Le cancer de l’ovaire est le cancer gynécologique avec la plus grande mortalité due à un diagnostique tardif au stade de maladie extensive péritonéale. Malgré les progrès de la chirurgie radicale et de la chimiothérapie les récurrences abdominales demeurent la cause la plus fréquente de mortalité. Il existe peu d’études de la maladie métastatique péritonéale. Notre hypothèse de travail est que les différences entre la maladie métastatique et la tumeur primaire sont primordiales dans la survenue d’une maladie résiduelle ou récurrente. Nous avons utilisé une approche exhaustive comprenant des études du transcriptome, des variations du nombre de copie (VNC) et des sequençages des exomes pour caractériser les différences entre lésions primaires, métastases péritonéales et métastases lymphatiques.Résultats: Notre étude démontre que les VNC varient de façon significative entre la tumeur primaire et la métastase peritonéale. Les différences d’expressions géniques bien que mineures permettent de retrouver les voies de signalisation primordiales pour le développement des métastases. Le séquençage des exomes montre très peu de différences en terme de polymorphisme. Par ailleurs la majorité des polymorphismes présents dans les métastases se retrouvent à une faible fréquence dans la tumeur primaire de façon concordante avec la théorie clonale. Conclusion: L’ensemble des résultats montre la possibilité d’une origine clonale de la maladie métastatique des cancers de l’ovaire comportant la majorité des anomalies au niveau des variations du nombre de copie. L’intégration de ces données permettrait d’optimiser les thérapeutiques ciblées. / Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. There are few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. Our hypothesis is that differences between the metastatic and primary lesions might be the cause of residual disease and, most importantly may have a role in post-chemotherapeutic recurrences. Methods: We conducted integrated genomics analysis on matched primary and metastatic tumors from 9 patients. In the papers presented here we analyze genome-wide Copy Number Variations (CNVs) using SNP Arrays targeting peritoneal metastasis differences, Gene expression differences using Microarrays also targeting peritoneal metastasis differences, and for some patients, Single Nucleotide Polymorphisms (SNPs) in genes through Exome sequencing.Results: Here we show that CNVs vary significantly between primary and metastatic tumors and include genes that have been considered potential chemotherapeutic targets based on primary tumor only data. Gene expression differences, while minor, showed highly statistically significant enrichment of genes in ovarian cancer critical pathways. In agreement with findings in other cancers, exome sequencing data revealed very few SNP differences of which most metastasis enriched SNPs were present at very low levels in the primary tumor. The results presented here should allow better design of therapies to target residual ovarian cancer disease.

Os transtornos do espectro autista (TEA) são condições neuropsiquiátricas caracterizadas por padrões comportamentais estereotipados, ausência ou limitação de comunicação verbal e de interação social recíproca. Diversos estudos têm mostrado que esses transtornos possuem etiologia genética complexa e heterogênea, o que dificulta a identificação dos fatores causais. Estima-se que cerca de 70% dos casos de TEA são idiopáticos. Portanto, com o objetivo de identificar mecanismos etiológicos associados aos TEA, utilizamos as seguintes estratégias: customização de uma lâmina de microarray CGH que possibilite a detecção não só de grandes CNVs, mas também de alterações menores do que 10 kbp, em exons e regiões UTR de genes potencialmente candidatos; a comparação entre os tipos de rearranjos detectados em pacientes sindrômicos e em não sindrômicos e, ainda, a investigação mais detalhada de uma família com indivíduos portadores de transtorno autista e síndrome de Asperger. Foram avaliados 103 portadores de TEA não sindrômicos e 18 sindrômicos, sendo as taxas de detecção de alterações potencialmente patogênicas, respectivamente, de 11,6% e 38,9%. Dentre as alterações detectadas 44,4% são menores do que 10 Kbp. Portanto, a estratégia de usar uma lâmina customizada, com alta densidade de sondas complementares aos exons e regiões não codificantes de genes potencialmente envolvidos na etiologia dos TEA, capaz de detectar tanto alterações grandes quanto pequenas, parece ser relevante na tentativa de elucidar o maior número de casos possíveis e melhor compreender esses transtornos. Além disso, essa lâmina também pode ser utilizada como uma ferramenta para auxiliar o diagnóstico clínico e o aconselhamento genético com um custo mais acessível em comparação a outras comerciais ou ao sequenciamento de última geração. Cerca de 33,3% das CNVs observadas afetam região UTR, sugerindo que mutações nessas regiões podem explicar uma proporção significativa dos casos nos quais não são detectadas alterações através de