Phylogenetic relationships of Cyprinidae (Teleostei : Cypriniformes) inferred from the partial S6K1 gene sequences and implication of indel sites in intron 1

The family Cyprinidae is widely distributed in East Asia, and has the important phylogenetic significance in the fish evolution. In this study, the 5' end partial sequences (containing exon 1, exon 2 and indel 1) of S6K1 gene were obtained from 30 representative species in Cyprinidae and outgroup using PCR amplification and sequencing. The phylogenetic relationships of Cyprinidae were reconstructed with neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. Myxocyprinus asiaticus (Catostomidae) was assigned to the outgroup taxon. Similar phylogenetic relationships within the family Cyprinidae were achieved with the four analyses. Leuciscini and Barbini were monophyletic lineages respectively with the high nodal supports. Leuciscini comprises Hypophthalmichthyinae, Xenocyprinae, Cultrinae, Gobioninae, Acheilognathinae and East Asian species of Leuciscinae and Danioninae. Monophyly of East Asian clade was supported with high nodal support. Barbini comprises Schizothoracinae, Barbinae, Cyprininae and Labeoninae. The monophyletic lineage consisting of Danio rerio, D. myersi, and Rasbora trilineata was basal in the tree. In addition, the large fragment indels in intron 1 were analyzed to improve the understanding of Cyprinidae relationships. The results showed that the large fragment indels were correlated with the relations among species. Some conserved regions in intron 1 were thought to be involved in the functional regulation. However, no correlation was found between sequence variations and species characteristic size.

HLA-DRB1 allele polymorphisms in genetic susceptibility to esophageal carcinoma

AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province. METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics. RESULTS: Allele frequency (AF) of HLA-DRB1*0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1*0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles. CONCLUSION: HLA-DRB1*0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1*0901 may be susceptible to esophageal carcinoma.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 13%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Heterozygous ATM mutations do not contribute to early onset of breast cancer

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

LNK mutations and myeloproliferative disorders

The lymphocyte adaptor protein (LNK) is one of a family of adaptor proteins involved cell signalling and control of B cell populations. It has a critical role in regulation of signalling in hematopoiesis. Lnk negatively regulates cytokine initiated cell signalling and it functions as a negative regulator of the mutant protein in myeloproliferative neoplasms JAK2V617F. A number of mutations in LNK have been described in a variety of myeloproliferative neoplasms some of which have been demonstrated to cause increased cellular proliferation. The majority of mutations occur in exon 2. In a small number of cases idiopathic erythrocytosis with subnormal erythropoietin levels LNK mutations have been found which may account for the clinical phenotype. Thus investigation for LNK mutations should be considered in the investigation of idiopathic erythrocytosis and perhaps other myeloproliferative neoplasms.

Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor alpha gene

Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants.

The role of molecular, clinical and socioeconomic factors in the long-term survival of axillary node negative breast cancer patients

The overall objective of this study was to investigate factors associated with long-term survival in axillary node negative (ANN) breast cancer patients. Clinical and biological factors included stage, histopathologic grade, p53 mutation, Her-2/neu amplification, estrogen receptor status (ER), progesterone receptor status (PR) and vascular invasion. Census derived socioeconomic (SES) indicators included median individual and household income, proportions of university educated individuals, housing type, "incidence" of low income and an indicator of living in an affluent neighbourhood. The effects of these measures on breast cancer-specific survival and competing cause survival were investigated. A cohort study examining survival among axillary node negative (ANN) breast cancer patients in the greater Toronto area commenced in 1 989. Patients were followed up until death, lost-to-follow up or study termination in 2004. Data were collected from several sources measuring patient demographics, clinical factors, treatment, recurrence of disease and survival. Census level SES data were collected using census geo-coding of patient addresses' at the time of diagnosis. Additional survival data were acquired from the Ontario Cancer Registry to enhance and extend the observation period of the study. Survival patterns were examined using KaplanMeier and life table procedures. Associations were examined using log-rank and Wilcoxon tests of univariate significance. Multivariate survival analyses were perfonned using Cox proportional hazards models. Analyses were stratified into less than and greater than 5 year survival periods to observe whether known markers of short-tenn survival were also associated with reductions in long-tenn survival among breast cancer patients. The 15 year survival probabilities in this cohort were: for breast cancerspecific survival 0.88, competing causes survival 0.89 and for overall survival 0.78. Estrogen receptor (ER) and progesterone receptor (PR) status (Hazard Ratio (HR) ERIPR- versus ER+/PR+, 8.15,95% CI, 4.74, 14.00), p53 mutation (HR, 3.88, 95% CI, 2.00, 7.53) and Her-2 amplification (HR, 2.66, 95% CI, 1.36, 5.19) were associated with significant reductions in short-tenn breast cancer-specific survival «5 years following diagnosis), however, not with long-term survival in univariate analyses. Stage, histopathologic grade and ERiPR status were the clinicallbiologieal factors that were associated with short-term breast cancer specific survival in multivariate results. Living in an affluent neighbourhood (top quintile of median household income compared to the rest of the population) was associated with the largest significant increase in long-tenn breast cancer-specific survival after adjustment for stage, histopathologic grade and treatment (HR, 0.36, 95% CI, 0.12, 0.89).

