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

Estudos de citogenética comparativa são rotineiramente desenvolvidos a partir da comparação dos padrões de bandamentos cromossômicos. Contudo, quando se trata de espécies que apresentam genomas altamente rearranjados, como no caso das espécies de Akodon, ou que são muito divergentes, a comparação de cromossomos por padrões de bandas torna-se inadequada. Como consequência, a pintura cromossômica tem se tornado o método de escolha assertivo, já que permite comparação genômica no nível citogenético. Nessa tecnologia sondas de um cromossomo inteiro de uma determinada espécie são hibridadas in situ em cromossomos de outra espécie, detectando as regiões homólogas entre os genomas. No presente estudo comparamos os cariótipos altamente diferenciados de algumas espécies de Akodon por meio de pintura cromossômica recíproca com uso de sondas espécie-específicas, obtidas a partir de cromossomos separados por citometria de fluxo. Os resultados revelaram homologia completa entre os complementos de Akodon sp. n. (ASP), 2n=10, A. cursor (ACU), 2n=15, A. montensis (AMO), 2n=24 e A. paranaensis (APA), 2n=44 e evidenciaram com precisão muitos rearranjos cromossômicos entre os complementos das espécies. Rearranjos Robertsonianos e em tandem, inversões pericêntricas e/ou reposicionamento de centrômero, inversão paracêntrica, translocações, inserções e existência de sítios frágeis nos complementos foram observados. A pintura cromossômica empregando o conjunto de sondas de APA para 21 autossomos mais cromossomos X e Y evidenciou oito segmentos sintênicos compartilhados entre A. montensis, A. cursor e Akodon sp. n., cinco associações exclusivas para A. cursor e seis para Akodon sp. n. Foi também detectada homologia completa dos cromossomos X, com exceção da região heterocromática de ASP X, e até mesmo dos cromossomos Y que geralmente não apresenta sinal hibridação entre diferentes espécies de mamíferos. Esses dados indicam que essas espécies intimamente relacionadas passaram por um processo recente de intensa diferenciação autossômica, no qual se conservou a homologia total, por exceção de Akodon sp. n., entre os cromossomos sexuais. Pertencente a tribo Akodontini, Deltamys Thomas 1917 é um táxon pouco estudado e que é raramente capturado. Utilizando-se de caracteres morfológicos ou genéticos, alguns autores consideram Deltamys como um gênero pleno enquanto outros acreditaram que esse devesse ser referido como subgênero ou sinônimo de Akodon. A única espécie formalmente descrita é Deltamys kempi que apresenta cariótipo básico com 2n=37 nos machos e 2n=38 nas fêmeas, NF=38, com sistema de determinação de sexo do tipo X1X1X2X2: X1X2Y. Uma característica citogenética que distingue Deltamys de Akodon é a presença de um pequeno par metacêntrico marcador em Akodon. Um cariótipo com 2n=40 e NF=40; XX: XY foi relacionado ao gênero Akodon porém, como em Deltamys kempi , esse complemento não apresenta o pequeno par metacêntrico. As análises filogenéticas de máxima parcimônia e máxima verossimilhança baseadas em sequências do gene mitocondrial do citocromo b evidenciaram o monofiletismo dos espécimens de Akodon sp. 2n=40, e o monofiletismo de Deltamys kempi. Além disso, revelaram que Akodon sp. é grupo irmão de Deltamys kempi, estando mais relacionado a este último gênero do que às demais espécies de Akodon, sugerindo a inclusão dos espécimens portadores do cariótipo com 2n=40 em Deltamys. Dessa forma, o gênero Deltamys se mostrou mais diverso do que até então se tinha conhecimento, agrupando duas linhagens: Deltamys kempi , 2n=37-38 ; X1X1X2X2: X1X2Y e Deltamys sp. 2n=40, XX: XY, que apresentam entre sí uma marcante divergência genética que chega a 12, 1%. Um cariótipo com 2n=50, NF=48 foi descrito para espécimes de Thaptomys Thomas,1916, coletados em Una, estado da Bahia, Brasil, que são indistinguíveis morfologicamente dos Thaptomys nigrita que apresentam 2n=52, NF=52, encontrados em outras localidades brasileiras. Foi então proposto que esse novo cariótipo com 2n=50 pertencesse a uma espécie críptica de Thaptomys nigrita, uma vez que os rearranjos cromossômicos observados somados a distância geográfica pode representar uma barreira reprodutiva entre as duas formas. Com o intuito de se estabelecer as relações entre indivíduos de Thaptomys com os dois números diplóides, análises filogenéticas moleculares foram realizadas com o uso de dezoito sequências do gene mitocondrial do citocromo b provenientes de espécimes cariotipados coletados ao longo da distribuição geográfica do gênero. Dois clados principais, Nordeste (A) que agrupa espécimens com 2n=50 e Sudeste (B) que agrupa espécimes com 2n=52, foram recuperados por análises de máxima parcimônia e máxima verossimilhança. As relações filogenéticas intra-genéricas corroboram os distintos números diplóides, e mostram os cariótipos com 2n=50 e 2n=52 como clados irmãos entre si separados pela cladogênese basal de Thaptomys . No presente trabalho são apresentados estudos de filogenia molecular e citogenética para o gênero monotípico de roedor fossorial Blarinomys Thomas 1896. Foram realizadas análises de máxima parcimônia e máxima verossimilhança com base em sequências do gene mitocondrial citocromo b , para amostra de 11 exemplares de B. breviceps provenientes de nove localidades de quatro estados do território brasileiro. Todas as topologias recuperaram duas linhagens principais: um clado Nordeste (A) e outro Sudeste. O clado sudeste agrupou dois clados irmãos, B e C. A divergência de sequência entre os indivíduos variou de: 4,7- 8,0% entre os clados nordeste e sudeste; 4,3-5,7% entre os clados B e C; 6,1-8,0% entre os clados nordeste; e B, e 4,7-6,4% entres os clados nordeste e C. Dentro dos clados a divergência variou de 0- 4,2% no clado nordeste, foi de 0,7% no clado B, e variou de 0,1- 1,3% no clado C. Variação entre espécimes da mesma região geográfica foi de 0-1,3%. Os estudos de citogenética de cinco exemplares revelaram alta diversidade cariotípica com cinco números diplóides distintos: 2n=52 (48A+2Bs, XY), 2n=43 (37A+4Bs, XX), 2n=37 (34A+1B, XY), 2n=34 (32A, XX) e 2n=31 (27A+2Bs, XX) e mesmo número de braços autossômicos (NF=50), excluindo-se os cromossomos sexuais e os