Messplattformen, die die genomweite Bestimmung von Kopienzahlaberrationen in einem Experiment ermöglichen. Die bei der Analyse entstehenden Datensätze sind komplex und erfordern automatische Methoden zur Unterstützung der Analyse und Interpretation der Messergebnisse. In dieser Promotionsarbeit wurden Methoden entwickelt, welche die Analyse von Array-CGH- und SNP-Array-Messungen ermöglichen. Diese Methoden wurden für die Auswertung umfangreicher Datensätze von malignen Non-Hodgkin-Lymphomen verwendet. Dabei wurden Lymphome der Entitäten Burkitt-Lymphom, diffus großzelliges B-Zell-Lymphom, Mantelzelllymphom, primäres ZNS-Lymphom und peripheres T-Zell-Lymphom – nicht anderweitig spezifiziert – analysiert. Für die untersuchten Lymphom-Entitäten konnten hierbei zahlreiche neue rekurrente Kopienzahlaberrationen sowie uniparentale Disomien gezeigt werden, die neue Einblicke in die Pathogenese der jeweiligen Erkrankungen erlauben. Darüber hinaus erfolgte ein Vergleich beider Messplattformen anhand eines Datensatzes mit gepaarten Array-CGH- und SNP-Array-Daten. Für die eingesetzten Plattformen (2800k-BAC-Array vs. Affymetrix 250k-Sty-SNP-Array) konnte eine circa zwölffach höhere effektive Auflösung der SNP-Array-Plattform gezeigt werden. Die wesentlichen Ergebnisse dieser Arbeit sind in sieben Publikationen eingeflossen.:Inhaltsverzeichnis Abkürzungsverzeichnis Tabellenverzeichnis Abbildungsverzeichnis 1. Einführung 1.1 Biologischer Hintergrund 1.1.1 Aberrationen der DNA-Kopienzahl und Tumorentstehung 1.1.2 Lymphome 1.2 Motivation und Rationale für die Arbeit 1.3 Array-CGH Analyse 1.4 SNP-Array-Analyse 1.5 Vergleich von Array-CGH und SNP-Array-Analyse 1.6 Assoziationen von DNA-Kopienzahlaberrationen mit RNA-Expression, Lymphomentität sowie klinischen und phänotypischen Faktoren 2.Publikationen 2.1 Publikation 1: “Development and implementation of an analysis tool for array-based comparative genomic hybridization” Methods Inf Med. 2007;46(5):608-13 2.2 Publikation 2: “Recurrent loss of the Y chromosome and homozygous deletions within the pseudoautosomal region 1: association with male predominance in mantle cell lymphoma” Haematologica. 2008 Jun;93(6):949-50 2.3 Publikation 3: “GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma” Br J Haematol. 2009 Feb;144(3):317-31 2.4 Publikation 4: “Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma.” Leukemia. 2009 Oct;23(10):1875-84 2.5 Publikation 5: “High resolution SNP array genomic profiling of peripheral T cell lymphomas, not otherwise specified, identifies a subgroup with chromosomal aberrations affecting the REL locus” Br J Haematol. 2010 Feb;148(3):402-12 2.6 Publikation 6: “Detection of genomic aberrations in molecularly defined Burkitt\''s lymphoma by array-based, high resolution, single nucleotide polymorphism analysis” Haematologica. 2010 Dec;95(12):2047-55 2.7 Publikation 7: “Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma” Blood. 2012 Feb 23;119(8):1882-7 2.8 Kennzeichnung des Eigenanteils für alle eingeschlossenen Publikationen 3. Diskussion und Ausblick 4. Zusammenfassung 5. Referenzen 6. Eigene Publikationen 7. Erklärung 8. Danksagung 9. Curriculum vitae

Deficiência intelectual é uma condição heterogênea e complexa, diagnosticada em 1-3% da população mundial. Desequilíbrios cromossômicos e variações no número de cópias (CNVs) são as causas mais frequentes de DI e, até recentemente, a maior parte desse desequilíbrio era averiguado por análises citogenéticas convencionais. Antes da utilização de microarrays cromossômicos (CMA), a causa etiológica da DI ainda permanecia desconhecida em ~60% dos pacientes. A aplicação de CMA tem revolucionado o diagnóstico da DI e de muitas outras doenças congênitas, permitindo explicar a etiologia molecular de parte da DI através da identificação de CNVs patogênicas. Nos países desenvolvidos, CMA é considerado como primeiro teste para avaliar pacientes com múltiplas anomalias congênitas, DI e/ou autismo. Contudo, nos países em desenvolvimento, a detecção de alterações ainda é feita principalmente por métodos citogenéticos convencionais. O objetivo desse estudo foi identificar, através do uso de SNP arrays, o espectro de anomalias cromossômicas presente em uma amostra de 40 pacientes com DI idiopática moderada e grave, apresentando ou não aspectos dismórficos e anomalias congênitas. Em especial, essa coorte de pacientes, em sua maioria (~2/3), não havia sido previamente cariotipada. Embora mundialmente desde 2010 a recomendação seja de realizar arrays antes de cariótipo, a maioria dos pacientes relatados em estudos já havia sido cariotipada antes de array ser oferecido a eles como teste. Foram identificadas alterações raras em 18 pacientes (45%). Em 12 (30%) desses Pacientes, as CNVs eram sabidamente patogênicas; esta taxa diagnóstica está muito acima da taxa de detecção reportada na literatura (~20%) e possíveis