Caractérisation cytogénétique et clinique des gènes de fusion impliquant MLL dans les leucémies

Le gène MLL (Mixed-Lineage Leukemia), un homologue du gène trithorax de la Drosophile, localisé à la bande chromosomique 11q23, est fréquemment réarrangé dans plusieurs types de leucémies, essentiellement suite à des translocations chromosomiques. Dans les différentes translocations chromosomiques, la partie N-terminale de MLL est fusionnée avec les séquences d’un gène partenaire. Malgré le grand nombre de partenaires de fusion rapportés, peu de fusions MLL ont été bien caractérisées sur le plan moléculaire. De plus, l’impact pronostique de plusieurs fusions moins fréquentes n’est pas bien établi. L’objectif de mon projet est de caractériser plusieurs translocations MLL qui ont été détectées dans 39 spécimens leucémiques collectés par la Banque de cellules leucémiques du Québec (www.bclq.gouv.qc.ca), et d’établir une corrélation entre les résultats de la cytogénétique et différents paramètres biologiques et cliniques des leucémies respectives. L’identification des gènes partenaires de fusion (GPF) dans notre série (30 échantillons étudiés), a révélé la fusion de MLL à un gène partenaire très récurrent dans 26 leucémies: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; à un GPF modérément commun dans 1 leucémie : MLLT6(AF17); et à un partenaire rare de MLL dans 3 leucémies : GAS7 et AF15/CASC5 (2 cas). Nous avons poursuivi notre travail avec la caractérisation des points de cassure de deux fusions, soit MLL-ELL associée à un syndrome myéloprolifératif (une association rare), et MLL-GAS7 (une fusion rare de MLL), associée à une leucémie aiguë myéloïde. L’analyse des transcrits de fusion par RT-PCR et séquençage a révélé respectivement la fusion de l’exon 9 de MLL à l’exon 2 de ELL et des exons 7 ou 8 de MLL (deux transcrits) à l’exon 2 de GAS7. Ce travail permettra d’effectuer des études fonctionnelles et des projets de recherche translationnelle en utilisant ces spécimens de leucémies avec différents réarrangements de MLL, bien caractérisés sur le plan clinique et moléculaire.