supernumerários. Foram observados polimorfismos decorrentes de rearranjos Robertsonianos, além de variação de 0 a 4 cromossomos Bs, que são heterogêneos quanto a morfologia, constituição de heterocromatina e presença de sinais teloméricos intersticiais (ITS). ITS também foram observados na região pericentromérica de alguns pares autossômicos com dois braços em três dos exemplares. Foi realizada pintura cromossômica com sonda do cromossomo X de Akodon cursor (ACU X). Nossos dados revelaram uma diversidade até então desconhecida para Blarinomys , mostrando duas linhagens distintas correspondentes a regiões na Mata Atlântica e um extraordinário polimorfismo cromossômico. / Traditionally comparative cytogenetic studies are based mainly on banding patterns. Nevertheless, when dealing with species with highly rearranged genomes, as in Akodon species, or with other highly divergent species, cytogenetic comparisons of banding patterns prove to be inadequate. Hence, comparative chromosome painting has become the method of choice for genome comparisons at the cytogenetic level, since it allows complete chromosome probes of a species to be hybridized in situ onto chromosomes of other species, detecting homologous genomic regions between them. In the present study, we have explored the highly rearranged complements of the Akodon species using reciprocal chromosome painting through species-specific chromosome probes obtained by chromosome sorting. The results revealed complete homology among the complements of Akodon sp. n. (ASP), 2n=10, A. cursor (ACU), 2n=15, A. montensis (AMO), 2n=24 and A. paranaensis (APA), 2n=44 and extensive chromosome rearrangements have been detected within the species with high precision. Robertsonian and tandem rearrangements, pericentric inversions and/or centromere repositioning, paracentric inversion, translocations, insertions and fragile sites were observed. The chromosome painting using the APA set of 21 autosomes plus X and Y exhibited eight syntenic segments that are shared with A. montensis, A. cursor and Akodon sp. n. plus five exclusive associations for A. cursor and six for Akodon sp. n. Chromosomes X, except for the heterochromatin region of ASP X, and even chromosome Y that often present no hybridization signal when hybridized between species of mammals, shared complete homology among the species. These data indicate that all those closely related species have experienced a recent intensive process of autosomic differentiation, in wich, there is still complete maintenance, except for chromosome X of Akodon sp. n., of the sex chromosomes homologies. Member of the tribe Akodontini, Deltamys Thomas 1917 is a poorly studied and rarely collected taxon. Based on morphological or genetic characters, some authors considered Deltamys as a full genus while others regarded it as subgenus or synonym of Akodon. The single described species, Deltamys kempi presents a basic karyotype with 2n=37 in males and 2n=38 in females, FN=38, and with sex determination system of the type X1X1X2X2: X1X2Y. A cytogenetic character that distinguishes Deltamys from Akodon is the presence of a small metacentric pair marker in Akodon. A karyotype with 2n=40 and FN=40; XX: XY was related to the genus Akodon, but as in Deltamys kempi, this complement does not present the small metacentric pair. Phylogenetic analyses of maximum parsimony and maximum likelihood based on sequences of the mitochondrial gene cytochrome b evidenced the monophyly of a clade grouping specimens of Akodon sp. 2n=40 and monophyly of a clade containing specimens of Deltamys kempi. Besides that, the analyses showed that Akodon sp. is the sistergroup of Deltamys kempi, thus more related to this genus than to other species of Akodon and suggesting the placement of specimens with 2n=40 Deltamys. The genus Deltamys is, thus, more diverse than previously thought, grouping two lineages: Deltamys kempi, 2n=37-38 ; X1X1X2X2: X1X2Y and Deltamys sp. 2n=40, XX: XY, with a marked genetic divergence of 12,1% between them. A karyotype with 2n=50, FN=48 has been described for specimens of Thaptomys Thomas, 1916 collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita with 2n=52, FN=52 found in other Brazilian localities. It has been hence proposed that this new karyotype with 2n=50 could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosome rearrangements observed along with the geographic distance could represent a reproductive barrier between both forms. Molecular phylogenetic analyses using the cytochrome b sequences of eighteen karyotyped specimens of Thaptomys were performed attempting to establish the relationships among the individuals along the geographic distribution of the genus. Two major clades, Northeastern (A) with specimens with 2n=50 and Southeastern (B) with specimens with 2n=52, were reconstructed by maximum parsimony (MP) and maximum likelihood (ML). The intra-generic relationships recovered by phylogenetic analyses corroborated the distinct diploid numbers. The 2n=50 and 2n=52 karyotypes appeared as monophyletic separated by the basal cladogenesis of the genus, sister-group to each other. We present molecular phylogenetic and cytogenetic data on the monotypic fossorial rodent genus Blarinomys . Maximum parsimony and maximum likelihood based on cytochrome b gene sequences were performed for a sample of 11 individuals from nine localities of four states of Eastern Brazil. All topologies recovered two main lineages: a Northeastern (A) and a Southeastern clade. The Southeastern grouped two sister-clades B and C. Sequence divergence between individuals ranged from 4.7-8.0% between northeastern and southeastern clades, from 4.3-5.7% between clades B and C, from 6.1-8.0% between clades northeastern and B, and from 4.7-6.4% between clades northeastern and C. Within the clades, divergence varied from 0- 4.2% in the northeastern clade, was 0.7% in the clade B, and varied from 0.1- 1.3% in clade C. Variation among specimens from the same geographic regions ranged from 0-1.3%. Cytogenetic studies of five individuals revealed high karyotypic diversity with five distinct diploid numbers: 2n=52 (48A+2Bs,XY) from state of Bahia, and 2n=43 (37A+4Bs,XX), 2n=37 (34A+1B,XY), 2n=34 (32A,XX), and 2n=31 (27A+2Bs,XX) from state of São Paulo; and same number of autosomic arms (FN=50) excluding sex chromosomes and supernumeraries. Polymorphisms are due to Robertsonian rearrangements, in addition to the variation from none to four B chromosomes, which are heterogeneous regarding morphology, heterochromatin constitution and presence of interstitial telomeric signals (ITS). ITSs were also observed in the pericentromeric regions of some biarmed autosomic pairs of three specimens. Our results revealed a high unknown diversity for Blarinomys , showing two distinct lineages corresponding to regions of the Atlantic Rainforest, besides an extraordinary chromosomal polymorphism.