A presença de DNA fetal livre de células no plasma materno possibilitou o surgimento de novas tecnologias para o diagnóstico pré natal não invasivo. Existem técnicas de genotipagem fetal já estabelecidas em investigações de sequências exclusivas fetais, mas a sua aplicação para a identificação humana em testes de paternidade ainda é pouco conhecida. A metodologia de genotipagem fetal não invasiva foi padronizada com o uso de três lócus InDels e iniciadores inserção-específicos. Entretanto, para que se alcance o poder satisfatório, é necessário investigar elevado número de lócus informativos. Para suprir a ausência de informações suficientes sobre lócus deste tipo, são descritas, no presente trabalho, as condições laboratoriais para PCR e as frequências alélicas de um conjunto de lócus InDels, ainda inéditos, em uma amostra representativa da população urbana brasileira, visando aumentar a robustez e a confiabilidade da genotipagem fetal não invasiva na investigação de paternidade. Foram selecionados 20 lócus, um em cada cromossomo. Em um dos lócus polimórficos foi encontrado um alelo novo, não registrado no banco de dados do NCBI. O poder de discriminação e o poder de exclusão dos 16 lócus polimórficos combinados (0,999 e 0,937, respectivamente) tiveram valores dentro do esperado para um conjunto com essa quantidade de lócus bialélicos. Alguns lócus apresentaram elevado número de indivíduos que não responderam à amplificação, indicando a necessidade de reajustes na padronização dos procedimentos laboratoriais e a possibilidade de variabilidade das sequências complementares aos iniciadores empregados, o que exigirá o sequenciamento completo das regiões. Para a maioria dos lócus, entretanto, os parâmetros forenses atingiram valores esperados e sugerem que podem ser úteis na composição de painel robusto e eficiente para ser usado na genotipagem fetal não invasiva. / The presence of cell-free fetal DNA in maternal plasma allows for new non-invasive prenatal diagnosis technologies to develop. Even though there are already well-established fetal genotyping techniques in fetal-exclusive sequence investigations, their application to human identification in paternity tests is not yet well-known. Non-invasive fetal genotyping methodology was previously standardized using three InDel loci and insertion-specific primers. However, in order to attain satisfactory power, it is necessary to investigate large number of informative loci. To compensate for the absence of sufficient information about this type of locus, this work describes the PCR conditions and determinates allelic frequencies for a set of unpublished InDel loci, in a representative group of Brazilian population, raising the robustness and reliability of non-invasive fetal genotyping in paternity investigation. We selected 20 loci, one in each chromosome. Not previously registered on NCBI database allele was found in one of the polymorphic loci. Discrimination and exclusion power of the 16 polymorphic loci combined (0.999 and 0.937, respectively) had expected values for an ensemble with such an amount of biallelic loci. Some loci showed many non-amplified individuals, vincing the need of corrections in the standardizing process and the possible variability of the complementary sequences to the primers used. However, for most loci, the forensic parameters reached the expected values and suggest that they may be useful in the more robust panel for utilization in non-invasive fetal genotyping.