causas desta discrepância são discutidas. Outros 6 Pacientes (15%) apresentaram variantes raras de significado incerto (variants of unknown significance - VUS). Um aspecto adicional investigado foram os mecanismos envolvidos na formação de alguns dos rearranjos estruturais; enquanto nosso foco inicial era o uso de arrays para detecção de CNVs, se tornou evidente no decorrer do projeto que o padrão dos SNPs obtido nos arrays revelava, a partir do DNA, informação valiosa sobre a estrutura dos cromossomos e a composição heterogênea de células em uma amostra (mosaicismo). Esses resultados são discutidos em detalhes em duas situações: (1) A descrição de uma deleção terminal 1p36, associada a dissomia uniparental (UPD) em mosaico de segmentos de 1pter de diferentes tamanhos. Sugerimos que essa composição reflita eventos recorrentes de captura de telômero, embora processo similar nunca tenha sido descrito, e propomos um possível mecanismo responsável por originar esse desequilíbrio complexo. (2) Três dos nossos pacientes apresentam 4 cópias ou uma combinação de 3-4 cópias de segmentos proximais, na maior parte superpostos, de 15q11q13. Possíveis mecanismos de origem desses rearranjos são discutidos / Intellectual disability (ID) is a complex and heterogeneous condition affecting about 1-3% of the general population. Chromosomal imbalances and copy-number variations (CNVs) have been recognized as the most frequent causes of ID and, until recently, most of these imbalances were diagnosed by cytogenetic analysis. Before the application of microarray analysis (CMA), the underlying cause of ID remains unknown in ~60% of patients. The use of CMA has revolutionized the diagnosis of ID and several other congenital disorders, and have made it possible to identify pathogenic CNVs that could explain the molecular etiology of ID. In developed countries, CMA is considered the first-tier technique for the analysis of patients with multiple congenital anomalies, ID, and/or autism spectrum disorders. However, in developing nations, detection of alterations is still performed mainly by conventional cytogenetic techniques. The aim of this study was identifying, using a high-density resolution SNP microarray, chromosomal imbalances in a total of 40 patients presented with moderate-to-severe ID, associated or not with dysmorphic features and congenital anomalies. Particularly, most of the patients in the cohort (~2/3) was not karyotyped previously. Although CMA has been recommended as the first-tier test since 2010 all over the world, the majority of the patients in the reported studies were karyotyped before CMA was offered as a diagnostic test. Rare CNVs were detected in 18 patients (45%). Among those patients, 12 (30%) carried pathogenic CNVs. This yield is much higher than reported in the literature (~20%), and possible causes for this discrepancy are discussed. Six patients (15%) carried variant of unknown significance (VUS). Furthermore, mechanisms involved in structural rearrangements found in some patients were investigated. Even though the main focus of this dissertation was the detection of CNVs using high resolution SNP arrays, throughout the course of this project it was clear that the SNP patterns found could reveal crucial information about the structure of chromosomes and the heterogeneous composition of cells (mosaicism). Those results are discussed in detail in two situations: (1) One description of a terminal 1p36 deletion, associated with mosaic uniparental disomy (UPD) of different sized 1pter segments. We hypothesized that this composition reflects recurrent telomere capture events, although a similar process has never been described so far, and proposed a possible mechanism responsible for originating this complex imbalance. (2) Three of our patients carried four copies or a four-three copies-combination of a proximal, partially overlapping, 15q11q13 segment. Possible mechanisms responsible for this complex rearrangement are discussed

Head and neck squamous cell carcinomas (HNSCCs) most commonly develop in older patients (≥60 years of age) with a history of tobacco and alcohol use. However, young individuals (≤45 years of age) can also develop HNSCC, often without common risk factors. Increasing evidence shows that Human Papillomavirus (HPV) infection is associated with particular HNSCC sites (e.g. oropharynx). We assessed the Roche Linear Array HPV Genotyping Test in several lesions and then examined the prevalence of HPV in HNSCCs from young and older patients. HPV infection was most prevalent in oropharyngeal cancers (16/22, 73%), rarely found in oral cavity cancers (2/53, 4%), and other head and neck sites (1/17, 6%). HPV positive tumors were associated with patients that were >40 and <60 years old (p=0.02). The absence or shortened time of carcinogen exposure from common risk factors and the development of oral