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-04-24T14:44:00Z No. of bitstreams: 2 Marcos_Cochak2017.pdf: 1598683 bytes, checksum: e921541055d1b3f686918cb26de4db2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-24T14:44:00Z (GMT). No. of bitstreams: 2 Marcos_Cochak2017.pdf: 1598683 bytes, checksum: e921541055d1b3f686918cb26de4db2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-07-28 / population, responsible for spontaneous abortions, problems related to infertility, and a large number of congenital anomalies that cause psychosocial and economic impact in families and also in the health system. They are present in about 1% of the liveborn, 2% of the conceptions known in women over the age of 35 years and almost all the abortions occurred in the first trimester of gestation. These anomalies involve changes in the set or structure of the chromosomes and are referred to as syndromes, such as Down syndrome or trisomy 21 is the best known, corresponding to the fourth most frequent cause of congenital anomalies. Chromosomal changes may be inherited or de novo originated, having biological influence or associated with environmental factors such as exposure to physical and chemical agents such as industrial wastes and agrochemicals. Diagnosis is made through karyotype analysis, and their knowledge is the basis for subsequent clinical treatment, prognosis, and risk-of-recurrence data for genetic counseling. Thus, the objective of this research was to analyze, through a retrospective study, human chromosomal disorders from regions of Brazil in the period of ten years (2005 to 2015), and correlates them with the use and occupation of soil through MaxEnt (maximum entropy) a predictive model for evaluation of association of occurrence of cases of chromosomal alterations as a function of soil use and occupation. In order to do so, a documentary study was carried out in the karyotype database of samples sent to the cytogenetic study of a national clinical laboratory. Of the 43,672 karyotype results, 83% (n= 36,435) were normal, 52% (n= 18,946) female and 48% (n= 17,489) male. Karyotypes with chromosomal abnormalities were found in 17% (n= 7,237), where 52% (n= 3763) female and 48% (n= 34740) male, were the most frequent aneuploidies (77% 5.558), structural changes totaled 16% (n = 1,163) and concomitant numerical and structural changes 7% (n= 516). 79.2% of the alterations involved autosomal chromosomes and 20.3% sex chromosomes, and 0.48% involved both autosomal and sexual. Among the altered cases, 9% (n= 668) were detected in mosaic. Characterizing chromosomal diseases by regions of Brazil, the highest proportion was in the South region, with 6 cases changed/100,000 inhabitants, followed by the Midwest and North regions, with 4 cases/100,000 inhabitants. The Northeast and Southeast regions presented the lowest proportions (3 cases changed/100,000 inhabitants and 2 altered cases/100,000 inhabitants respectively). When characterizing chromosomal diseases by regions of Brazil, it was possible to observe that in absolute numbers the highest frequencies occurred in the North and Northeast regions. Structural autosomal alterations were more frequent in the Southeast region, and numerical and structural concomitants were more frequent in the Northern region. Changes in mosaics were more frequent in the South, Northeast and Center-West regions, and those of single lineage were significantly more frequent in the Southeast region. Regarding land use and occupation, urbanized areas had a higher probability of occurrence of chromosomal diseases (50 to 90%), followed by areas using permanent crops (40 to 50%). This research demonstrates the prevalence of chromosomal diseases and their geographic distributions in Brazil being of great value, since studies of this genre are scarce in Brazil and, can serve as a tool to identify the incidence and recurrence risk of chromosomal diseases, enabling genetic counseling and information for the elaboration of public policies that improve the patients quality of life. / As alterações cromossômicas são doenças genéticas representativas na população, responsáveis por abortamentos espontâneos, problemas relacionados à infertilidade e grande número de anomalias congênitas, que causam impacto psicossocial e econômico nas famílias e no sistema de saúde. Estão presentes em cerca de 1% dos nativivos, 2% das concepções conhecidas em mulheres com idade acima de 35 anos e quase a totalidade dos abortos ocorridos no primeiro trimestre de gestação. Estas anomalias envolvem alterações no conjunto ou estrutura dos cromossomos e são denominadas como síndromes, dentre elas a de Down, ou trissomia do 21, é a mais conhecida, correspondendo à quarta causa mais frequente de anomalias congênitas. As alterações cromossômicas podem ser herdadas ou originadas de novo, tendo influência biológica ou associadas a fatores ambientais, como a exposição a agentes físicos e químicos, como os resíduos industriais e agrotóxicos. O diagnóstico é feito através da análise do cariótipo, e o seu conhecimento é a base para o tratamento clínico subsequente, prognóstico e dados sobre o risco de recorrência para o aconselhamento genético. Assim, o objetivo desta pesquisa foi analisar através de estudo retrospectivo as cromossomopatias humanas oriundas de regiões brasileiras no período de dez anos (2005 a 2015), e correlacioná-las com o uso e ocupação de solo através do programa MaxEnt (máxima entropia), um modelo preditivo para avaliação de associação de ocorrência de casos em função do uso e ocupação do