outros testes genômicos, visto que a maioria desses ainda não exploram adequadamente regiões não codificantes. Entre os pacientes autistas não sindrômicos verificou-se que a maioria dos genes afetados por CNVs estão envolvidos em duas principais funções biológicas - sinapses glutamatérgicas e orientação axonal, sugerindo que TEA não sindrômico pode ser causado por disfunção em genes diferentes envolvidos em processos fisiológicos comuns. Diferente do que observamos entre pacientes não sindrômicos, detectamos mais de uma alteração em um mesmo indivíduo ou alterações que englobam mais de um gene entre os pacientes sindrômicos, reforçando o modelo oligogênico para alguns casos de TEA. Por fim, os dados obtidos no estudo da família com portadores de síndrome de Asperger e transtorno autista sugere que a gravidade do quadro clínico possa estar relacionada ao número de mutações e possivelmente por duas mutações diferentes em ambos os alelos de um mesmo gene. Nossos resultados, além de apoiar o envolvimento dos genes MDGA2, FHIT, HTR2A, SHANK2, GRIA3, ZNF778, PRKCα, CDH15, DIAPH3, GCH1, GRM5, MARK1, SLC17A6, IMMP2L, BZRAP1, SYNGAP1, ANK3, MAP1A, GABRR2 e LAMC3 nos TEA também sugere novos genes candidatos: LRRC7, LRRIQ3, CADPS1, NUFIP, SEMA3A, SNAP29, MBD2, GAD2, DGKH e PARD3 / The autism spectrum disorders (ASD) are neuropsychiatric conditions typically characterized by social deficits, communication difficulties, stereotyped or repetitive behaviors and interests. Several studies have shown that these disorders have a complex and heterogeneous genetic etiology, which makes difficult to identify the causal factors. Approximately 70% of cases are idiopathic. In order to identify etiological mechanisms associated with ASD, we have used the following strategies: customized a microarray CGH platform that allows detection not only of large CNVs, but also alterations smaller than 10 kbp in exons and UTR regions of potential candidate genes, the comparison between the types of rearrangements detected in syndromic and non-syndromic patients and further, more detailed investigation of a family segregating both autistic disorder and Asperger syndrome. We evaluated 103 nonsyndromic and 18 syndromic patients by the custom-designed array and the detection rate of possibly pathogenic alterations were, respectively, 11.6% and 38.9%. Among these CNVs, 44.4% are smaller than 10 kbp. Therefore, the strategy of using a custom-designed array, enriched with probes targeted to genes potentially involved in the ASD etiology and able to detect both large and small CNVs, seems to be relevant in an attempt to elucidate the largest number of cases and to better understand these disorders. Furthermore, this platform can also be used as a tool to support the clinical diagnosis and genetic counseling with a more affordable cost compared to conventional other or next-generation sequencing. Approximately 33.3% of the observed CNVs affect UTR region, suggesting that mutations in non-coding regions might explain a significant proportion of ASD cases negative for most genomic screenings, which still do not explore adequately these regions. Among nonsyndromic autistic patients we found that most of the genes affected by CNVs are involved in two main biological functions - glutamatergic synapses and axonal guidance, suggesting that nonsyndromic ASD can be caused by dysfunction in different genes of a few common physiological processes. In contrast to our findings in nonsyndromic patients, we detected more than one alteration in a single individual or alterations that involve more than one gene among the syndromic patients, reinforcing the oligogenic model for some cases of ASD. Finally, the data obtained in the study of the family segregating both Asperger syndrome and autistic disorder suggests that the severity of ASD seems to be modulated by the number of hits and possibly by hits in both alleles of the same gene. Our results support the involvement of genes MDGA2, FHIT, HTR2A, SHANK2, GRIA3, ZNF778, PRKCα, CDH15, DIAPH3, GCH1, GRM5, MARK1, SLC17A6, IMMP2L, BZRAP1, SYNGAP1, ANK3, MAP1A, GABRR2 and LAMC3 in ASD etiology and also suggests new candidates: LRRC7, LRRIQ3, CADPS1, NUFIP, SEMA3A, SNAP29, MBD2, GAD2, DGKH and PARD3

Orientadores: Antonia Paula Marques de Faria, Maricilda Palandi de Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-30T05:34:46Z (GMT). No. of bitstreams: 1 Henrique_PamelaPontes_M.pdf: 8320960 bytes, checksum: 1d8ff3e1253bf81cc18b4afd51572860 (MD5) Previous issue date: 2015 / Resumo: A deficiência intelectual ligada ao X (DILX) é uma das causas genéticas mais frequentes de deficiência intelectual (DI), ocorrendo em 10 a 12% de todos os homens afetados, provavelmente pelo maior número de genes identificados no cromossomo X em comparação a qualquer segmento autossômico. Cerca de 100 genes seriam determinantes de DILX, porém mesmo com o conhecimento do papel de vários deles, há aspectos a serem elucidados, como a contribuição de cada um na determinação da DI ou ainda as correlações genótipo-fenótipo, cuja