squamous cell carcinoma (OSCC) at an early age suggest aberrant genetic events that are different than those in OSSCs from older patients. We used Affymetrix SNP 6.0 arrays to genomically profile oral tumors from young and older patients. Tumors from young patients showed different regions/genes of copy number alterations than those from older patient tumors. An increase of regions of loss of heterozygosity (LOH) in tumors from older patients was observed, and there was a high prevalence of copy number neutral LOH on chromosome 9 in tumors from young and older patients. These data suggest different genetic mechanisms in these patient groups. We have previously shown that HNSCCs from younger patients exhibited a high incidence of microsatellite instability (MSI), a marker of defective mismatch repair (MMR). Deregulated mRNA levels of hPMS1, hPMS2 and hMLH1 were observed and absent/low expression of hPMS1, hPMS2 and hMLH1 protein levels were observed in >50% of OSCCs. No mutations were observed in hPMS1 and hPMS2 and no significant differences of MSI or LOH were observed across genomic loci between tumors of young and older patients. The role of these genetic mechanisms in oral cancer appears complex; studies such as ours should further improve our knowledge of the molecular mechanisms leading to early-onset oral carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) most commonly develop in older patients (≥60 years of age) with a history of tobacco and alcohol use. However, young individuals (≤45 years of age) can also develop HNSCC, often without common risk factors. Increasing evidence shows that Human Papillomavirus (HPV) infection is associated with particular HNSCC sites (e.g. oropharynx). We assessed the Roche Linear Array HPV Genotyping Test in several lesions and then examined the prevalence of HPV in HNSCCs from young and older patients. HPV infection was most prevalent in oropharyngeal cancers (16/22, 73%), rarely found in oral cavity cancers (2/53, 4%), and other head and neck sites (1/17, 6%). HPV positive tumors were associated with patients that were >40 and <60 years old (p=0.02). The absence or shortened time of carcinogen exposure from common risk factors and the development of oral squamous cell carcinoma (OSCC) at an early age suggest aberrant genetic events that are different than those in OSSCs from older patients. We used Affymetrix SNP 6.0 arrays to genomically profile oral tumors from young and older patients. Tumors from young patients showed different regions/genes of copy number alterations than those from older patient tumors. An increase of regions of loss of heterozygosity (LOH) in tumors from older patients was observed, and there was a high prevalence of copy number neutral LOH on chromosome 9 in tumors from young and older patients. These data suggest different genetic mechanisms in these patient groups. We have previously shown that HNSCCs from younger patients exhibited a high incidence of microsatellite instability (MSI), a marker of defective mismatch repair (MMR). Deregulated mRNA levels of hPMS1, hPMS2 and hMLH1 were observed and absent/low expression of hPMS1, hPMS2 and hMLH1 protein levels were observed in >50% of OSCCs. No mutations were observed in hPMS1 and hPMS2 and no significant differences of MSI or LOH were observed across genomic loci between tumors of young and older patients. The role of these genetic mechanisms in oral cancer appears complex; studies such as ours should further improve our knowledge of the molecular mechanisms leading to early-onset oral carcinomas.

Nephroblastoma is the most prevalent pediatric kidney tumor, which occurs primarily in younger children with the average age at diagnosis of 42,5 months for girls and 36,5 months for boys. Even though its treatment is currently very succesful and the overall survival rate reaches over 90 %, there are still more things to be discovered and improved. An important role for the right choice of treatment plays not only the histology of tumor, but also the chromosomal changes present at tumor. Some of them (for example 1q gain, simultaneous deletion of 1p and 16q, TP53 deletion) were confirmed as negative prognostic markers because they are associated with an increased risk of relapse or with anaplastic type of nephroblastoma that is included in a high risk group. These changes are therefore used together with the tumor histology for stratification of nephroblastomas. Some of these changes were found in a heterogeneous state (only in a part of the cells) in nephroblastoma, which also complicates the treatment of the patient and which cannot be solved when only one sample is taken from the tumor. In this work we concentrated on the detection of chromosomal changes present in nephroblastomas of 44 patients and their associations with clinical data. We have proved some of the known associations (22q...