solo. Para tanto, foi realizado um estudo documental, no banco de dados dos resultados de cariótipos, de amostras enviadas para o estudo citogenético de um laboratório clínico de abrangência nacional. Dos 43.672 resultados de cariótipo, 83% (n=36.435) foi normal, sendo 52% (n=18.946) do sexo feminino e 48% (n=17.489) masculino. Cariótipos com alterações cromossômicas foram encontradas em 17% (n=7.237), onde 52% (n=3763) do sexo feminino e 48% (n=3474) do sexo masculino, sendo as aneuploidias as mais frequentes (77%; n=5.558). As alterações estruturais somaram 16% (n=1.163) e alterações numéricas e estruturais concomitantes, 7% (n=516). Das alterações, 79.2% envolvia cromossomos autossômicos, 20,3% cromossomos sexuais e 0,48% envolvia ambos autossômicos e sexuais. Dentre os casos alterados, 9% (n=663) foi detectado em mosaico. Caracterizando as cromossomopatias por regiões do Brasil, a maior proporção de alterações por habitantes foi na região Sul, com 6/100.000, seguida pelas regiões Centro-Oeste e Norte, com 4/100.000. As regiões Nordeste e Sudeste apresentaram as menores proporções, 3/100.000 e 2/100.000, respectivamente. Ao caracterizar as frequências por regiões do Brasil, foi possível observar que em número absoluto, as maiores ocorreram nas regiões Norte e Nordeste. As alterações autossômicas estruturais na região Sudeste, e numéricas e estruturais concomitantes na região Norte. Alterações em mosaicos foram mais frequentes nas regiões Sul, Nordeste e Centro-Oeste e as de linhagem única foram mais significativamente na região Sudeste. Em relação ao uso e ocupação de solo, as áreas urbanizadas apresentaram uma maior probabilidade de ocorrência de cromossomopatias (50 a 90%), e seguida por áreas de lavouras permanentes (40 a 50%). Esta pesquisa demonstra as prevalências das alterações cromossômicas e suas distribuições geográficas no Brasil sendo de grande valia, pois estudos deste gênero são escassos no País e, poderão servir de ferramenta para identificar o risco de incidência e recorrência de doenças cromossômicas, possibilitando o aconselhamento genético e informações para a elaboração de políticas públicas que melhorem a qualidade de vida dos pacientes.

A incidência dos tumores adrenocorticais na região sul do Brasil é 10-15 vezes maior que a incidência mundial. Mutações no gene supressor tumoral p53, localizado na região 17p13.1 têm sido identificadas em diversos tumores humanos. Uma distinta mutação germinativa, Arg337His, localizada no domínio de tetramerização da proteína supressora tumoral P53 foi identificada em 35 de 36 crianças da região sul do Brasil. No presente trabalho, investigamos a presença da mutação Arg337His em 71 pacientes não relacionados, 41 adultos e 30 crianças, portadores de tumores adrenocorticais benignos e malignos. Adicionalmente, análise de perda de heterozigose do locus p53, mapa de deleção do cromossomo 17 e instabilidade cromossômica foram estudados em DNA genômico destes pacientes. Nenhum dos pacientes estudados apresentava histórico familial compatível com a síndrome de Li-Fraumeni. Sequenciamento automático permitiu a identificação da mutação Arg337His, em DNA extraído a partir de sangue periférico e/ou tecido tumoral, em 29 (24 crianças e 5 adultos) dos 71 pacientes. Nas 10 famílias em que foi possível analisar o DNA genômico de ambos os pais verificamos que a mutação Arg337His tem caráter hereditário. Por outro lado, esta mutação não foi encontrada em DNA de 160 indivíduos do grupo controle, não relacionados, analisados por sequenciamento automático e/ou digestão enzimática. A análise pareada de DNA gênomico de sangue periférico e de tecido tumoral revelou perda de heterozigose para o locus p53 em 18 de 21 (86%) pacientes portadores da mutação Arg337His. Não observamos correlação entre a presença desta mutação e o comportamento maligno dos tumores. O estudo de dois marcadores polimórficos intragênicos do p53, pelo programa de análise de tamanho de fragmento GeneScan, evidenciou um mesmo haplótipo associado à mutação Arg337His em 91% dos pacientes com tumores adrenocorticais, configurando uma origem comum para esta mutação. O estudo de 6 marcadores polimórficos ao longo do cromossomo 17 (D17S926, VNTRP53, D17S1856, D17S942, D17S1351 e D17S928) em DNA genômico pareado de 29 pacientes demonstrou uma freqüência elevada (81%) de perda do cromossomo 17 em associação à mutação Arg337His. Não observamos correlação entre a perda do cromossomo 17 e a agressividade tumoral nestes pacientes. Instabilidade cromossômica envolvendo os cromossomos 2, 9 e 11 nos 17 pacientes que perderam o cromossomo 17 foi identificada em 47%, 47% e 71%, respectivamente. Perda dos cromossomos 2 e 11 foi evidenciada em tumores benignos e malignos. A perda do cromossomo 9 foi evidenciada exclusivamente nos tumores malignos, assim como a perda concomitante de 3 ou mais cromossomos. Em conclusão, confirmamos uma freqüência elevada da mutação Arg337His em crianças brasileiras com tumores adrenocorticais benignos e malignos. Esta mutação também foi encontrada no grupo de adultos, embora em menor freqüência. Não houve correlação entre sua presença e o comportamento maligno dos tumores adrenocorticais. Efeito fundador para a mutação Arg337His e inativação bialélica do p53, caracterizada pela presença da mutação Arg337His e a perda do cromossomo 17 foram demonstradas na maioria dos casos analisados. Finalmente, a instabilidade cromossômica envolvendo três ou mais cromossomos contribuiu para o diagnóstico de carcinoma adrenocortical / The incidence of adrenocortical tumors in the South region of Brazil is 10 to 15 times higher than the worldwide one. Mutations in the tumor suppressor p53 gene, located in chromosome 17p13.1, have been described in different human tumors. A germline mutation, Arg337His, in the tetramerization domain of the tumor suppressor P53 was identified in 35 of 36 children from the South region of Brazil. In the present study we have searched for Arg337His mutation in genomic DNA of 71 non-related patients, 41 adults and 30 children, with benign or malignant adrenocortical tumors. Additionally, we also analyzed the loss of heterozigosity of p53 locus, deletion mapping of chromosome 17 and chromosome instability, in genomic DNA of these