análise depende da investigação genética em indivíduos com DI idiopática. Entre os métodos que permitem a investigação molecular dessa condição destaca-se a Multiplex Ligation-dependent Probe Amplification (MLPA) por sua rapidez, sensibilidade e baixo custo. O objetivo do presente estudo foi investigar alterações em genes do cromossomo X pela técnica de MLPA em pacientes do sexo masculino com atraso global do desenvolvimento ou DI de origem indeterminada. Foram investigados 107 indivíduos com o kit SALSA MLPA P106 MRX probemix (MRC-Holland), 104 deles apresentaram resultado na faixa de normalidade e em três foram identificadas alterações do número de cópias interpretadas como duplicações. O paciente P13 apresentou alteração no gene HUWE1, que atua no controle da diferenciação neural e tem mutações descritas em algumas famílias com DI de moderada a grave; no paciente P139 foram identificadas alterações nos genes SCL6A8 e GDI, ambas confirmadas pela análise por Real Time Polymerase Chain Reaction (qPCR); mutações no primeiro são incluídas entre as síndromes de deficiência de creatina, com fenótipos variando de DI leve e atraso de fala até DI grave, convulsões e alterações de comportamento no sexo masculino, enquanto no segundo se associam à DILX inespecífica; já no paciente P39 foi detectada alteração no gene ARX, relacionado a mais de uma condição classificada como DILX sindrômica, que não foi confirmada. Como apenas alguns éxons relacionados à DILX foram investigados, não se afasta a eventual ocorrência de rearranjos localizados em regiões não abordadas pelo kit utilizado. Contudo, a técnica utilizada se mostrou uma opção de custo relativamente baixo e fácil reprodutibilidade, sendo viável para aplicação em algoritmos de investigação da DI. Os resultados reforçam a relevância da DILX entre as causas de DI, justificando a inclusão de testes moleculares específicos para a elucidação diagnóstica dessa condição / Abstract: X-linked intellectual disability (XLID) is one of the most frequent genetic causes of intellectual disability (ID), occurring in 10-12% of all affected men, probably because the larger number of identified genes on the X chromosome related to this condition than in any other autosomal segment. Although about 100 genes have been considered as determinant of XLID, the the role of several of these genes remains yet be elucidated despite the knowledge on the function of several of them. For instance, the contribution of each gene in determining the ID and the genotype-phenotype correlation depend on the genetic investigation of affected individuals. The Multiplex Ligation-dependent Probe Amplification (MLPA) is among the methods that allow molecular investigation of this condition because it is rapid and low cost and presents high sensitivity. The aim of this study was to investigate copy number variations in X-linked genes by MLPA technique in males with global developmental delay or ID of undetermined origin. A hundred and seven individuals were investigated using SALSA MLPA P106 MRX kit (MRC-Holland) and alterations were confirmed by Real Time Polymerase Chain Reaction (qPCR). A normal invariant pattern was observed in 104 out of 107 individuals, and three showed variations that have been interpreted as duplications. Patient P13 showed increased signal for HUWE1 gene, which plays a role in the control of neural differentiation. HUWE1 mutations have been described in families with moderate or severe ID. Patient P139 showed increased signals corresponding to regions of SCL6A8 and GDI1 genes. The former is included among genes involved in the creatine deficiency syndrome whose phenotype can range from mild ID and speech delay to severe ID, convulsions and behavior changes in males, and the latter is involved with non-syndromic XLID. Conversely, the variation in ARX gene, which is associated to more than one condition classified as syndromic XLID, observed in MLPA analysis for patient P39 was not confirmed in the qPCR assay. As only a few exons related to XLID were investigated, it does not rule out the possible occurrence of rearrangements located in regions not covered by the kit used. However, the technique employed was an easily reproducible, relatively low cost option, manageable for application in ID research algorithms. The results reinforce the importance of XLID among the causes of ID, justifying the inclusion of specific molecular tests for the laboratory diagnosis of this condition / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