Étude des déterminants moléculaires de la signalisation des récepteurs couplés aux protéines G et développement d'outils pour l'étude de l'effecteur bêta-arrestine.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines membranaires du génome humain. Ils transmettent les signaux extracellulaires provenant de plusieurs stimuli comme les odeurs, les ions, les hormones et les neurotransmetteurs, à l'intérieur des cellules. En se liant aux RCPGs, ces molécules contribuent à la stabilisation des changements conformationnels activateurs qui se propagent jusqu'au domaine intracellulaire des récepteurs. Ces derniers engagent ensuite un ou plusieurs effecteurs, comme les protéines G hétérotrimériques et les β-arrestines (βarrs), qui activent une cascade d'événements moléculaires menant à la réponse cellulaire.Récemment, la publication de structures cristallines de RCPGs liant des ligands diffusibles a offert une opportunité de raffiner à une résolution atomique les modèles des mécanismes de transduction des signaux. Dans la première partie de cette thèse, nous avons donc exploré les déterminants de la signalisation du récepteur prototypique β2-adrénergique (β2AR), induite par les β-bloqueurs. En ne tenant compte que de leur efficacités sur le β2AR dans les voies de l'adénylate cyclase (AC) et des protéines kinases activées par les facteurs mitogéniques (MAPK), les β-bloqueurs peuvent être classés en 3 groupes distincts (agoniste inverse AC / agoniste MAPK, antagoniste neutre AC / agoniste MAPK et agoniste inverse AC / agoniste inverse MAPK). Afin de déterminer le lien entre leur efficacité et leur mode de liaison, nous avons réalisé des expériences d'arrimages moléculaires in silico entre des β-bloqueurs de chacun des groupes et la structure cristalline du β2AR liée au carazolol. De manière intéressante, les ligands à l'intérieur d'un groupe partagent un mode de liaison, alors que ceux des ligands entre les groupes divergent, suggérant que le mode de liaison des β-bloqueurs pourrait être utilisé pour prédire leur l'efficacité. En accord avec cette hypothèse, nous avons prédit et confirmé l'efficacité agoniste MAPK du carazolol, un inverse agoniste AC du β2AR se liant au récepteur de manière similaire au groupe inverse agoniste AC / agoniste MAPK. De manière intéressante, le groupement aryl des ligands agonistes inverses agonistes AC / agoniste MAPK, le seul groupement chimique variable de ce groupe, est prédite pour lier la région des 3e et 5e hélices transmembranaires (TM3 et TM5). Nous avons donc émis l'hypothèse que cette région pourrait être un déterminant de l'efficacité de ces ligands. En accord avec cette dernière, la mutation de 2 résidus (T118I, S203A) localisés proches du site de liaison des groupements aryls des β-bloqueurs, prévient l'efficacité agoniste inverse de l'ICI-118551 sur la voie de l'AC sans affecter l'efficacité d'un agoniste, indiquant que cette région est importante pour la transmission de l'effet agoniste inverse, du moins sur la voie de l'AC. Les βarrs sont des protéines d'échafaudage qui coordonnent la formation de complexes avec plusieurs dizaines d'effecteurs de signalisation. Originalement identifiées pour leur rôle dans la désensibilisation et l'internalisation des RCPGs, elles sont aussi d'importants effecteurs de la signalisation des RCPGs indépendante des protéines G hétérotrimériques. Cependant, contrairement aux protéines G hétérotrimériques, il n'existe que peu d'outils pour les étudier. Ainsi, la deuxième partie de la thèse est dédiée au développement d'outils pour l'étude des βarrs. À cette fin, nous avons d'abord tenté de transposer une méthode de mesure de l'interaction entre 2 protéines par la technologie de transfert d'énergie de bioluminescence par résonance (BRET) en microscopie et chez des souris transgéniques afin de mesurer de manière subcellulaire et dans un contexte natif l'engagement de la βarr à des RCPGs. Ainsi, nous avons établi les preuves de principe que le BRET peut être utilisé pour localiser l'interaction entre la βarr et le récepteur de la vasopressine de type 2 (V2R) sur une cellule au microscope et pour détecter l'interaction entre la βarr et le β2AR sur des tissus de souris transgéniques exprimant ces protéines fusionnées avec des partenaires BRET. Finalement, il n'existe aucun inhibiteur pharmacologique ciblant les βarrs. Ainsi, grâce à la combinaison d'approches de criblage virtuel sur un modèle de la structure des βarrs et d'essais de validation cellulaire, nous avons développé un inhibiteur pharmacologique des βarrs. À l'aide de cet outil, nous avons confirmé l'implication des βarrs dans l'activation des MAPK par le V2R, mais aussi montré un nouveau rôle des βarrs dans le recyclage du β2AR. Les connaissances et outils développés dans cette thèse permettront de mieux comprendre les déterminants moléculaires de la signalisation des RCPGs et entre autres, grâce à des nouvelles approches pour étudier le rôle cellulaire et physiologique des βarrs.

