Made available in DSpace on 2014-07-29T15:10:31Z (GMT). No. of bitstreams: 1 claudio carlos.pdf: 4222908 bytes, checksum: 9eff1b9f349ce5868c10ca1d5b440898 (MD5) Previous issue date: 2009-10-21 / The larynx is a structure of the upper aerodigestive tract responsible for the production of sounds as well as protecting the lower airways and helping during the normal act of swallowing. Any pathology which affects the larynx can impose several challenges that disrupt its normal physiological function, and consequently and directly resulting in reduction of the patient s quality of life. Among the different diseases that affect the larynx, cancer is one of the most serious. The squamous cell carcinoma (SCC) of the larynx is a multifactorial disease, influenced by environmental factors and individual behavioral, habits, and susceptibility. The current study describes the molecular and genetic assessment of 20 patients with the squamous cell carcinoma of the larynx. In summary, the study strategy included the analyses of the genetic polymorphism of codon 72 of the TP53, the detection and genotyping of HPV genome, assessing genomic instability (MIS and LOH) and random chromosomal imbalances using PCR and CGH approaches. Regarding to the polymorphism of the TP53 gene, arginine homozygous genotypes (p53AA) and arginine-proline heterozygous genotypes (p53PA) were found in 65% (13/20) and 35% (7 / 20) of cases, respectively. HPV genome was found in association with tumor cells in 20% of SCC cases. HPV 16, 11, and 45 were the genotypes identified. Moreover, co-infection of HPV 11 and 45 was also observed. Both samples found positive for HPV genome were associated with p53AA at the TP53 gene. Our results corroborated the data published in the literature that described an increased susceptibility to the virus-induced carcinogenesis of the larynx in arginine homozygous genotypes. Here in we report genomic instability for a panel of 8 microsatellite markers distributed in 5 chromosomes. One locus (D8S135) was found in homozygous for all cases. On the other hand, RH-92600 was not included in the analysis mainly because it was also found homozygous for most cases. We found 76.2% (244/320) of informative loci. Thus the mean frequency of MIS was 9.0% (22/244) and LOH was 6.1% (15/244). The frequency of MIS was found higher than the LOH, suggesting that the tumorigenesis of SCC of the larynx follows a mechanism of mutator phenotype. Using CGH, chromosomal gains were observed in 1q21qter. On the other hand, chromosomal losses occurred in chromosome 3p21p28, 11q23, 16p16, 16q12, 17p13pter and 22q13. The techniques of comparative genomic hybridization have improved the understanding of genetic alterations in squamous cell carcinoma of the larynx, especially for characterizing the relationship between early precursors of laryngeal carcinomas, as well as to identify genomic regions that may contain oncogenes and tumor suppressor genes involved in the tumorigenesis of this anatomical site. / A laringe é uma estrutura tubular do trato aero digestivo com a principal função de formação de sons, além de estar relacionada com a proteção das vias aéreas inferiores e deglutição dos alimentos. Qualquer patologia que acomete este órgão pode ocasionar diversos problemas em sua função fisiológica normal, com influência plena e direta no decréscimo da qualidade de vida do indivíduo. Das diversas doenças que afetam a laringe, o câncer apresenta-se como uma das mais graves. O carcinoma de células escamosas da laringe apresenta-se como uma doença multifatorial e epigenética sendo influenciada por fatores ambientais, comportamentais e inerentes ao indivíduo. Este estudo avaliou 20 indivíduos portadores do carcinoma das células escamosas da laringe. Dentre dos parâmetros avaliados, destacam-se o polimorfismo do códon 72 do gene do gene TP53, a detecção e genotipagem do genoma de HPV, a instabilidade genômica (LOH e MIS) e alterações cromossômicas não balanceadas. Em relação ao polimorfismo de TP53, os resultados foram 65% (13/20) de homozigotos (TP53AA) e 35% (7/20) de heterozigotos (TP53PA). Em 20% dos pacientes foi observada a presença de genoma HPV associada às células tumorais, tendo sido genotipados os subtipos virais HPV 16 em uma amostra e HPV 11 e 45 na outra amostra, caracterizando uma co-infecção. Os dois casos positivos para HPV apresentaram genótipo homozigoto (TP53AA) para o gene TP53. Os resultados