O transtorno do espectro autista (ASD) é uma doença clinica e geneticamente heterogênea, com mecanismo etiológico ainda pouco conhecido. Assim, os principais objetivos deste trabalho foram descrever as características clínicas e genéticas de pacientes brasileiros com ASD, bem como determinar o risco de recorrência e a herdabilidade. Verificamos que a maioria das comorbidades avaliadas tem prevalência similar àquelas anteriormente descritas. A hipotonia exibiu maior prevalência no sexo feminino. A ausência de fala apresentou prevalência significativamente maior no grupo de pacientes com comorbidades, sendo que a gravidade da fala foi positivamente correlacionada com a presença das crises convulsivas. A herdabilidade estimada foi de 76% e o risco de recorrência ~5%. As alterações citogenéticas e os casos positivos para a Síndrome do X-Frágil explicaram cerca de 8% dos casos de ASD da nossa amostra. As CNVs nas regiões estudadas foram detectadas em 2,7% da amostra. Nós verificamos que há penetrância incompleta do ASD para as regiões. O estudo mais detalhado dos dois casos de duplicação da região 15q13.3, envolvendo somente o gene CHRNA7, mostrou que um dos pacientes (F5240) exibiu uma segunda CNV, possivelmente patogênica. A análise in silico sugeriu que genes que interagem diretamente com o CHRNA7 podem conter mutações patogênicas e, juntamente com a duplicação do 15q13.3, possivelmente estão envolvidos na etiologia do ASD. Este estudo mostrou que é necessário fazer uma ampla caracterização genética dos pacientes, para possibilitar o estudo dos possíveis mecanismos moleculares envolvidos na causa do ASD / Autism Spectrum Disorder (ASD) is a clinically and genetically heterogeneous disease and its etiological mechanisms are still poorly understood. The main objectives of this study were to describe the clinical and genetic features of Brazilian patients with ASD, and to determine the recurrence risk and heritability. Great part of the comorbidities assessed here had comparable prevalence to those of previous works. The hypotonia was significantly prevalent in the female sex. Absent speech was significantly more frequent in patients with comorbidities, and severity of speech problems was positively correlated with presence of seizures. Heritability was estimated as 76% and the recurrence risk as approximately 5%. Cytogenetic alterations and positive results for Fragile X Syndrome explain about 8% of the ASD etiology of our sample. The CNVs at the chromosomal regions 15q11-q13, 16p11.2 and 22q13 were present in 2.7% of the sample. Incomplete penetrance of ASD was observed for the 16p and 15q regions. Further investigation of the two cases with duplication of the region 15q13.3, involving only the CHRNA7 gene, revealed that one of them (F5240) exhibited a second possible pathogenic CNV. In silico analysis suggested that genes interacting directly with the CHRNA7 could harbor pathogenic mutations and, together with the duplication at 15q13.3, could be involved in the ASD etiology. This study showed the necessity of a broad genetic characterization of patients with ASD, to enable the elucidation of possible molecular mechanisms related to ASD etiology

We developed a pipeline to identify the copy number polymorphisms (CNPs) in the Northern Swedish population using whole genome sequencing (WGS) data. Two different methodologies were applied to discover CNPs in more than 1,000 individuals. We also studied the association between the identified CNPs with the expression level of 438 plasma proteins collected in the same population. The identified CNPs were summarized and filtered as a population copy number matrix for 1,021 individuals in 243,987 non-overlapping CNP loci. For the 872 individuals with both WGS and plasma protein biomarkers data, we conducted linear regression analyses with age and sex as covariance. From the analyses, we detected 382 CNP loci, clustered in 30 collapsed copy number variable regions (CNVRs) that were significantly associated with the levels of 17 plasma protein biomarkers (p < 4.68×10-10).

INTRODUÇÃO: Os defeitos cardíacos congênitos (DCC) são um grupo de anormalidades estruturais mais prevalentes ao nascimento e uma das principais causas de morbidade e mortalidade infantil. Os fatores genéticos são importantes na etiologia dos DCC. Estudos têm mostrado a contribuição da variação no número de cópias (CNV) na gênese das malformações cardíacas. A deleção 22q11.2 é a causa mais comum de microdeleção humana e está relacionada com defeito cardíaco (DCC) conotruncal. O MLPA (Multiplex Ligation-dependent Probe Amplification) é um método eficaz para detectar microdeleções/microduplicações em pacientes com DCC. OBJETIVO: Detectar a presença da variação no número de cópias gênicas em pacientes portadores de cardiopatia conotruncal pela técnica de MLPA e associar ao fenótipo do paciente. MÉTODOS: Foram avaliados 39 pacientes (23 do sexo masculino, 16 do sexo feminino) com idade entre 2 dias e 19 anos (mediana de 6 anos), todos com cardiopatia conotruncal, a maioria dos pacientes (56%) apresentavam tetralogia de Fallot. Avaliação clínica e laboratorial foi realizada em todos os pacientes. O cariótipo foi normal em todos pacientes. MLPA foi realizada com os kits P064, P036/P070 e P250. RESULTADOS: Foram detectadas CNVs em sete pacientes: deleção 22q11.2, duplicação 22q11.2, duplicação 15q11.2, duplicação 20p12.2, deleção 19p, duplicação 15q e duplicação 8p23.2 com duplicação 10p12.31. As cardiopatias encontradas nestes pacientes foram: dupla via de saída de ventrículo direito (2), coartação da