Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O desenvolvimento de mudanças neuropatológicas características da doença de Alzheimer (DA) nos portadores de síndrome de Down (SD) a partir dos 40 anos de idade e a alta taxa de indivíduos com SD em famílias nas quais a DA está presente sugerem uma susceptibilidade comum entre as duas doenças. Além disso, mulheres jovens (<35 anos na concepção) que tiveram filhos com a SD têm um risco cinco vezes maior de desenvolverem DA do que a população em geral. Esta alta suscetibilidade compartilhada poderia envolver um processo de envelhecimento acelerado, que levaria à SD em filhos de mães jovens e a um risco aumentado de demência nestas mães. Até o momento, os genes APOE, PSEN1 e TNF-&#945;, envolvidos direta ou indiretamente na deposição cerebral de beta amilóide e reconhecidos dentre os determinantes genéticos para a DA, parecem também intervir na patogênese da demência na SD. Neste estudo, analisamos a freqüência dos alelos &#949;2, &#949;3 e &#949;4 do gene APOE e dos polimorfismos -48C>T no gene PSEN1 e -850C>T no gene TNF-&#945; em mães de portadores da SD em comparação com mães de indivíduos cromossomicamente normais. Para isto, amostras de DNA foram extraídas a partir do sangue periférico de 114 mães de portadores da trissomia livre do cromossomo 21 com idade inferior a 35 anos no momento da concepção e de 110 mães controle. Para a triagem dos polimorfismos dos alelos do gene APOE e dos polimorfismos PSEN1 -48C>T e TNF-&#945; -850C>T foi realizada a técnica de PCR seguida da digestão dos produtos amplificados com as endonucleases de restrição HhaI, HgaI e HincII, respectivamente. Os produtos digeridos foram analisados através de eletroforese em géis de poliacrilamida corados por prata. A análise estatística através do teste exato de Fisher revelou que não houve diferenças significativas entre as distribuições genotípicas observadas nas amostras caso e controle tanto para os alelos do gene APOE, quanto para os polimorfismos PSEN1-48C>T e TNF-&#945; -850C>T (c2 =4,26; g.l. = 4; P =0,37; c2 =0,22; g.l. = 2; P =0,89; c2 = 4,24; g.l. = 2; P = 0,12, respectivamente). Diferenças significativas também não foram observadas tanto na análise da interação gênica multiplicativa entre os alelos dos genes APOE, PSEN1 e TNF-&#945; como na interação entre estes alelos e polimorfismos participantes do metabolismo do folato (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC-1 80G>A, TC 776C>G e CBS 844ins68). No Brasil, este é o primeiro relato da investigação destes genes entre as mães de indivíduos com SD. Nossos resultados sugerem que os alelos APOE e4, PSEN1-48 C e TNF-&#945; -850T não são fatores de risco independentes para a DA nestas mães. Entretanto, em virtude da complexidade tanto da SD como da DA, a análise de variantes em outros genes é de fundamental importância para melhor elucidar o aparecimento insidioso de DA em mães jovens que tiveram filhos com SD. / The development of neuropathological changes of Alzheimer's disease (AD) in patients with Down syndrome (DS) from 40 years old and the high rate of DS births in families of DA individuals suggest a common susceptibility between the two diseases. Besides a five-fold higher risk for AD has been reported in young mothers of DS individuals. This shared genetic susceptibility involving DS and AD could be due to an accelerated ageing process, leading to the birth of a DS child in the offspring of young mothers and to an increased risk of dementia among them. Between the genetic risk factors for AD, APOE, PSEN1 and TNF-&#945; genes are directly or indirectly involved in cerebral amyloid deposition and seem to play a role in the pathogenesis of dementia