HLA-A Typing in formalin-fixed paraffin embedded tissue samples : towards potential retrospective analysis

El Antígeno Leucocitario Humano (HLA en inglés) ha sido descrito en muchos casos como factor de pronóstico para cáncer. La característica principal de los genes de HLA, localizados en el cromosoma 6 (6p21.3), son sus numerosos polimorfismos. Los análisis de secuencia de nucleótidos muestran que la variación está restringida predominantemente a los exones que codifican los dominios de unión a péptidos de la proteína. Por lo tanto, el polimorfismo del HLA define el repertorio de péptidos que se unen a los alotipos de HLA y este hecho define la habilidad de un individuo para responder a la exposición a muchos agentes infecciosos durante su vida. La tipificación de HLA se ha convertido en un análisis importante en clínica. Muestras de tejido embebidas en parafina y fijadas con formalina (FFPE en inglés) son recolectadas rutinariamente en oncología. Este procedimiento podría ser utilizado como una buena fuente de ADN, dado que en estudios en el pasado los ensayos de recolección de ADN no eran normalmente llevados a cabo de casi ningún tejido o muestra en procedimientos clínicos regulares. Teniendo en cuenta que el problema más importante con el ADN de muestras FFPE es la fragmentación, nosotros propusimos un nuevo método para la tipificación del alelo HLA-A desde muestras FFPE basado en las secuencias del exón 2, 3 y 4. Nosotros diseñamos un juego de 12 cebadores: cuatro para el exón 2 de HLA-A, tres para el exón 3 de HLA-A y cinco para el exón 4 de HLA-A, cada uno de acuerdo las secuencias flanqueantes de su respectivo exón y la variación en la secuencia entre diferentes alelos. 17 muestran FFPE colectadas en el Hospital Universitario de Karolinska en Estocolmo Suecia fueron sometidas a PCR y los productos fueron secuenciados. Finalmente todas las secuencias obtenidas fueron analizadas y comparadas con la base de datos del IMGT-HLA. Las muestras FFPE habían sido previamente tipificadas para HLA y los resultados fueron comparados con los de este método. De acuerdo con nuestros resultados, las muestras pudieron ser correctamente secuenciadas. Con este procedimiento, podemos concluir que nuestro estudio es el primer método de tipificación basado en secuencia que permite analizar muestras viejas de ADN de las cuales no se tiene otra fuente. Este estudio abre la posibilidad de desarrollar análisis para establecer nuevas relaciones entre HLA y diferentes enfermedades como el cáncer también.

Enfermedad de Creutzfeldt-Jakob esporádica: estudio clínico, patológico y molecular de un caso

Generalidades. Las encefalopatías espongiformes transmisibles son enfermedades neurodegenerativas ocasionadas por la acumulación anormal de una variante mal plegada de la proteína priónica, lo cual induce la formación de conglomerados proteicos resistentes a la degradación. Además, son responsables de la disfunción sináptica, daño neuronal y de la sintomatología clásica acompañante. Esta proteína de membrana es codificada por el exón 2 del gen PRNP, ubicado en el brazo corto del cromosoma 20 y parece estar involucrada en la trasmisión sináptica, la transducción de señales, la actividad antioxidante de la superoxidodismutasa, neuroplasticidad y sobrevida celular. Un polimorfismo en el codón 129 se asocia con una susceptibilidad diferencial a la enfermedad Creutzfeldt-Jakob esporádica. Objetivo. Estudio clínico, patológico y molecular de un caso de una mujer de 58 años con diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica. Métodos y resultados. Se presenta el caso de una mujer en quien aparece un trastorno depresivo del afecto con demencia progresiva y sintomatología general. Al final de la enfermedad, el cuadro progresó a un déficit neurológico focalizado en el área visual. La RMN mostró hiperintensidades inespecíficas córtico-subcorticales en el núcleo estriado; en el EEG se encontró pérdida de ritmos de fondo, patrón de descargas periódicas generalizadas y complejos trifásicos; en la biopsia cerebral postmorten se evidenció pérdida severa de la población neuronal en todas las capas, vacuolas en el neuropil, en el soma neuronal y en la glía. El análisis de secuencia del gen PRNP, a partir de extracción de DNA de sangre periférica, identificó homocigosis para metionina en el codón 129. La paciente fallece a los 3 meses del inicio de la sintomatología. Conclusión. Por epidemiología, curso clínico y exámenes paraclínicos se confirma el diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica.La determinación del genotipo para los polimorfismos de riesgo se convierte en una herramienta útil para complementar por medios moleculares el diagnóstico y para profundizar la comprensión de la fisiopatología de la enfermedad de Creutzfeldt-Jakob, tanto para formas esporádicas como para la nueva variante.