Made available in DSpace on 2016-08-19T18:16:00Z (GMT). No. of bitstreams: 1 FABIO FRANCA SILVA.pdf: 387985 bytes, checksum: b614ae131f06a658fbdac4196b4d623c (MD5) Previous issue date: 2009-04-06 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous pregnancy losses before the 20th week of gestation, a situation that occurs in 1 to 2% of women in reproductive age. Genetic, anatomical, endocrine, infectious and immunologic factors through mechanisms that relate to the Major Histocompatibility Complex (MHC) and the presence of certain HLA (Human Leukocyte Antigens) are associated to RSA. HLA gene is located on the short arm of chromosome 6 between the 6p21.31 and 6p21.33 regions. This gene is inherited in haplotypes and expressed in codominance, having influence on modulation and induction of mother tolerance during the pregnancy. The aim of this study was to compare the allelic frequencies of HLA-A*, HLA-B* and HLA-DRB1* loci in women with and without RSA. It was a case-control study in 200 women (100 for each group) between 18 and 35 years of age. All samples were typified by the PCR-SSOP method (Polymerase Chain Reaction-Sequence Specific Oligonucleotide Probes). The most frequent alleles observed in the group of women with and without RSA were: HLAA* 02 (56%) and (49%), HLA-DRB1*13 (31%) and (39%) respectively - there was no statistical significative difference when compared among the groups for this alleles; HLA-A*24 (12%) e (25%), (OR: 0.41; 95% CI: 0.18-0.92; p=0.028); HLA-A*34 (8%) e (1%), (OR: 8.61; 95% CI: 1.06-187.04; p=0.034); HLA-B*35 (16%) e (41%) (OR: 0.27; 95% CI: 0.13 0.56; p=0.0002). The most frequent haplotypes observed in the group of women with and without RSA were: A*02DRB1*16 (12%) e (2%) (OR: 6.68; 95% CI: 1.36 44.52; p=0.012) respectively. In this research, DRB1* locus in women with RSA was in linkage disequilibrium (p=0.01.). The high frequency of HLA-A*02 and HLA-DRB1*13 alleles in this study was due to the wide distribution of this allele in the population of Maranhão. HLA-A*24 e HLA-B*35 alleles were considered as a protection factor and HLA-A*34 allele was considered as a risk factor to RSA. The A*02DRB1*16 haplotype was the most frequent and considered as a risk factor to RSA. In order to confirm the observed results in this research, a study involving a higher sample size is necessary as well as genetic epidemiology researches to shed light on the role of HLA antigens and/or its connection to other genes as a risk factor. / Abortamento Espontâneo Recorrente (AER) caracteriza-se por duas ou mais perdas conceptuais espontâneas e consecutivas antes da 20ª semana de gestação, acometendo entre 1% e 2% das mulheres em idade reprodutiva. Fatores genéticos, anatômicos, endócrinos, infecciosos e imunológicos, por meio de mecanismos que relacionam o Complexo Principal de Histocompatibilidade (CPH) e a frequência de determinados antígenos HLA (Antígeno Leucocitário Humano), estão associados ao AER. O gene HLA localiza-se no braço curto do cromossomo 6 entre as regiões 6p21.31 e 6p21.33, é herdado em bloco e expresso em co-dominância. O mesmo exerce uma grande influência na modulação e indução da tolerância materna durante a gestação. Esta pesquisa teve como objetivo verificar as frequências alélicas dos loci HLA-A*, -B* e - DRB1* em mulheres com e sem AER. Realizou-se um estudo caso-controle em 200 mulheres (100 para cada grupo), entre 18 e 35 anos de idade. Todas as amostras foram tipificadas pelo método PCR-SSOP (Reação em cadeia da Polimerase Sondas de Oligonucleotídios de Sequências Especificas). Os alelos mais frequentes tanto em mulheres com e sem AER foram, respectivamente: HLA-A*02 (56%) e (49%), HLADRB1* 13 (31%) e (39%)-embora sem resultado estatisticamente significante; HLAA* 24 (12%) e (25%), (OR: 0,41; 95% IC: 0,18-0,92; p=0,028); HLA-A*34 (8%) e (1%), (OR: 8,61; 95% IC: 1,06-187,04; p=0,034); HLA-B*35 (16%) e (41%) (OR: 0,27; 95% IC: 0,13 0,56; p=0,0002). Os haplótipos mais frequentes em mulheres com e sem AER foram, respectivamente: A*02DRB1*16 (12%) e (2%) (OR 6,68; 95% IC: 1,36 44,52; p=0,012). No presente estudo, apenas o locus DRB1* apresentou desequilíbrio de ligação significante (p=0,01) em mulheres com AER. A elevada frequência dos alelos HLA-A*02 e HLA-DRB1*13 é justificada pela ampla distribuição desses alelos na população maranhense. Os alelos HLA-A*24 e HLA-B*35 apresentaram-se como um fator de proteção e o alelo HLA-A*34 um fator de risco para AER. Para as associações haplotípicas, o haplótipo A*02DRB1*16 foi mais frequente em mulheres com AER, sendo um fator de risco para este grupo. Para a ratificação dos resultados deste trabalho, faz-se necessário aumentar o número amostral, bem como estudos de epidemiologia genética para o melhor entendimento do papel dos antígenos HLA e/ou sua ligação a outros genes como fator de risco para o AER.