in DS. In this study, we analyzed the frequency of APOE alleles &#949;2, &#949;3 e &#949;4 and the polymorphisms -48C>T in the PSEN1 gene and -850C>T in the TNF-&#945; gene in DS young mothers in comparison to mothers who had had chromosomicaly normal children. With this purpose, DNA samples were extracted from 114 mothers of DS individuals (full trisomy 21) with less than 35 years-old at conception, and from 110 control mothers. For the screening of the APOE alleles, PSEN1 -48C>T and TNF-&#945; -850C>T polymorphisms, polymerase chain reaction (PCR) was accomplished, followed by the digestion of the amplification products with the restriction endonucleases HhaI, HgaI and HincII, respectively. The digested products were analysed by polyacrilamide gels electrophoresis followed by silver staining. No significant differences were observed between the genotype distributions observed in control and case samples with respect APOE alleles, PSEN1 -48C>T and TNF-&#945; -850C>T polymorphisms (Fisher exact test: c2 =4,26; g.l. = 4; P =0,37; c2 =0,22; g.l. = 2; P =0,89; c2 = 4,24; g.l. = 2; P = 0,12, respectively). No differences were also observed in the gene multiplicative interaction analysis for APOE, PSEN1 and TNF-&#945; alleles and in the interaction of these alleles with folate pathway polymorphisms (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC-1 80G>A, TC 776C>G e CBS 844ins68). This is the first study reporting the investigation of these genes in Brazilian DS mothers. Our findings suggest that APOE &#949;4, PSEN1 -48C and TNF-&#945; -850T alleles are not independent risk factors for AD in young DS mothers. However, considering the complexity of both DS and AD, polymorphisms in other genes should be investigated to evaluate the actual relationship between the genetic risk factors and AD in DS young mothers.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O desenvolvimento de mudanças neuropatológicas características da doença de Alzheimer (DA) nos portadores de síndrome de Down (SD) a partir dos 40 anos de idade e a alta taxa de indivíduos com SD em famílias nas quais a DA está presente sugerem uma susceptibilidade comum entre as duas doenças. Além disso, mulheres jovens (<35 anos na concepção) que tiveram filhos com a SD têm um risco cinco vezes maior de desenvolverem DA do que a população em geral. Esta alta suscetibilidade compartilhada poderia envolver um processo de envelhecimento acelerado, que levaria à SD em filhos de mães jovens e a um risco aumentado de demência nestas mães. Até o momento, os genes APOE, PSEN1 e TNF-&#945;, envolvidos direta ou indiretamente na deposição cerebral de beta amilóide e reconhecidos dentre os determinantes genéticos para a DA, parecem também intervir na patogênese da demência na SD. Neste estudo, analisamos a freqüência dos alelos &#949;2, &#949;3 e &#949;4 do gene APOE e dos polimorfismos -48C>T no gene PSEN1 e -850C>T no gene TNF-&#945; em mães de portadores da SD em comparação com mães de indivíduos cromossomicamente normais. Para isto, amostras de DNA foram extraídas a partir do sangue periférico de 114 mães de portadores da trissomia livre do cromossomo 21 com idade inferior a 35 anos no momento da concepção e de 110 mães controle. Para a triagem dos polimorfismos dos alelos do gene APOE e dos polimorfismos PSEN1 -48C>T e TNF-&#945; -850C>T foi realizada a técnica de PCR seguida da digestão dos produtos amplificados com as endonucleases de restrição HhaI, HgaI e HincII, respectivamente. Os produtos digeridos foram analisados através de eletroforese em géis de poliacrilamida corados por prata. A análise estatística através do teste exato de Fisher revelou que não houve diferenças significativas