Variabilidade genética no Complexo Principal de Histocompatibilidade (MHC) de três espécies de mamíferos marinhos da costa do Rio grande do Sul

O MHC (Major Histocompatibility Complex) é um sistema genético importante para a manutenção de espécies ameaçadas, uma vez que baixa variabilidade para locos MHC tem sido associada a uma menor capacidade de resposta a doenças e diminuição do sucesso reprodutivo. Deste modo, pesquisas sobre a variabilidade genética do MHC têm demonstrado ser bastante informativas em estudos populacionais voltados para aspectos referentes à conservação. No presente trabalho foi investigada a variabilidade genética do MHC para três espécies de mamíferos marinhos (toninha, baleia franca austral e lobo marinho sul-americano) do sul do Brasil, com intensa mortalidade provocada por atividades humanas atuais ou passadas. As amostras foram coletadas de animais mortos encalhados na costa, de animais capturados acidentalmente por barcos pesqueiros, e também através de um sistema de biópsia. A região variável do exon 2 do gene DQB do MHC foi amplificada por PCR (Polymerase Chain Reaction) em 109 amostras de toninhas (Rio de Janeiro n=32, Rio Grande do Sul n=52, Argentina n=25), 35 amostras de lobo marinho sul-americano e 30 amostras de baleia franca austral, utilizando-se um par de primers heterólogos. O fragmento resultante de 172 pares de bases foi analisado quanto ao polimorfismo de seqüência através da técnica de SSCP (Polimorfismo de Conformação de Fita Simples) em todas as amostras de toninha e de lobo marinho sul-americano e 14 amostras de baleia franca austral. Dificuldades associadas à amplificação resultaram em padrões de SSCP pouco informativos para as amostras de lobo marinho sul-americano e baleia franca austral Todas as amostras de toninha apresentaram um padrão de pelo menos 4 bandas por indivíduo. As 4 bandas de um único indivíduo do Rio Grande do Sul foram seqüenciadas, tendo sido possível verificar que 2 seqüências relacionadas ao genes DQB estão sendo amplificadas com estes primers. Pelas análises de SSCP foi possível detectar ausência de variabilidade para as amostras de toninha provenientes do Rio de Janeiro e diferenciá-las da população da Argentina, que é polimórfica. A população do Rio Grande do Sul parece apresentar níveis intermediários de variação em relação aos extremos da distribuição da espécie. Analisando as três populações amostradas, conclui-se que a espécie apresenta baixos níveis de variabilidade para o loco DQB, a exemplo do que é reportado para os genes de MHC de outros mamíferos marinhos.

Análise de haplótipos intragênicos ao gene MJD1 em pacientes com doença de Machado-Joseph