patients. None of the patients had a familial history of Li-Fraumeni syndrome. Automatic sequencing identified the Arg337His mutation in genomic DNA from peripheral leukocytes and/or tumor tissues in 29 (24 children and 5 adults) of these 71 patients. In 10 families in which the study of both parent\'s DNA was possible, the Arg337His mutation was inherited from one of the parents. Sequencing analysis and/or enzymatic restriction showed that this mutation was not present in DNA of 160 non-related control subjects. Paired analysis of genomic DNA of peripheral leukocytes and tumor tissue revealed loss of heterozigosity of p53 locus in 18/21 (86%) patients with Arg337His mutation. There was no correlation between the presence of this mutation and the malignant behavior of these tumors. The study of two intragenic polymorphic markers of p53 through GeneScan software showed the association of the same haplotype with the Arg337His mutation in 91% of patients with adrenocortical tumors, indicating a common origin of this mutation. The study of 6 polymorphic markers along chromosome 17 (D17S926, VNTRP53, D17S1856, D17S942, D17S1351, D17S928) in paired genomic DNA of 29 patients showed an increased frequency (81%) of chromosome 17 loss in association with the presence of the Arg337His mutation. We did not observe any correlation between the loss of chromosome 17 and aggressive tumor behavior in these patients. In the 17 patients who lost chromosome 17, chromosome instability of chromosomes 2, 9 and 11 was identified in 47%, 47% e 71%, respectively. Loss of chromosomes 2 and 11 was observed in benign and malignant tumors, whereas the loss of chromosome 9 was observed exclusively on malignant tumors. Similarly, the concomitant loss of 3 or more chromosomes was only observed in malignant tumors. In conclusion we confirmed an increased frequency of Arg337His mutation in Brazilian children with benign or malignant adrenocortical tumors. This mutation was also found in the adult group, although at a lower frequency. There was no correlation between the presence of the mutation and the malignant behavior of adrenocortical tumor. We demonstrated a founder effect for this mutation and also a biallelic inactivation of p53 characterized by the presence of the Arg337His mutation and the loss of chromosome 17 in most of the cases studied. Finally, chromosome instability involving 3 or more chromosomes contributed for the diagnosis of adrenocortical carcinoma in these

Rearranjos cromossômicos estruturais estão potencialmente associados ao desenvolvimento de doenças genéticas devido à disrupção, inativação ou alteração da dosagem gênica. O objetivo deste projeto foi realizar a caracterização genômica de duas pacientes e seus familiares portadores de rearranjo cromossômico estrutural envolvendo o braço curto do cromossomo 4 e o braço longo do cromossomo 12, associando técnicas de citogenética clássica (bandamento GTG), citogenética molecular (FISH) e citogenômica (array-CGH), para definição diagnóstica e maior conhecimento sobre os fatores envolvidos na correlação cariótipo-genótipo-fenótipo. Foram avaliados seis indivíduos, duas pacientes, primas em primeiro grau que apresentavam alterações fenotípicas, assim como seus familiares, portadores de translocação aparentemente equilibrada e fenótipo normal. Apesar das duas pacientes apresentarem alteração cromossômica comum, derivativo do cromossomo 4 [der(4)], foram observados achados fenotípicos distintos. A investigação permitiu a definição do diagnóstico de deleção 4p16 e trissomia 12qter para as duas pacientes com fenótipo alterado e cariótipo 46,XX,der(4)t(4;12)(p16;q24.3), a definição precisa dos pontos de quebra em 4p16.3 e 12q24.31->q24.33, assim como a determinação da origem parental do rearranjo e a definição do diagnóstico citogenético final de quatro portadores de translocação aparentemente equilibrada e cariótipo t(4;12)(4pter->4p16.3::2q24.31->12qter;12qter->12q24.31::4p16.3->4pter),direcionando o aconselhamento genético para a família. Nas duas pacientes, a técnica de array-CGH (Plataforma 2x400K, Agilent®) detectou uma diferença sutil de tamanho entre as perdas e ganhos referentes aos cromossomos envolvidos no rearranjo, sendo diagnosticado em P1 uma perda de 2.707.221 pb na citobanda 4p16.3, além de um ganho de 12.405.205 pb em 12q24.31->q24.33. A paciente 2 apresentou uma perda de 2.710.969 pb em 4p16.3 e um ganho de 12.393.885 pb em 12q24.31->q24.33. Ambas as regiões de desequilíbrio genômico incluem genes que podem ser relevantes para manifestação fenotípica observada nas pacientes, entre eles: WHSC1, NELFA, LETM1, FGFRL1 e SPON2. Os resultados da investigação citogenômica indicaram, ainda, a presença de translocação equilibrada nos quatro indivíduos portadores, não sendo detectadas perdas e/ou ganhos genômicos nas regiões dos pontos de quebra cromossômica. Os resultados obtidos na investigação do padrão de metilação dos genes FGFRL1 e SPON2 não permitiram afirmar que uma provável repressão da expressão gênica devido ao imprinting materno e paterno esteja associada às características fenotípicas distintas observadas nas duas pacientes. Embora tenha sido possível a indicação de genes correlacionados ao fenótipo das pacientes, a correlação entre a alteração genética e o fenótipo das mesmas pode depender da ação sinérgica dos mais de 190 genes envolvidos neste rearranjo cromossômico estrutural familiar. / Structural chromosomal rearrangements are potentially associated with the development of genetic disorders due to disruption, inactivation or gene dosage alterations. The objective of this project was to perform the genomic characterization of a familial structural chromosomal rearrangement involving the short arm of chromosome 4 and the long arm of chromosome 12 in two patients and carriers. The experimental approach involved using a combination of classical cytogenetic techniques (GTG banding), molecular cytogenetics (FISH) and cytogenomics (array-CGH), to provide a diagnostic definition and a better understanding of how changes in the karyotype and genotype may be associated with the phenotype. Six individuals were evaluated, two patients with phenotypic abnormalities, as well as the carriers of an apparently