aorta, tetralogia de Fallot (3) e transposição de grandes artérias. Os achados clínicos extracardíacos encontrados nestes pacientes foram dismorfismo facial, dente neonatal, atrofia e displasia cerebral, atresia duodenal, dificuldade de aprendizado, insuficiência velofaríngea, aplasia de timo, refluxo gastroesofágico, hérnia umbilical, asma, infecções de vias aéreas frequente, déficit de crescimento e somente três apresentavam retardo no desenvolvimento neuropsicomotor (dup 15q11.2, dup 15q, del 22q11.2). As características clínicas foram compatíveis com o relatado na literatura associado com a microdeleção/microduplicação encontrada. Nenhuma destas alterações foram herdadas de seus pais testados em seis casos. CONCLUSÃO: O uso do MLPA possibilitou a detecção de CNVs em pacientes com DCC. O diagnóstico precoce das CNVs em pacientes com DCC auxilia na prevenção de morbidade e diminuição da mortalidade nestes pacientes, contudo em um país com regiões com poucos recursos laboratoriais genéticos uma avaliação clínica minuciosa em todo paciente com DCC é imprescindível para direcionar qual melhor exame deve ser realizado / INTRODUCTION: Congenital heart defects (CHD) are a group of structural abnormalities most prevalent birth and a major cause of infant morbidity and mortality. Genetic factors are important in the etiology of CHD. Studies have shown the contribution of copy number variation (CNV) in the genesis of cardiac malformations. The deleletion 22q11.2 is the most common cause of human microdeletion and is related conotruncal cardiac defect (DCC). The MLPA (Multiplex Ligation-dependent Probe Amplification) is an effective method to detect microdeletions/micoduplications in patients with CHD. PURPOSE: Detect the presence of gene copies number variation in the patients with conotruncal heart defect by MLPA technique and associate the phenotype of the patient. METHODS: 39 patients (23 males, 16 females) aged 2 days old - 19 years old (median= 6 years old) with conotruncal cardiac defect were evaluated. Tetralogy of Fallot was more prevalent heart defect (56%). All patients were evaluated clinical and laboratory. Karyotypes were normal in all pacients. MLPA was performed with the P064, P036/P070 and P250 kits. RESULTS: CNVs were detected in seven patients: 22q11.2 deletion, 22q11.2 duplication, 15q11.2 duplication, 20p12.2 duplication, 19p deletion, 15q duplication and 8p23.2 duplication with 10p12.31 duplication. The congenital heart defect found in these patients were: double outlet right ventricle (2), coarctation of the aorta, tetralogy of Fallot (3) and transposition of the great arteries. Clinical findings in these patients were facial dysmorphism, neonatal tooth, brain atrophy and dysplasia, duodenal atresia, learning disabilities, velopharyngeal insufficiency, thymic aplasia, gastroesophageal reflux, umbilical hernia, asthma, frequent infections of the airways , failure to thrive, and only three had delayed psychomotor development (dup 15q 11.2, dup 15q, del 22q11.2) The clinical features were consistent with those reported in the literature associated with the microdeletion /microduplication found. None of these alterations were inherited from six parents tested. CONCLUSIONS: MLPA was effective to detect CNVs in patients with CHD. Early diagnosis of CNVs in patients with CHD assists in preventing morbidity and decreased mortality in these patients, however, in a country with regions with few genetic laboratory resources a thorough clinical evaluation in all patients with CHD is essential to direct which should be further analyzed performed

INTRODUÇÃO: Os defeitos cardíacos congênitos (DCC) são um grupo de anormalidades estruturais mais prevalentes ao nascimento e uma das principais causas de morbidade e mortalidade infantil. Os fatores genéticos são importantes na etiologia dos DCC. Estudos têm mostrado a contribuição da variação no número de cópias (CNV) na gênese das malformações cardíacas. A deleção 22q11.2 é a causa mais comum de microdeleção humana e está relacionada com defeito cardíaco (DCC) conotruncal. O MLPA (Multiplex Ligation-dependent Probe Amplification) é um método eficaz para detectar microdeleções/microduplicações em pacientes com DCC. OBJETIVO: Detectar a presença da variação no número de cópias gênicas em pacientes portadores de cardiopatia conotruncal pela técnica de MLPA e associar ao fenótipo do paciente. MÉTODOS: Foram avaliados 39 pacientes (23 do sexo masculino, 16 do sexo feminino) com idade entre 2 dias e 19 anos (mediana de 6 anos), todos com cardiopatia conotruncal, a maioria dos pacientes (56%) apresentavam tetralogia de Fallot. Avaliação clínica e laboratorial foi realizada em todos os pacientes. O cariótipo foi normal em todos pacientes. MLPA foi realizada com os kits P064, P036/P070 e P250. RESULTADOS: Foram detectadas CNVs em sete pacientes: deleção 22q11.2, duplicação 22q11.2, duplicação 15q11.2, duplicação 20p12.2, deleção 19p, duplicação 15q e duplicação 8p23.2 com duplicação 10p12.31. As cardiopatias encontradas nestes pacientes foram: dupla