A doença de Machado-Joseph (DMJ), ou ataxia espinocerebelar tipo 3 (SCA3), é uma desordem neurodegenerativa autossômica dominante, originalmente descrita em famílias de ancestralidade portuguesa-açoriana. Incordenação generalizada da marcha, dos membros e da fala podem estar presentes nos pacientes afetados por essa doença. O início das manifestações clínicas ocorre, em geral, entre os 30 e os 40 anos, apresentando uma progressão bastante lenta. O tempo médio de sobrevida depois do início da doença é de 14 a 17 anos. O gene associado à doença, denominado MJD1, foi identificado em 1994 e localiza-se no cromossomo 14. Este gene se caracteriza por apresentar uma repetição nucleotídica CAG na região 5` do exon 2. O número destas repetições é polimórfico na população, sendo que indivíduos normais apresentam de 12 a 37 repetições CAG, enquanto os afetados pela DMJ podem apresentar de 61 a 84 repetições. Um recente estudo mundial de haplótipos demonstrou a presença de dois diferentes haplótipos em famílias de origem açoriana, que são específicos à ilha de origem. Em famílias da porção continental de Portugal, os dois haplótipos foram encontrados. A maioria das famílias não portuguesas também compartilha o mesmo haplótipo encontrado em famílias originárias da ilha de Flores, mas três outros haplótipos foram encontrados nessas famílias. Até o momento, a hipótese mais forte declara que a difusão da mutação original está diretamente ligada à imigração portuguesa-açoriana No presente estudo, esta hipótese foi testada através da análise de disequilíbrio de ligação de três polimorfismos intragênicos (A669TG/C669TG, C987GG/G987GG, TAA1118/TAC1118). O polimorfismo na posição 669 foi identificado por PCR seguido de SSCP e confirmado por sequenciamento direto. Os polimorfismos nas posições 987 e 1118 foram detectados por PCR alelo-específico. Os resultados obtidos indicaram que o haplótipo intragênico A-C-A estava associado ao alelo com a expansão na maioria dos pacientes (92%). Estes resultados confirmam achados em outros estudos. Isto indica que uma única mutação na DMJ foi introduzida em várias populações, seguida de um efeito fundador local, e indicando também que provavelmente esta mutação é muito antiga. E, para finalizar, baseado neste estudo e comparando com dados gerados no estudo mundial de haplótipos, pode-se especular que a origem da mutação associada a DMJ nos pacientes do sul do Brasil é proveniente da ilha de Flores.

Methylation status of the SOCS 1 and JUNB genes in chronic myeloid leukemia patients

Alteração no padrão de metilação gênica pode contribuir para a progressão da leucemia mielóide crônica (LMC). Neste estudo, o padrão de metilação no exon 2 do gene SOCS- 1 e região promotora de ambos SOCS- 1 e JUNB foram avaliadas em pacientes com LMC. O padrão de metilação desses genes foi analisado usando a técnicamethylation- specific polymerase chain reaction (MSP) em 30 amostras de pacientes com LMC, 30 amostras desses mesmos pacientes após transplante de medula óssea (TMO) e 30 amostras controle de indivíduos saudáveis. As amostras de pacientes com LMC apresentaram o seguinte padrão de metilação: gene JUNB (3.3%), região promotora do gene SOCS- 1 (6.6%) e exon2 do gene SOCS- 1 (46.6%). Amostras dos indivíduos saudáveis apresentaram metilação somente no exon 2 do gene SOCS- 1 (10%, P = 0.002). Após o transplante, os pacientes apresentaram alterações no padrão de metilação da região promotora do gene SOCS- 1 (6.6%), no exon2 do gene SOCS- 1 (46.6%) e na região promotora do gene JUNB (16.6%). Metilação das regiões promotoras dos genes SOCS- 1 e JUNB não é um evento frequente em LMC. em contraste, metilação no exon 2 do gene SOCS- 1 apresenta- se como um evento frequente, suscetível a alterações no padrão de metilação após TMO.

Association of the Leptin Gene with Carcass Characteristics in Nellore Cattle

Advances in DNA technology have created biotechnological tools that can be used in animal selection and new strategies for increasing herd productivity and quality. The objective of the present work was to associate the genotypes of leptin gene exon 2 polymorphisms with productive traits in Nellore cattle. Blood was collected from Nellore males and PCR-RFLP reactions were performed with the restriction enzymes ClaI and Kpn2I. The gene frequencies resulting from digestion by ClaI were 0.60 and 0.40 for allele A and T, respectively; the genotypic frequencies were AA = 0.20 and AT = 0.80. The gene frequencies from digestion by Kpn2I were 0.81 for allele C and 0.194 for allele T; the genotypic frequencies were CC = 0.62 and CT = 0.38. The populations in both cases were not in Hardy-Weinberg equilibrium (p > 0.05), and the TT genotype was not found. Significant associations were noted between leptin gene exon 2 polymorphisms and five productive traits in Nellore cattle: carcass fat distribution, the intensity of red muscle coloration, pH, marbling, and post-slaughter fat thickness. © 2013 Copyright Taylor and Francis Group, LLC.