balanced 4p;12q translocation with normal phenotypes. Although the two patients showed a common chromosomal abnormality, the derivative chromosome 4 [der (4)], they presented distinct phenotypic findings. The investigation provided a definition of the diagnosis of 4p16 deletion and trisomy 12qter for the two patients with abnormal phenotypes and a karyotype 46,XX,der(4)t(4;12)(p16;q24.3). In addition a precise definition of the breakpoints at 4p16.3 and 12q24.31->q24.33, and the parental origin of the rearrangement was determined. A precise definition of the cytogenetic diagnosis of four carriers with an apparently balanced translocation and karyotype t(4;12)(4pter->4p16.3::2q24.31->12qter; 12qter 12q24.31->4pter::4p16.3), facilitated the genetic counseling for the family. In both patients, the array-CGH technique (2x400K Platform, Agilent®) detected a subtle difference in size between losses and gains in the chromosomal regions involved in the rearrangement. Patient 1 presented a loss of 2,707,221 bp in the cytoband 4p16.3, and a gain of 12,405,205 bp in 12q24.31->q24.33. Patient 2 had a loss of 2,710,969 bp in 4p16.3 and a gain of 12,393,885 bp in 12q24.31 -> q24.33. Both regions of genomic imbalance included genes that may be relevant to phenotypic findings observed in our patients, including: WHSC1, NELFA, LETM1, FGFRL1 and SPON2. Genomic findings also confirmed the presence of a balanced translocation in four carriers, with no genomic losses and/or gains in the regions of chromosome breakpoints. The results of the investigation of the methylation pattern of FGFRL1 and SPON2 genes could not demonstrate that repression of gene expression due to maternal and paternal imprinting was associated with the distinct phenotypes observed in the two patients. Although it has been possible to indicate genes related to the phenotype of the patients, the correlation between the genetic alteration and phenotype may depend on the synergistic action of multiple genes from more than the 190 involved in this familial chromosomal rearrangement.

L'expression des gènes peut être contrôlée à différents niveaux : transcriptionnel post-transcriptionnel, traductionnel et post­-traductionnel. A ce jour la majorité des études se sont concentrées au niveau transcriptionnel, néanmoins l'importance des mécanismes de régulation post-transcriptionnels se fait de plus en plus évidente. Chez les procaryotes cette régulation post-transcriptionnelle est assurée par les ARN régulateurs dont le mécanisme d'action passe par l'interaction directe avec les ARN messagers ou les protéines. Grâce à l'essor des analyses transcriptomiques haut-débit (RNA-seq), l'identification de ces ARN est devenue accessible, en revanche leur caractérisation fonctionnelle demeure toujours un défi. Nous avons identifié de nombreux ARN régulateurs candidats chez Agrobacterium fabrum C58 (anciennement Agrobacterium tumefaciens C58). Cette bactérie commune du sol devient phytopathogène lorsqu'elle porte le plasmide Ti (pour Tumor inducing). Elle est alors responsable de la maladie dite de la galle du collet qui se traduit par la formation de tumeurs chez les plantes. Ces travaux de thèse ont eu pour objectif de caractériser fonctionnellement des ARN régulateurs présent sur le plasmide Ti. Deux candidats ont été étudiés en combinant prédiction de cibles et analyses phénotypiques. Le premier, nommé RNA1111, a été caractérisé tant que régulateur de la virulence. Quant au deuxième, nommé QfsR, nous avons démontré qu'il régulait des gènes responsables du transfert conjugatif du plasmide Ti et de la production du signal de quorum sensing associé, mais également des gènes chromosomiques responsables de la motilité et de la production de succinoglycane. En utilisant un système rapporteur, nous avons également démontré que QfsR agissait via une interaction directe avec les ARN messagers des gènes cibles. QfsR représente le premier exemple d'ARN régulateur plasmidique régulant des cibles chromosomiques. L'existence d'un tel régulateur chez un plasmide présent transitoirement au sein des populations d'Agrobacterium illustre le dialogue entre plasmide et chromosome / Gene expression can be controlled at different levels: transcriptional, post-transcriptional, translational and post-translational. To date, the majority of studies have been concentrated on the transcriptional level, but the importance of post-transcriptional regulation mechanisms is becoming more and more evident. In prokaryotes this post-transcriptional regulation is ensured by regulatory RNAs whose mechanism of action passes through direct interaction with messenger RNAs or proteins. With development of high-throughput transcriptomic analyzes (RNA-seq), the identification of these RNAs has become accessible, but their functional characterization remains challenging. We have identified many candidate regulatory RNAs in Agrobacterium fabrum C58 (formerly Agrobacterium tumefaciens C58). This common bacterium of the soil becomes phytopathogenic when carrying the plasmid Ti (for Tumor inducing). It is then responsible for the so-called grown gall disease which results in the formation of tumors in plants. The objective of this thesis was to characterize functionally regulatory RNAs present on the Ti plasmid. Two candidates were studied by combining target prediction and phenotypic analysis. The first, named RNA1111, was characterized as a regulator of virulence. As for the second, named QfsR, we demonstrated that it regulates genes responsible for the conjugative transfer of the Ti plasmid and the production of the associated quorum sensing signal, as well as chromosomal genes responsible for the motility and succinoglycan production. Using a reporter system, we also demonstrated that QfsR was acting via direct interaction with the messenger RNAs of the target genes. QfsR represents the first example of plasmid regulatory RNA regulating chromosomal targets. The existence of such a regulator in a plasmid transiently present in the populations of Agrobacterium illustrates the dialogue between the plasmid and the chromosome