via de saída de ventrículo direito (2), coartação da aorta, tetralogia de Fallot (3) e transposição de grandes artérias. Os achados clínicos extracardíacos encontrados nestes pacientes foram dismorfismo facial, dente neonatal, atrofia e displasia cerebral, atresia duodenal, dificuldade de aprendizado, insuficiência velofaríngea, aplasia de timo, refluxo gastroesofágico, hérnia umbilical, asma, infecções de vias aéreas frequente, déficit de crescimento e somente três apresentavam retardo no desenvolvimento neuropsicomotor (dup 15q11.2, dup 15q, del 22q11.2). As características clínicas foram compatíveis com o relatado na literatura associado com a microdeleção/microduplicação encontrada. Nenhuma destas alterações foram herdadas de seus pais testados em seis casos. CONCLUSÃO: O uso do MLPA possibilitou a detecção de CNVs em pacientes com DCC. O diagnóstico precoce das CNVs em pacientes com DCC auxilia na prevenção de morbidade e diminuição da mortalidade nestes pacientes, contudo em um país com regiões com poucos recursos laboratoriais genéticos uma avaliação clínica minuciosa em todo paciente com DCC é imprescindível para direcionar qual melhor exame deve ser realizado / INTRODUCTION: Congenital heart defects (CHD) are a group of structural abnormalities most prevalent birth and a major cause of infant morbidity and mortality. Genetic factors are important in the etiology of CHD. Studies have shown the contribution of copy number variation (CNV) in the genesis of cardiac malformations. The deleletion 22q11.2 is the most common cause of human microdeletion and is related conotruncal cardiac defect (DCC). The MLPA (Multiplex Ligation-dependent Probe Amplification) is an effective method to detect microdeletions/micoduplications in patients with CHD. PURPOSE: Detect the presence of gene copies number variation in the patients with conotruncal heart defect by MLPA technique and associate the phenotype of the patient. METHODS: 39 patients (23 males, 16 females) aged 2 days old - 19 years old (median= 6 years old) with conotruncal cardiac defect were evaluated. Tetralogy of Fallot was more prevalent heart defect (56%). All patients were evaluated clinical and laboratory. Karyotypes were normal in all pacients. MLPA was performed with the P064, P036/P070 and P250 kits. RESULTS: CNVs were detected in seven patients: 22q11.2 deletion, 22q11.2 duplication, 15q11.2 duplication, 20p12.2 duplication, 19p deletion, 15q duplication and 8p23.2 duplication with 10p12.31 duplication. The congenital heart defect found in these patients were: double outlet right ventricle (2), coarctation of the aorta, tetralogy of Fallot (3) and transposition of the great arteries. Clinical findings in these patients were facial dysmorphism, neonatal tooth, brain atrophy and dysplasia, duodenal atresia, learning disabilities, velopharyngeal insufficiency, thymic aplasia, gastroesophageal reflux, umbilical hernia, asthma, frequent infections of the airways , failure to thrive, and only three had delayed psychomotor development (dup 15q 11.2, dup 15q, del 22q11.2) The clinical features were consistent with those reported in the literature associated with the microdeletion /microduplication found. None of these alterations were inherited from six parents tested. CONCLUSIONS: MLPA was effective to detect CNVs in patients with CHD. Early diagnosis of CNVs in patients with CHD assists in preventing morbidity and decreased mortality in these patients, however, in a country with regions with few genetic laboratory resources a thorough clinical evaluation in all patients with CHD is essential to direct which should be further analyzed performed

L'autisme est un trouble du développement du système nerveux central défini par des altérations des interactions sociales et de la communication, et par des comportements restreints et répétitifs. Sa prévalence est actuellement évaluée jusqu'à 1% dans la population générale. L'autisme est caractérisé par une grande hétérogénéité sur les plans phénotypiques et génétiques. À ce jour, plus de 300 gènes candidats ont été caractérisés soit par des variations du nombre de copies (CNV) et/ou des variations nucléotidiques (SNV). Leur identification a permis de mettre en évidence une contribution significative de mutation de novo, ainsi que l'implication de voies physiopathologiques cibles, en particulier la densité post-synaptique (PSD). / Autism is a developmental disorder of the central nervous system defined by impairments in social interaction and communication, and by restricted and repetitive behavior. Its prevalence is currently estimated at around 1% in the general population. Autism is characterized by a wide heterogeneity at both phenotypic and genetic level. To date, more than 300 candidate genes were characterized either by copy number variations (CNV) and/or nucleotide variations (SNV). Their identification has highlighted a significant contribution of de novo mutations, as well as the involvement of targeted pathophysiological pathways, particularly post-synaptic density (PSD).