entre as distribuições genotípicas observadas nas amostras caso e controle tanto para os alelos do gene APOE, quanto para os polimorfismos PSEN1-48C>T e TNF-&#945; -850C>T (c2 =4,26; g.l. = 4; P =0,37; c2 =0,22; g.l. = 2; P =0,89; c2 = 4,24; g.l. = 2; P = 0,12, respectivamente). Diferenças significativas também não foram observadas tanto na análise da interação gênica multiplicativa entre os alelos dos genes APOE, PSEN1 e TNF-&#945; como na interação entre estes alelos e polimorfismos participantes do metabolismo do folato (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC-1 80G>A, TC 776C>G e CBS 844ins68). No Brasil, este é o primeiro relato da investigação destes genes entre as mães de indivíduos com SD. Nossos resultados sugerem que os alelos APOE e4, PSEN1-48 C e TNF-&#945; -850T não são fatores de risco independentes para a DA nestas mães. Entretanto, em virtude da complexidade tanto da SD como da DA, a análise de variantes em outros genes é de fundamental importância para melhor elucidar o aparecimento insidioso de DA em mães jovens que tiveram filhos com SD. / The development of neuropathological changes of Alzheimer's disease (AD) in patients with Down syndrome (DS) from 40 years old and the high rate of DS births in families of DA individuals suggest a common susceptibility between the two diseases. Besides a five-fold higher risk for AD has been reported in young mothers of DS individuals. This shared genetic susceptibility involving DS and AD could be due to an accelerated ageing process, leading to the birth of a DS child in the offspring of young mothers and to an increased risk of dementia among them. Between the genetic risk factors for AD, APOE, PSEN1 and TNF-&#945; genes are directly or indirectly involved in cerebral amyloid deposition and seem to play a role in the pathogenesis of dementia in DS. In this study, we analyzed the frequency of APOE alleles &#949;2, &#949;3 e &#949;4 and the polymorphisms -48C>T in the PSEN1 gene and -850C>T in the TNF-&#945; gene in DS young mothers in comparison to mothers who had had chromosomicaly normal children. With this purpose, DNA samples were extracted from 114 mothers of DS individuals (full trisomy 21) with less than 35 years-old at conception, and from 110 control mothers. For the screening of the APOE alleles, PSEN1 -48C>T and TNF-&#945; -850C>T polymorphisms, polymerase chain reaction (PCR) was accomplished, followed by the digestion of the amplification products with the restriction endonucleases HhaI, HgaI and HincII, respectively. The digested products were analysed by polyacrilamide gels electrophoresis followed by silver staining. No significant differences were observed between the genotype distributions observed in control and case samples with respect APOE alleles, PSEN1 -48C>T and TNF-&#945; -850C>T polymorphisms (Fisher exact test: c2 =4,26; g.l. = 4; P =0,37; c2 =0,22; g.l. = 2; P =0,89; c2 = 4,24; g.l. = 2; P = 0,12, respectively). No differences were also observed in the gene multiplicative interaction analysis for APOE, PSEN1 and TNF-&#945; alleles and in the interaction of these alleles with folate pathway polymorphisms (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC-1 80G>A, TC 776C>G e CBS 844ins68). This is the first study reporting the investigation of these genes in Brazilian DS mothers. Our findings suggest that APOE &#949;4, PSEN1 -48C and TNF-&#945; -850T alleles are not independent risk factors for AD in young DS mothers. However, considering the complexity of both DS and AD, polymorphisms in other genes should be investigated to evaluate the actual relationship between the genetic risk factors and AD in DS young mothers.

No description available.