La déficience intellectuelle (DI) est définie par un QI < 70. La DI, répartie en formes non syndromiques et en formes syndromiques, est observée dans 3 % de la population. Des anomalies chromosomiques sont identifiées dans 15 % des DI syndromiques. Les translocations chromosomiques réciproques (TR) apparemment équilibrées sont observées chez 1 individu sur 1000 et seul 6 % des patients avec une TR de novo apparemment équilibrée ont une DI. Plusieurs mécanismes chromosomiques peuvent expliquer la DI syndromique associée à une TR : (i) un microremaniement déséquilibré identifié par l'utilisation de techniques plus résolutives, (ii) la formation d'un gène de fusion, (iii) un effetde position, (iv) la modification d’une région soumise à une empreinte parentale, (v) une interruption d'un gène au niveau d'un ou des deux points de cassure, (vi) une mutation génique sans rapport avec la TR, (vii) ou encore une cause acquise ou multifactorielle. Nous rapportons l'étude de 12 patients avec DI et porteurs d'une TR de novo apparemment équilibrée. L'analyse systématique par puces à ADN de ces individus a été réalisée avec une résolution de 25 kb. Un déséquilibre infracytogénétique au niveau des points de cassure ou ailleurs dans le génome a été observé chez 3/12 patients. Chez les 9 patients sans anomalies sur puces à ADN, nous avons étudié les points de cassure des remaniements de novo apparemment équilibrés. En dehors de la technique de marche sur le chromosome par FISH, deux autres approches ont été mises en oeuvre : (i) l'Array-Painting qui correspond à l'hybridation sur puces à ADN de chacun des dérivés chromosomiques préalablement séparés par Cytométrie en Flux, (ii) et le séquençage haut débit (WGS - Whole Genome Sequencing). Grâce à l'Array-Painting, nous avons identifié (i) chez 2 patients, des interruptions de gènes pouvant expliquer leur phénotype, à savoir les gènes : KIF1A, AUTS2 et EPHA6 ; (ii) et chez 1 patiente, un point de cassure entraînant une dérégulation de la transcription du gène MEF2C. L'étude par WGS a permis (i) chez 1 patiente, de diagnostiquer un déséquilibre plus complexe que celui observé par puce à ADN ; (ii) chez 2 patients, de mettre en évidence unchromothripsis, qui pourrait avoir un impact dans les pathologies constitutionnelles par interruption de gènes et/ou par effet de position ; (iii) et chez 2 autres patients, de caractériser précisément les points de cassure. Ainsi, grâce aux résultats obtenus par ces différentes techniques, plusieurs mécanismes physiopathologiques responsables de DI sont mis en évidence permettant un conseil génétique adéquat. Cependant, aucun mécanisme chromosomique commun ne peut être identifié hormis le chromothripsis observé chez patients. Finalement, ce travail nous permet principalement de comparer les techniques mises en oeuvre qui se sont avérées complémentaires. En conclusion, nous proposons une démarche diagnostique pour explorer un remaniement chromosomique apparemment équilibré chez des patients à phénotype anormal / Intellectual disability (ID) is defined by an IQ <70. ID, observed in 3% of the population, and displays heterogeneous origins, including acquired etiology (toxicologic, pathologic, traumatic) or genetic disorders with non-syndromic and syndromic forms. Numerical or structural chromosomal abnormalities are observed in 15% of patients with ID. Reciprocal balanced chromosomal translocations (RT) are observed in one individual in 1000. However, only 6% of patients carrying a de novo apparently balanced RT present ID. The relation between these balanced rearrangements and ID could be explained by different mechanisms namely (i) subtle rearrangement, (ii) gene fusion, (iii) position effect, (iv) disturbance of parental imprinting, (v) gene disruption at the breakpoints, (vi) mutation in gene unrelated to the translocation, (vii) or acquired or multifactorial cause. We report a chromosomal study of 12 patients with DI and carrying a de novo apparently balanced reciprocal translocation. A systematic analysis by microarrays was performed in all individuals (using a resolution of 25 kb). For three patients, a microdeletion was observed at the breakpoints or elsewhere in the genome. For the 9 remaining cases, we hypothesize that the phenotype is due to a disruption of gene(s) located at the breakpoint(s). In this context, we studied the breakpoints of the apparently balanced de novo rearrangements in these patients. Outside FISH walking, two approaches have been implemented namely Array-Painting, which combines flow chromosome sorting in an attempt to isolate derivative chromosomes from each other and DNA microarrays as well as Whole Genome Sequencing (WGS). Using Array-Painting, we identified (i) in 2 patients, a gene disruptions: in the KIF1A, AUTS2 and EphA6 genes; (ii) and in 1 patient, a breakpoint resulting in deregulation of transcription of the gene MEF2C. The WGS technology has permitted (i) in 1 patient, to diagnose more complex imbalance than that observed by micro-array; (ii) in 2 patients, to show a chromothripsis, (iii) and 2 other patients, to characterize precisely breakpoints. In conclusion, taking together, these results highlight different physiopathological mechanisms responsible for DI allowing adequate genetic counseling. However, no common chromosomal mechanism can be identified except for chromothripsis observed in 2 patients. In addition, this work allows us especially to compare the used techniques which seem to be complementary. Finally, we propose a pipeline to elucidate the etiology of the abnormal phenotype in patients carrying an apparently balanced rearrangement