La prise en charge du mélanome métastatique a été bouleversée par les thérapies ciblées comme les inhibiteurs de RAF (RAFi) et les inhibiteurs de MEK (MEKi). Les RAFi permettent dans le mélanome BRAF V600 muté d’améliorer la survie mais leur effet est limité en monothérapie par l’hyperactivation paradoxale de la voie des MAPK dans les cellules non BRAF V600 mutée qui est responsable de l’apparition de mélanomes primitifs induits. Dans ce travail nous montrons la différence entre les modifications transcriptomiques induites par les RAFi dans des lignées cellulaires de mélanome BRAF mutées par rapport à celles induites dans des lignées non BRAF mutées ce qui a permis d’identifier des gènes potentiellement impliqués dans le développement de mélanomes induits. L’efficacité des RAFi est également limitée dans le temps par la survenue de résistances acquises. Nous montrons qu’une charge allélique mutée (CAM) de BRAF V600 élevée est potentiellement associée à une bonne réponse au traitement par RAFi et que la plasticité cellulaire induite par ZEB1 est associée à la résistance aux RAFi. Il n’existe pour le moment pas de thérapie ciblée approuvée dans le mélanome métastatique NRAS muté. Les MEKi sont limités par des résistances qui ne leur ont pas permis d’améliorer significativement la survie des patients. Nous montrons qu’une amplification de NRAS et la mutation MEK P124L sont responsables de résistance aux MEKi dans le mélanome NRAS muté. Nous avons testé le tramétinib (MEKi) en association avec d’autres thérapies ciblées dans des PDX de mélanome NRAS muté afin de mettre en évidence des combinaisons qui améliorent l’efficacité anti tumorale des MEKi dans cette indication / Targeted therapies like RAF inhibitors (RAFi) and MEK inhibitors (MEKi) have completely changed the therapeutic landscape in metastatic melanome. RAFi offer increased survival in patients with BRAF V600 mutated metastatic melanoma but they are limited in their use as single agents by an hyperactivation of the MAPK signaling pathway in non BRAF mutated cells that is responsible for the developpement of treatment induced primary melanomas. In this work we show the differences in the transcriptomic modifications induced by RAF inhibitos in BRAF mutated melanoma cell lines and NRAS mutated cell lines and we identified potential genes implicated in the development of treatment induced melanomas. The anti-tumor efficacity of RAFi is also limited by the acquired resistances that appear after several months of treatment. We show that a high BRAF V600E mutant allelic burden is potentially associated with a good response to RAFi and that ZEB1 induced cell plasticity is responsible for resistance to RAFi. No targeted therapy is approuved for NRAS mutated melanoma. Innate and acquired resistances limit the developpment of MEKi in this setting. We show that NRAS amplification and a MEK P124L mutation are responsible for resistance in NRAS melanoma. Finally, we tested five targeted therapies in combination with trametinib in NRAS mutated PDXs to identify combinations that improve the anti-tumoral effect of MEKi in this setting

<p>In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.</p><p>The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.</p>

In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA. The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

En 1975, Wilson et King ont proposé que l'évolution opère non seulement via des changements affectant la structure des protéines, mais aussi via des mutations qui modifient la régulation génétique. L'étude des éléments régulateurs de l'expression génétique a un rôle important dans la compréhension de l'expression de différentes maladies et de la réponse thérapeutique. Nous avons développé un algorithme bio- informatique qui nous permet rapidement de trouver des sites de régulation génétique à travers tout le génome et pour une grande quantité de gènes. Notre approche consiste à trouver des sites polymorphes (SNPs) qui sont en déséquilibre de liaison avec le débalancement allélique (AI) afin de cartographier la région régulatrice et le site responsable. Notre méthode est avantageuse par rapport à d'autres méthodes, car elle n'a pas besoin des données « phasées». De plus, les données de débalancement allélique ne sont pas affectées par des facteurs externes étant donné qu'ils sont mesurés dans la même cellule. Nous avons démontré que notre approche est fiable et qu'elle peut détecter des sites loin du gène. De plus, il peut être appliqué à des données de génotypage sans avoir besoin de les « phaser » . / Wilson and King (1975) proposed that evolution frequently operates through mutations affecting genetic regulation. Likewise, it is expected that genetic variation responsible for inter-individual differences will be due to variation in regulatory sites. Identifying such sites is thus important in the genetic and medical research. We have developed a new bioinformatics algorithm to find genome-wide regulatory sites for a big number of genes. Individuals carrying different alleles at a regulatory site will exhibit allelic imbalance(AI) due to differential expression of the two copies the same locus. Our approach consists of searching polymorphic sites (SNPs) in linkage disequilibrium with AI in order to map regulatory regions. We have detected many SNPs associated to the regulation of different genes pointed in previous studies. We have also found regulatory regions far from the transcription start site (TSS). The major advantage of this method is that phased data is not needed. In addition, AI data has the benefit of not being affected by external factors since it is measured in the same cell. The results show that our approach is reliable and it can detect sites far from the gene.