L’avènement du séquençage haut débit permet actuellement d’étudier et d’analyser la part de lacomposante génétique des maladies complexes médiée par les variants rares. Cependant, leurinterprétation représente un défi majeur. En effet, le séquençage de milliers d’exomes et degénomes a révélé la complexité du polymorphisme humain et notamment la surreprésentation devariants rares. Et malgré le développement de logiciels d’analyse ainsi que de différentes bases dedonnées, la priorisation des variants rares reste difficile. Dans le cadre de cette thèse, nous avons focalisé nos analyses sur les variations génétiques rares impliquées dans la maladie d’Alzheimer (MA). D’un point de vue génétique, la MA répond dans une majorité des cas à un déterminisme multifactoriel mais une minorité des cas sont des formes précoces à transmission autosomique dominante. La caractérisation des gènes PSEN1, PSEN2 et APP responsables des formes mendéliennes de MA a permis de formuler l’hypothèse de la cascade amyloïde en plaçant le peptide amyloïde (Aβ) au centre du processus physiopathologique. Afin de détecter de nouveaux facteurs de risque génétique dans la survenue de la MA, nous avons réalisé une étude d’association à partir de données de séquençage d’exomes de 522 cas atteints de formes précoces de MA et 584 contrôles. Les premières analyses ont porté sur les variants mononucléotidiques ainsi que les courtes insertions/délétions et ont permis de mettre en évidence un enrichissement en variants rares prédits délétères dans le gène ABCA7 chez les individus malades. Notre attention s’est ensuite portée sur les variations du nombre de copies (CNVs). L’absence de récurrence à l’échelle d’un gène nous a amené à travailler sur une liste de gènes. En nous focalisant sur l’hypothèse amyloïdergique, nous avons construit une liste de 342 gènes impliqués dans le métabolisme et la toxicité du peptide Aβ. Grâce à cette stratégie, nous avons ainsi réussi à mettre en évidence un enrichissement de CNVs rares intersectant ce réseau centré sur le peptide Aβ. Le résultat majeur de cette étude de CNVs a été la mise en évidence d’une duplication du locus 17q21.31 chez 5 patients atteints d’une maladie neurodégénérative similaire à une maladie d’Alzheimer. Les patients porteurs présentent un diagnostic clinique de MA, des biomarqueurs et une imagerie métabolique en faveur d’une neurodégénérescence de type Alzheimer. En revanche, l’imagerie amyloïde et l’analyse neuropathologique n’ont pas révélé de pathologie amyloïde et sont donc en faveur d’une tauopathie pure. L’étude des CNVs a également révélé une délétion partielle du gène PSEN1, emportant les exons 9 et 10, pour laquelle nous avons pu réaliser des études fonctionnelles. Nous avons ainsi pu déterminer que la protéine mutante favorisait la production de peptides amyloïdes plus longs, ces derniers étant des médiateurs majeurs de la neurotoxicité d’Aβ. / Next-generation sequencing allows studying and analyzing the genetic component part of complexdiseases mediated by rare variants. However, their interpretation represents a major challenge.Indeed, the sequencing of thousands of exomes and genomes revealed the human polymorphismcomplexity and in particular the overrepresentation of rare variants. Despite the development ofsoftwares and variant databases, the prioritization of rare variants remains arduous. My thesis subject was focused on the involvement of rare variants in Alzheimer's disease (AD). From a genetic point of view, AD is caused, in most cases, by a multifactorial determinism, but a minority of cases are autosomal dominant early-onset forms (ADEOAD). The characterization of mutations in the PSEN1, PSEN2 and APP genes as a cause of these Mendelian forms of AD led to the formulationof the amyloid cascade hypothesis, stating that the amyloid-β peptide (Aβ) is triggering the pathophysiological process. In order to detect new genetic risk factors involved in AD, we performed an association study using exome sequencing data from 522 cases with early-onset Alzheimer Disease and 584 controls. The first analyzes focused on single nucleotide variants and short insertions / deletions, and revealed an enrichment in cases of variants that are predicted to be deleterious in the ABCA7 genes. We then then focused on copy number variations (CNVs). The lack of recurrence at the gene-level incited us to work on a gene list. By focusing on the amyloidogenic hypothesis, we built a list of 342 genes involved in the metabolism and toxicity of the Aβ peptide. Thanks to this strategy, we found an enrichment of rare CNVs intersecting this Aβ network in cases.The main result of this CNV study was the identification of a duplication of the 17q21.31 locus in 5patients with a neurodegenerative disease similar to Alzheimer's disease. These patients have aclinical diagnosis of AD, as well as biomarkers and metabolic imaging consistent with an ADneurodegeneration. However, amyloid imaging and neuropathological analysis did not reveal anyamyloid pathology, and were therefore pointing to a pure tauopathy. This CNV study also revealed a partial deletion of the PSEN1 gene, overlapping exons 9 and 10, for which we performed functional studies. We demonstrated that the mutant protein enhanced the production of longer amyloid peptides, the latter being major mediators of Aβ neurotoxicity.