L'étude comparative de l'architecture des génomes mammaliens a révélé l'association des séquences répétées et des réarrangements. Cette thèse porte sur la dynamique et le rôle dans les remaniements de deux types de séquences répétées: les clusters ribosomiques et les satellites. Ces séquences sont analysées par une approche cytogénomique (FISH, CO-FISH) dans le genre Mus connu pour sa diversité chromosomique, et pour lequel les phylogénies moléculaires et chromosomiques sont disponibles.1) La distribution chromosomique des clusters ribosomiques, établie chez 19 espèces, a permis de reconstruire les états ancestraux des clusters. Cette analyse montre que les clusters (24%) sont associés à des points de cassures, mais présentent également une grande labilité en l'absence de réarrangements. De plus, une forte association entre les clusters et les centromères est mise en évidence. 2) Le sous-genre Mus se caractérise par un caryotype très conservé excepté chez une sous-espèce de la souris domestique (M. musculus domesticus), qui est connue pour son extraordinaire radiation chromosomique impliquant les séquences satellites du centromère. Afin de rechercher les spécificités génomiques responsables de ce patron d'évolution contrasté, la dynamique évolutive des séquences satellites a été analysée chez 11 taxons. Révélant des différences qualitatives entre taxons, cette étude a permis de proposer un scénario évolutif de ces séquences. Toutefois, aucune des caractéristiques étudiées (composition, orientation) n'est propre à M. m. domesticus, et ne permet de rendre compte de sa plasticité chromosomique. De même, chez cette dernière, aucun lien entre la quantité de séquences satellites et la fréquence d'implication des chromosomes dans les réarrangements n'est mis en évidence.Cette étude confirme que les séquences répétées participent à l'évolution chromosomique, mais ne constituent pas à elles seules l'élément clef de cette dernière. / Comparative analyses of the architecture of mammalian genomes have highlighted the association between repetitive sequences and rearrangements. This thesis focuses on the evolutionary dynamics of two repeat sequences (ribosomal clusters and satellites) and explores their role in chromosomal change. These sequences are analyzed by a cytogenomic approach (FISH, CO-FISH) in the genus Mus that is known for its chromosomal diversity and for which molecular and chromosomal phylogenies are available.1) The chromosomal distribution of ribosomal clusters, established in 19 species, allowed us to reconstruct the ancestral states of clusters. This analysis demonstrated that 24% of clusters were associated with breakpoints, whereas others showed high lability in the absence of rearrangements. Moreover, a strong association between clusters and centromeres was retrieved.2) The subgenus Mus is characterized by a highly conserved karyotype except for one subspecies of the house mouse (M. musculus domesticus), that displays an extraordinary chromosomal radiation involving centromeric satellite sequences. To determine the genomic traits related to this difference in rate, the evolutionary dynamics of satellite sequences was analyzed in 11 taxa. From the qualitative differences evidenced between taxa, an evolutionary scenario of these sequences is proposed. None of the studied features (composition, orientation) of these sequences was found to be specific to M. m. domesticus, and could explain its chromosomal plasticity. Similarly, in the latter, no relationship between satellite sequence quantity and the rearrangement frequency of chromosomes was found.This study confirms that although repeated sequences are involved in chromosomal evolution, they aren't in themselves the key element of the latter.

TIF1γ est une protéine nucléaire de 1127 acides aminés possédant deux activités : une activité d'E3-ubiquitine ligase et des fonctions de régulateur transcriptionnel. TIF1γ exerce majoritairement ses fonctions dans les processus de développement embryonnaire et de différenciation cellulaire, notamment via son implication dans la voie de signalisation du TGFβ. Le rôle anti-tumoral de TIF1γ a été mis en évidence dans plusieurs modèles murins et son expression est diminuée dans de nombreuses tumeurs humaines de diverses origines tissulaires. Néanmoins, les mécanismes moléculaires et cellulaires par lesquels TIF1γ exerce ses fonctions oncosuppressives sont méconnus. Dans ces travaux, nous avons pu mettre en évidence le rôle inhibiteur de TIF1γ sur la transition épithélio-mésenchymateuse (EMT, Epithelial-to- Mesenchymal Transition) médiée par le TGFβ in vivo, permettant ainsi de limiter les propriétés agressives des cellules tumorales. De plus, nous avons décrit l'implication de TIF1γ dans la progression de la mitose et le point de contrôle du fuseau mitotique : les cellules n'exprimant plus TIF1γ présentent de nombreuses anomalies nucléaires ainsi qu'une forte aneuploïdie associée à une résistance aux agents ciblant les microtubules, molécules classiquement utilisées en chimiothérapie. De plus, nous avons pu corréler la faible expression de TIF1γ à une augmentation de l'instabilité chromosomique dans différentes tumeurs humaines. Ainsi, nos travaux ont permis de mettre en évidence le phénotype cellulaire induit par la perte de TIF1γ dans les cellules tumorales : instabilité chromosomique, résistance aux traitements chimiothérapeutiques et acquisition de propriétés invasives / TIF1γ / TRIM33 (Transcriptional Intermediary Factor 1γ / TRIpartite Motif-containing 33) is a 1,127 amino acids nuclear protein with two biochemical activities: an E3-ubiquitin ligase activity and transcriptional regulatory functions. TIF1γ is ubiquitously expressed in many organisms and exerts its functions mainly in the processes of embryonic development and cell differentiation, particularly through its involvement in the TGFβ signaling pathway. The oncosuppressive functions of TIF1γ have been demonstrated in several mouse models and its expression is reduced in many human tumors of various tissue origins. Nevertheless, the molecular and cellular mechanisms driving TIF1γ anti-tumoral activities are unknown. In this work, we highlight its inhibitory role on TGFβ-mediated EMT (Epithelial-to-Mesenchymal Transition) in vivo, thus limiting the aggressive properties of tumor cells. In addition, we describe TIF1γ involvement in mitotic progression and the Spindle Assembly Checkpoint (SAC): TIF1γ deleted cells display many nuclear abnormalities, aneuploidy and resistance to spindle microtubule-disrupting agents, which are drugs classically used in chemotherapeutic treatments. Finally, we correlated the low expression level of TIF1γ to an increased rate of chromosomal instability in different human tumors. Thus, our work has highlighted the tumor suppressor role of TIF1γ: its deletion in tumor cells induce chromosomal instability, resistance to chemotherapeutic treatments and acquisition of invasive properties

No description available.