Made available in DSpace on 2016-06-02T20:21:29Z (GMT). No. of bitstreams: 1 4293.pdf: 642637 bytes, checksum: 6b7a62fa5cf6f1f58e418c8414593c06 (MD5) Previous issue date: 2012-02-29 / Financiadora de Estudos e Projetos / Tenderness is the main trait appreciated by consumers of bovine meat, however Nellore animals that comprise the largest part of Brazilian cattle, have lower tenderness when compared with European animals. In this way, it is essential understand the variability of genes associated to tenderness as well as their mechanisms of allelic expression, considering that deviations of expression depending on the parental origin of the allele have been described for some genes, and these phenomena must be understood to be incorporated in animal breeding programs. Therefore, the aim of this study was to evaluate the variability of SNPs in candidate genes for tenderness and investigate the expression pattern of their alleles. For this purpose, genotypes of SNPs in CAST, CAPN1, DGAT1, leptin, KCNJ11 and IGFBP3 genes were determined in 30 sires, in which the genes DGAT1, leptin and IGFBP3 were not polymorphic. After this, polymorphic SNPs in population of sires were genotyped in their offspring by TaqMan® assay in real time PCR, followed by allelic expression analysis. In the expression analysis, to discriminate each allele of the gene it was utilized one SNP, or two in the case of KCNJ11. All genes presented biallelic expression in adult muscular tissue. However, with exception of CAPN1 for which the allelic expression difference was not significant, the allelic expression ratio was significantly different of 1 for SNP of CAST (1.3±0.03) and the two SNPs of KCNJ11 SNP 2126T>C (1.2±0.04) and SNP 2942C>T (1.46±0.04). Differential allelic expression (DAE) found in SNP 2126T>C of KCNJ11 and in CAST were not influenced by parental origin of allele, but the differences in allelic expression for SNP 2942C>T of gene KCNJ11 showed parental origin effect and influence by genotype of the other SNP of KCNJ11. It was conclude that the CAST, CAPN1 and KCNJ11 are polymorphic in this population, they are not imprinted, but CAST and KCNJ11 showed differential allelic expression. / A maciez é o principal atributo apreciado pelos consumidores da carne bovina, no entanto, animais da raça Nelore, que compõem a maior parte do rebanho brasileiro, apresentam carne menos macia, quando comparados a animais de origem europeia. Assim, é fundamental compreender a variabilidade de genes associados à maciez além de seus mecanismos de expressão alélica, já que desvios da expressão em função da origem parental do alelo têm sido descritos para alguns genes e tais fenômenos precisam ser compreendidos para serem incorporados nos programas de melhoramento animal. Portanto, o objetivo deste trabalho foi avaliar a variabilidade de SNPs contidos em genes candidatos a influenciar a maciez da carne e compreender o padrão de expressão de seus alelos. Para isso, os genótipos para SNPs contidos nos genes CAST, CAPN1, DGAT1, leptina, KCNJ11 e IGFBP3 foram determinados em uma população de 30 touros, nos quais os genes DGAT1, leptina e IGFBP3 não se mostraram polimórficos. Os SNPs polimórficos na população de touros foram então genotipados na progênie, por ensaio TaqMan® em PCR em tempo real, seguida da avaliação da expressão alélica dos mesmos. Na análise de expressão utilizou-se um SNP para discriminar cada alelo do gene, ou dois SNPs, no caso do KCNJ11. O padrão de expressão de todos os genes foi bialélico no tecido muscular adulto. Entretanto, com exceção do CAPN1, para o qual a diferença de expressão alélica não foi significativa, as razões de expressão alélica foram significativamente diferentes de 1 para os genes CAST (1,3±0,03) e os dois SNPs analisados do KCNJ11 SNP 2126T>C (1,2±0,04) e SNP 2942C>T (1,46±0,04). A expressão alélica diferencial (EAD) encontrada no SNP 2126T>C do KCNJ11 e no CAST não foram influenciadas pela origem parental do alelo, mas a diferença de expressão alélica no SNP 2942C>T do gene KCNJ11 apresentou efeito de origem parental, além da influencia do genótipo do outro SNP do KCNJ11. Concluiu-se então que os genes CAST, CAPN1 e KCNJ11 são polimórficos na população, não são imprinted, mas os genes CAST e KCNJ11 apresentam expressão alélica diferencial.