Deficiência intelectual é uma condição heterogênea e complexa, diagnosticada em 1-3% da população mundial. Desequilíbrios cromossômicos e variações no número de cópias (CNVs) são as causas mais frequentes de DI e, até recentemente, a maior parte desse desequilíbrio era averiguado por análises citogenéticas convencionais. Antes da utilização de microarrays cromossômicos (CMA), a causa etiológica da DI ainda permanecia desconhecida em ~60% dos pacientes. A aplicação de CMA tem revolucionado o diagnóstico da DI e de muitas outras doenças congênitas, permitindo explicar a etiologia molecular de parte da DI através da identificação de CNVs patogênicas. Nos países desenvolvidos, CMA é considerado como primeiro teste para avaliar pacientes com múltiplas anomalias congênitas, DI e/ou autismo. Contudo, nos países em desenvolvimento, a detecção de alterações ainda é feita principalmente por métodos citogenéticos convencionais. O objetivo desse estudo foi identificar, através do uso de SNP arrays, o espectro de anomalias cromossômicas presente em uma amostra de 40 pacientes com DI idiopática moderada e grave, apresentando ou não aspectos dismórficos e anomalias congênitas. Em especial, essa coorte de pacientes, em sua maioria (~2/3), não havia sido previamente cariotipada. Embora mundialmente desde 2010 a recomendação seja de realizar arrays antes de cariótipo, a maioria dos pacientes relatados em estudos já havia sido cariotipada antes de array ser oferecido a eles como teste. Foram identificadas alterações raras em 18 pacientes (45%). Em 12 (30%) desses Pacientes, as CNVs eram sabidamente patogênicas; esta taxa diagnóstica está muito acima da taxa de detecção reportada na literatura (~20%) e possíveis causas desta discrepância são discutidas. Outros 6 Pacientes (15%) apresentaram variantes raras de significado incerto (variants of unknown significance - VUS). Um aspecto adicional investigado foram os mecanismos envolvidos na formação de alguns dos rearranjos estruturais; enquanto nosso foco inicial era o uso de arrays para detecção de CNVs, se tornou evidente no decorrer do projeto que o padrão dos SNPs obtido nos arrays revelava, a partir do DNA, informação valiosa sobre a estrutura dos cromossomos e a composição heterogênea de células em uma amostra (mosaicismo). Esses resultados são discutidos em detalhes em duas situações: (1) A descrição de uma deleção terminal 1p36, associada a dissomia uniparental (UPD) em mosaico de segmentos de 1pter de diferentes tamanhos. Sugerimos que essa composição reflita eventos recorrentes de captura de telômero, embora processo similar nunca tenha sido descrito, e propomos um possível mecanismo responsável por originar esse desequilíbrio complexo. (2) Três dos nossos pacientes apresentam 4 cópias ou uma combinação de 3-4 cópias de segmentos proximais, na maior parte superpostos, de 15q11q13. Possíveis mecanismos de origem desses rearranjos são discutidos / Intellectual disability (ID) is a complex and heterogeneous condition affecting about 1-3% of the general population. Chromosomal imbalances and copy-number variations (CNVs) have been recognized as the most frequent causes of ID and, until recently, most of these imbalances were diagnosed by cytogenetic analysis. Before the application of microarray analysis (CMA), the underlying cause of ID remains unknown in ~60% of patients. The use of CMA has revolutionized the diagnosis of ID and several other congenital disorders, and have made it possible to identify pathogenic CNVs that could explain the molecular etiology of ID. In developed countries, CMA is considered the first-tier technique for the analysis of patients with multiple congenital anomalies, ID, and/or autism spectrum disorders. However, in developing nations, detection of alterations is still performed mainly by conventional cytogenetic techniques. The aim of this study was identifying, using a high-density resolution SNP microarray, chromosomal imbalances in a total of 40 patients presented with moderate-to-severe ID, associated or not with dysmorphic features and congenital anomalies. Particularly, most of the patients in the cohort (~2/3) was not karyotyped previously. Although CMA has been recommended as the first-tier test since 2010 all over the world, the majority of the patients in the reported studies were karyotyped before CMA was offered as a diagnostic test. Rare CNVs were detected in 18 patients (45%). Among those patients, 12 (30%) carried pathogenic CNVs. This yield is much higher than reported in the literature (~20%), and possible causes for this discrepancy are discussed. Six patients (15%) carried variant of unknown significance (VUS). Furthermore, mechanisms involved in structural rearrangements found in some patients were investigated. Even though the main focus of this dissertation was the detection of CNVs using high resolution SNP arrays, throughout the course of this project it was clear that the SNP patterns found could reveal crucial information about the structure of chromosomes and the heterogeneous composition of cells (mosaicism). Those results are discussed in detail in two situations: (1) One description of a terminal 1p36 deletion, associated with mosaic uniparental disomy (UPD) of different sized 1pter segments. We hypothesized that this composition reflects recurrent telomere capture events, although a similar process has never been described so far, and proposed a possible mechanism responsible for originating this complex imbalance. (2) Three of our patients carried four copies or a four-three copies-combination of a proximal, partially overlapping, 15q11q13 segment. Possible mechanisms responsible for this complex rearrangement are discussed