Abstract Breast cancer is the most common cancer among women, and inherited predisposition is one of the major recognized causes of increased breast cancer risk. Only about half of the hereditary cases are explained by mutations in the known susceptibility genes, including the DNA damage response genes BRCA1, BRCA2 and PALB2, leaving the majority still uncovered. Identification of the missing genetic predisposing factors is important for more effective diagnostics and counseling of the risk families, and also for better understanding of the etiology and cellular characteristics of breast cancer. The first aim of this study was to investigate the cancer associations of six rare germline copy number variant (CNV) deletions, which were previously identified in breast cancer patients by a genome-wide microarray approach. The second aim was to identify novel susceptibility alleles, both protein-truncating variants and missense mutations, by next-generation sequencing (NGS) of nearly 800 DNA damage response genes in 189 hereditary breast cancer patients. The cancer-associations of all selected candidate alleles (6 CNVs, 39 protein-truncating variants and 35 missense mutations) were studied by case-control approach using DNA samples from several hundred breast cancer patients and healthy controls. The prevalence of the studied CNVs did not significantly differ between the cases and controls, but when studying the associations with specific clinical parameters, deletion in the CYP2C19 gene showed enrichment in the breast cancer patients with hormonally triple-negative tumors (p=0.021). As CYP2C19 functions in estrogen metabolism, the results indicate that disturbance of hormonal balance due to enzyme defects may predispose specifically to the estrogen receptor-negative subtype of breast cancer. Two protein truncating-variants, TEX15 c.7253dupT and FANCD2 c.2715+1G>A showed significant breast cancer association in the Northern Finnish cohort (p=0.018 and p=0.036, respectively). Similarly, two of the studied missense variants, RECQL p.Ile156Met (p=0.043) and POLG p.Leu392Val (p=0.010), were enriched in the breast cancer cases. Thus, this study provided novel connections between increased breast cancer risk and inherited mutations in TEX15, FANCD2 and POLG genes, and further supported the recently established role of RECQL as a breast cancer susceptibility gene. / Tiivistelmä Rintasyöpä on naisten yleisin syöpä, ja perinnöllinen alttius on yksi merkittävimmistä sairastumisriskiin vaikuttavista tekijöistä. Tunnetuimpia alttiustekijöitä ovat mutaatiot BRCA1-, BRCA2- ja PALB2-DNA-vauriovastegeeneissä, mutta ne yhdessä muiden altistavien geenimutaatioiden kanssa selittävät kuitenkin vain noin puolet perinnöllisistä rintasyöpätapauksista. Uusien alttiusgeenien löytäminen mahdollistaa tehokkaamman diagnostiikan ja korkeassa syöpäriskissä olevien sukujen perinnöllisyysneuvonnan, sekä auttaa ymmärtämään syvemmin rintasyövän etiologiaa ja syntymekanismeja solutasolla. Tämän väitöskirjan ensimmäisenä päämääränä oli tutkia tarkemmin aiemmin genominlaajuisella mikrosirumenetelmällä rintasyöpäpotilailta tunnistettujen harvinaisten perinnöllisten DNA-kopiolukuvariaatioiden (CNV) yhteyttä rintasyöpäriskiin. Toisena tavoitteena oli tunnistaa uusia rintasyöpäalttiusalleeleja, sekä proteiinitrunkaatioita että missense-mutaatioita, hyödyntämällä uuden sukupolven sekvensointitekniikkaa, jonka avulla tutkittiin mutaatioita lähes 800 DNA-vauriovastegeenistä 189 pohjoissuomalaiselta rintasyöpäpotilaalta. Valittujen kandidaattialleelien (6 deleetion aiheuttavaa CNV:tä, 39 proteiinitrunkaatiota ja 35 missense-mutaatiota) yhteyttä rintasyöpään tutkittiin tapaus-verrokkimenetelmällä käyttäen DNA-näytteitä usealta sadalta rintasyöpäpotilaalta ja terveeltä kontrollihenkilöltä. Tutkittujen CNV:iden esiintyvyydessä ei ollut merkitseviä eroja potilaiden ja kontrollien välillä, mutta tarkasteltaessa yhteyttä potilaiden kasvaimista saatuihin kliinisiin parametreihin, deleetio CYP2C19-geenissä oli yleisempi hormonaalisesti kolmoisnegatiivisissa rintatuumoreissa kuin muissa tuumorityypeissä (p=0.021). Koska CYP2C19 on estrogeenimetaboliaan osallistuva entsyymi, sen viallinen toiminta voi mahdollisesti altistaa erityisesti estrogeenireseptorinegatiiviselle rintasyövälle. Kaksi tutkituista proteiinitrunkaatioista, TEX15 c.7253dupT ja FANCD2 c.2715+1G>A, olivat rikastuneet perinnöllisessä rintasyöpäpotilasaineistossa verrattuna kontrolleihin (p=0.018 ja p=0.036). Myös kaksi missense-alleelia, RECQL p.Ile156Met (p=0.043) ja POLG p.Leu392Val (p=0.010), olivat yleisempiä rintasyöpäpotilailla. Tulokset osoittivat uuden yhteyden kohonneen rintasyöpäriskin ja perinnöllisten muutosten TEX15-, FANCD2- ja POLG-geenien välillä, sekä tukivat aiempia tutkimustuloksia, joiden mukaan RECQL on kohtalaisen riskin rintasyöpäalttiusgeeni.