Objective: Mutations in the parkin (PARK2) gene have been associated with autosomal recessive early-onset Parkinson's disease (EOPD) with various frequencies in different populations. The aim of the study is to describe phenotypic characteristics of Czech EOPD patients, to evaluate the influence of environmental risk factors, and to determine the frequency of parkin allelic variants in patients and healthy controls. Methods: A total of 70 EOPD patients (age at onset ≤ 40 years) and 75 controls were phenotyped and screened for the sequence variants and exon rearrangements in the parkin gene. Results: The main features in the phenotype of the patients' sample were: the absence of cognitive deficit, high occurrence of dystonia, depression, hyperhidrosis, an excellent response to dopaminergic therapy, early onset of dyskinesia and motor fluctuation. Patients with mutations in the parkin gene had significantly lower age at onset. The agricultural occupation and work with chemicals increased the risk of EOPD, however the coffee drinking appeared to be a protective factor. Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous...

O NER é uma via de reparo de DNA capaz de lidar com uma ampla variedade de lesões. Participam do NER diversas proteínas, entre elas a endonuclease XPG. Pacientes que possuem mutações no gene XPG apresentam a síndrome XP, e em alguns casos XP/CS. Investigamos a natureza genética de dois pacientes XP-G, que são irmãos e apresentam fenótipo moderado. As células destes pacientes demonstraram alta sensibilidade à luz UVC. Quando expostas a um agente oxidativo, apenas células XP-G/CS exibiram sensibilidade. Identificamos duas mutações missense no gene XPG desses pacientes, e comparamos com outras mutações existentes. Observamos que as mutações possuem um impacto negativo na funcionalidade de XPG. A proteína com a mutação p.Ala28Asp exibiu uma atividade residual em testes de complementação. Os resultados indicam que o fenótipo XP-G desses pacientes é causado por duas mutações missense em heterozigose composta, e que células portadoras dessas alterações exibem respostas diferenciadas frente aos estresses genotóxicos causados pela luz UV e pelo agente oxidativo utilizado. / NER is the most flexible of all known DNA repair mechanisms. XPG is an endonuclease that participates in the final steps of NER. Mutations in this gene may result in the human syndrome XP and, in some cases, in the XP/CS. We investigated the genetic nature in two XP-G patients, siblings and mildly affected. The cells from these patients demonstrated the high UV sensitivity typical of this syndrome. When exposed to an oxidative agent, only XP-G/CS cells exhibited sensitivity. We identified two missense mutations in the XPG gene of these patients, and a comparison with other known mutations is presented. These mutations have a negative impact in the function of XPG. The protein harboring the mutation p.Ala28Asp exhibited residual activity in complementation tests. These results indicate that the phenotype of XP-G patients is caused by two missense mutations in a compound heterozygous manner, and that the cells carrying these alterations exhibit different responses against genotoxic stress caused by the UV light and by the oxidative agent used.