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, &ldquo;The GeneChip® Mapping 500K Set&rdquo; 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 &ldquo;The GeneChip® Mapping 500K Set&rdquo; (Affymetrix), and the array-CGH platform &ldquo;CytoSure ISCA 4x180k (OGT)&rdquo;, 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

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, &ldquo;The GeneChip® Mapping 500K Set&rdquo; 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 &ldquo;The GeneChip® Mapping 500K Set&rdquo; (Affymetrix), and the array-CGH platform &ldquo;CytoSure ISCA 4x180k (OGT)&rdquo;, 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

Les syndromes myélodysplasiques (SMD) sont des hémopathies myéloïdes clonales évoluant vers une leucémie aiguë (LA). Les SMD et LA secondaires, survenant après traitement par chimiothérapie et/ou radiothérapie, ont un pronostic très péjoratif. Cependant seule une partie des sujets exposés aux traitements cytotoxiques développent un SMD secondaire, ce qui suggère une composante génétique dans la susceptibilité individuelle au risque de développer un SMD secondaire. Les objectifs de ce travail ont été d’identifier des polymorphismes génétiques de type SNP (Single Nucleotide Polymorphism) significativement associés à des caractères cliniques et biologiques des SMD tel leur caractère secondaire. Une « puce » à façon a sélectionné 384 SNP de fréquence allélique supérieure à 10 % impliqués dans la réparation de l’ADN, le métabolisme, le transport et la détoxication des xénobiotiques. L’ADN constitutionnel de 65 patients atteints de SMD primaire et secondaire a été recueilli et génotypé pour ces 384 SNP. La seule association significative par test exact de Fisher (p = 0,009 après correction de Benjamini-Hochberg) a été observée pour le caractère secondaire des SMD et la présence de l’allèle variant de MGMT (MéthylGuanine MéthylTransférase) sur deux SNP en déséquilibre de liaison, rs2308321 (Ile143Val) et rs2308327 (Lys178Arg). Nous avons recherché le caractère prédictif de la présence de l’allèle variant de MGMT pour le risque de SMD/LA secondaire chez des patientes ayant reçu un traitement cytotoxique pour un cancer du sein, et ayant développé une LA secondaire. Enfin, nous avons construit des lignées cellulaires stables, isogéniques, exprimant soit la forme sauvage soit la forme variante de MGMT. Les études fonctionnelles par tests de cytotoxicité, co-cultures à long terme et étude des demi-vies des protéines, sous traitement alkylant, montrent respectivement des différences de sensibilité, de prolifération ou de dégradation entre les formes variante et sauvage de MGMT. / Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders evolving toward acute myeloid leukaemia (AML). Therapy-related MDS and AML occur after chemo- and/or radiotherapy for previous cancer and have a very poor outcome. However, only a minimal proportion of patients exposed to anticancer drugs develop secondary MDS, suggesting a genetic component in individual susceptibility. The aim of our study was to identify gene polymorphisms significantly associated with MDS in patients. We have selected 384 SNPs (single nucleotide polymorphisms) with allele frequency >10% in genes involved in DNA repair and drug metabolism and transport. DNA extracts were obtained from blood and cheek samples from a population of 65 MDS patients, and the 384 SNPs were genotyped. We analysed the associations existing between each genotype and several pathological features, especially the treatment-related character of MDS. The Fisher exact test with Benjamini-Hochberg correction for multiple testing was applied for statistical analysis. The only significant association (p = 0.009 after correction) was observed for the treatment-related character of MDS and the presence of a variant allele in MGMT (methylguanine methyltransferase, a gene involved in DNA repair), characterised by two SNPs in complete linkage disequilibrium: rs2308321 (Ile143Val) and rs2308327 (Lys178Arg). An epidemiological study was performed to assess the predictive value of the variant allele in MGMT for the development of secondary acute leukaemia among patients treated for breast cancer. We have constructed isogenic stable cell lines expressing either the wild-type or the variant allele of MGMT. Functional studies (analysis of response to alkylating agents, allele quantification of mixed cultures of wild-type and variant cells and half-lives study of the proteins) show differences in sensitivity to DNA-damaging agents, proliferative capacity and MGMT rate of degradation between the wild-type and the variant MGMT.