Abstract Dupuytren’s disease is a Caucasian male-dominant disease that affects the palmar fascia. Incidence grows with age, but persons with strong diathesis seem to develop the disease at an earlier age than the majority of the diseased. Myofibroblasts are histopathologically the main cell type in DD tissue. Despite scientific research, the aetiology of the disease is still unrevealed. Only genetic susceptibility is generally accepted as predisposing to DD. Available treatment has thus far been unsatisfactory, because only symptoms can be cured to date.The disease recurs. With genetic susceptibility the recurrence rates are high (even up to 70%) and the time to recurrence is inevitably shorter. This behaviour is considered the aggressive type of DD. To be able to predict the behaviour of DD, whether it is an aggressive or conventional type, twenty-one Dupuytren samples were gathered and compared with five controls by means of immunohistochemical stainings. It was found that cellularity was better expresented in aggressive and recurred samples. Alfa-SMA and Ki-67 showed more activity in the aggressive tissue type of DD. Tenascin was vaguely expressed in aggressive-type samples. To compare the gene and protein expressions and to obtain a more profound understanding of the disease, a microarray technique was used. With a microarray it is possible to compare nucleotide pair hybridisations to resolve the genome of the tissues. In this study RT-PCR was used to examine mRNA levels to determine gene expression changes. Twelve DD palmar fascia samples were compared with three healthy control samples. Both myoglobin and ROR2, which we considered as the most valuable results, were found in the DD samples. ROR2 acts as a receptor or co-receptor for the Wnt system. The Wnt signalling pathway transfers signals from outside of the cell through cell surface receptors, and plays a significant role in proliferation processes such as in fibrotic conditions. To evaluate a possible chromosomal imbalance behind the aetiology of the disease, eighteen DD palmar fascia samples were compared with two reference samples. However, we were not able to detect any chromosomal imbalance in the DD samples. The method used was Oligonucleotide aCGH Agilent’s 60-mer oligonucleotide-based microarray according to the manufacturer’s instructions, which can reveal gains and losses of approximately 35 kilobases in the whole genome. The result does not exclude copy number changes entirely; a small presence of aberrant cells will not be detected if the change is less than 50%. In conclusion, we revealed elements in DD tissue that would enable us to predict the nature of the disease; whether the disease is aggressive with a stronger tendency to recur. Histological differences could be detected, and this can be used to benefit patients. As a new element, ROR2 was discovered in DD tissue.The genome-wide analysis with the 44K oligonucleotide-based array method revealed no changes of DNA number sequences. / Tiivistelmä Kämmenkalvon kuroumatauti eli Dupuytrenin kontraktuura on valkoihoisen miehen kämmenkalvon sairaus. Sairastumisen todennäköisyys lisääntyy ikääntymiseen liittyen, mutta vahva sukurasitus poikkeuksellisesti altistaa sairaudelle jo tavanomaista nuoremmalla iällä. Myofibroblastit ovat tärkein ja edustetuin solutyyppi Dupuytren kudoksessa. Huolimatta runsaasta tutkimustyöstä ei etiologiaa ole saatu vielä selvitettyä. Sukurasitus näyttää selkeästi altistavan taudille. Toistaiseksi kyetään hoitamaan ainoastaan sairauden aiheuttamat seuraukset, mutta ei perussyytä. Lisäksi tauti uusiutuu. Dupuytrenin sukurasitus lisää uusiutumista suurella todennäköisyydellä. Myös uusiutumisaika on tuolloin tavanomaista nopeampi, ja kyseessä katsotaan olevan ns.aggressiivisempi taudin muoto. Väitöskirjatyössäni pyrittiin löytämään mahdollisia tekijöitä, joiden perusteella voitaisiin ennustaan onko kyseessä aggressiivisempi vai tavanomainen taudin muoto. Tätä varten tutkittiin kaksikymmentä yksi Dupuytren kudosnäytettä ja viisi tervettä kämmenkalvon näytettä immunohistologisilla värjäyksillä, ja voitiin todeta, että soluisuus oli selkeästi koholla aggressiivisten ja taudin uusineiden potilaiden näytteissä. Tulos oli samanlainen myös alfa-SMA ja Ki-67 suhteen. Tenaskiiniä voitiin löytää edellisiä niukemmin aggressiivisista näytteistä. Dupuytrenin taudin luonteen lisäselvittelemiseksi geeni- ja proteiinitasolla tehtiin mikroarray, jossa emäsparien pariutumisen avulla selvitetään taudin genomia ja myös sitten tästä aiheutuvien proteiinien ilmentymistä. Kahtatoista Dupuytren potilaan kämmenkalvon kudosnäyttettä verrattiin kolmeen terveeseen verrokki kudosnäytteeseen ja voitiin todeta myoglobiinin ja ROR2:n selkeät pitoisuuden muutokset terveisiin näytteisiin verrattaessa. ROR2 toimii solujen välisten viestien välityksen reseptorina, eli siirtää signaalin solun ulkopuolelta sen sisäpuolelle solun pinnalla olevan kiinnittymiskohdan avulla. Sillä on selkeä merkitys ja tehtävä proliferatiivisissä tapahtumissa, kuten sidekudoksen lisääntymisessä. Mahdollisia kromosomin määrän muutoksia Dupuytren kudoksessa selviteltiin kahdeksantoista kudosnäytteen tutkimisella ja löydösten tulosta verrattiin sitten kahteen normaaliin verrokki kudosnäytteen tulokseen. Tutkimuksessa ei saatu selville kromosomien määrän muutosta, kun muutosten kokonaismäärä on vähäinen tai ainakin alle 50 % kokonaismäärää alhaisempi. Yhteenvetona voidaan todeta, että löytyi histologisia kudoselementtejä, joiden perusteella voidaan ennustaa, onko Dupuyrenin tauti aggressiivisempi ja todennäköisemmin uusiutuva luonteeltaan. ROR2 ei ole aikaisemmin yhdistetty Dupuytrenin kontraktuuraan. Dupuytren kudoksesta ei voitu 44K oligonukleotide mikroarray tekniikalla paljastaa geenimäärien muutoksia.