5 à 10% des cancers du sein sont héréditaires mais parmi ceux-ci seulement la moitié est expliquée par une altération constitutionnelle d’un gène de prédisposition connu tels que les gènes BRCA1 et BRCA2. L’importante hétérogénéité génétique qui caractérise les famillesBRCAx rend difficile la réalisation d’études familiales groupées et ne permet pas l’identification de nouveaux gènes de prédisposition au cancer du sein selon les méthodes classiques de liaison génétique ou d’association. Les techniques de séquençage de nouvelle génération (NGS) à l’échelle de l’exome ou du génome entier, autorisent en revanche l’étude de familles individuelles à la recherche de mutations constitutionnelles privées mais le nombre considérable de variants génétiques identifiés impose leur tri sur des critères de pathogénicité ou de récurrence. Un autre critère de tri peut être représenté par l’identification de régions candidates définies en fonction de réarrangements génomiques tumoraux communs à plusieurs tumeurs au sein d’une même famille. Le génotypage tumoral par puces SNP (pour single nucleotide polymorphism) permet en effet la détection d’haplotypes conservés dans des régions récurrentes de LOH (pour loss of heterozygosity) communes à plusieurs tumeurs familiales et donc l’identification de régions candidates suspectes d’abriter des mutations germinales dans des gènes de prédisposition au cancer. La combinaison de ces deux approches, génotypage tumoral puis NGS, a été appliquée à une série de 17 familles avec agrégation de cancers du sein pour lesquelles au moins deux échantillons tumoraux étaient disponibles. Aucun nouveau gène de prédisposition au cancer du sein n’a été identifié mais une mutation délétère constitutionnelle du gène ATM a ainsi été retrouvée, associée à une perte de l’allèle sauvage dans les 2 tumeurs d’une famille BRCAx. L’analyse de 17 tumeurs du sein supplémentaires provenant de 10 familles avec agrégation de cancers du sein et mutation constitutionnelle du gène ATM identifiée chez le cas index, a révélé que l’allèle sauvage d’ATM était fréquemment perdu dans ces tumeurs (>80% contre 20% attendu en situation sporadique ; p<0.001). Ce résultat plaide fortement en faveur de l’implication d’ATM dans la carcinogénèse de ces cancers du sein tel un gène suppresseur de tumeur et suggère que les mutations constitutionnelles d’ATM sont impliquées dans des formes familiales de cancer du sein. / Hereditary breast cancers (BCs) account for 5-10% of all diagnosed BCs, yet only 50% of such tumors arise in the context of a germline mutation in known tumor suppressor genes such as BRCA1 or BRCA2. The vast genetic heterogeneity which characterizes BRCAx families makes grouped studies impossible to perform. Next generation sequencing (NGS) techniques, however, allow individual families to be studied in order to identify private mutations. Single nucleotide polymorphism (SNP) arrays allow the detection of conserved haplotypes within recurrent regions of loss of heterozygosity, common to several familial tumors, therefore identifying genomic loci likely to harbor a germline mutation in cancer predisposition genes. The combination of both exome sequencing and SNP arrays for a series of 17 familial BC did not allow the identification of a novel BC predisposition gene, but revealed a germline ATM mutation associated with a loss of the wild-type allele in a BRCAx family. The analysis of 17 additional breast tumors from ten BC families in which a germline ATM mutation had been identified revealed a high frequency of wild-type allele loss in these tumors (>80% compared to the 20% expected in sporadic BC; p <0.001). This result argues strongly in favor of the involvement of ATM in the carcinogenesis of these tumors as a tumor suppressor gene and suggests that germline ATM mutations are involved in a subset of familial BC.