deste estudo corroboram os dados publicados na literatura pertinente, que correlacionam a susceptibilidade aumentada para CEC da laringe em indivíduos homozigotos para TP53Arg e a maior susceptibilidade destes à carcinogênese vírus-induzida. A instabilidade genômica foi avaliada utilizando um painel de 8 marcadores de microssatélites distribuídos em 5 cromossomos diferentes. Os loci D8S135 e RH-92600 foram considerados não informativos, pois se apresentaram homozigotos na maioria dos casos, enquanto que 76,2% (244/320) loci foram informativos. Contudo, a freqüência de MIS foi de 9% (22/244) e a freqüência de LOH de 6,1% (15/244). A freqüência observada de MIS foi maior do que a freqüência de LOH, sugerindo que o carcinoma das células escamosas da laringe segue o mecanismo fenótipo mutante . A hibridação genômica comparativa mostrou que a principal região cromossômica que apresentou ganhos foi em 1q21qter, enquanto que as perdas cromossômicas ocorreram em 3p21p28, 11q23, 16p16, 16q12, 17p13pter e 22q13. As técnicas de hibridação genômica comparativa têm contribuído para melhorar a compreensão das alterações genéticas no carcinoma das células escamosas da laringe, sobretudo para a caracterização da relação entre estágios precursores de câncer laríngeo, assim como, para a identificação de regiões genômicas que podem conter oncogenes e genes supressores tumorais.

A infertilidade afeta aproximadamente 15% dos casais, sendo atualmente reconhecido o envolvimento de fatores masculinos em metade dos casos. Alterações nas análises seminais são detectadas na maioria dos homens inférteis e a mais frequente é a baixa concentração de espermatozoides no ejaculado, conhecida como oligozoospermia. Vários estudos mostram uma forte relação entre fatores genéticos e a infertilidade, incluindo alterações cromossômicas e microdeleções do cromossomo Y, porém as causas da oligozoospermia ainda permanecem obscuras. O desenvolvimento de novas tecnologias de investigação vem possibilitando a detecção de alterações a nível genômico, como mutações e variações no número de cópias (CNVs). O presente trabalho teve por objetivo a caracterização genômica de homens com oligozoospermia sem causa definida, visando estabelecer correlação entre alterações no número de cópias e perdas de heterozigosidade (LOHs) e o fenótipo de infertilidade. Foram selecionados 18 pacientes após rigorosa avaliação clínica e investigação do histórico reprodutivo, sendo excluídos pacientes portadores de alterações cromossômicas e portadores de microdeleções do cromossomo Y. Seis homens comprovadamente férteis foram selecionados para o grupo controle. A investigação genômica de ambos os grupos, amostral e controle, foi realizada pela técnica de hibridação genômica comparativa em microarranjos (aCGH) utilizando a plataforma de resolução 180K (Agilent®,US), analisada pelo software Nexus 8.0. Foram detectadas alterações possivelmente patogênicas no cromossomo Y, no cromossomo X e em autossomos. Um ganho na região de AZFc envolvendo apenas os genes DAZ1 e DAZ4 foi detectado em nove pacientes e em quatro controles, sendo classificado como alteração benigna. Porém, alterações na região de AZFc possivelmente relacionadas ao fenótipo de oligozoospermia foram detectadas em três pacientes e incluíram extensas duplicações e deleções envolvendo, entre outros genes, as quatro cópias do gene DAZ. Após comparação de regiões selecionadas com a literatura e com diferentes bancos de dados genéticos, sugerimos que os genes PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 e ODF3B possam estar associados a alterações na produção espermática. Adicionalmente, entre os doze miRNAs presentes em regiões de LOH possivelmente relacionadas ao fenótipo de infertilidade, dez têm como alvo genes com funções relacionadas à espermatogênese e reprodução humana. Estudos adicionais a nível de expressão e sequenciamento gênico são necessários para confirmar a correlação entre o genótipo e o fenótipo de oligozoospermia. / Infertility affects about 15% of the couples, and it is currently recognized, that male factors are involved in about 50% of cases. Changes in seminal parameters are detected in most infertile men and the most common alteration, known as oligozoospermia, is a low concentration of sperm in the ejaculate. Several studies show a strong relationship between genetic factors and infertility, including chromosomal abnormalities and microdeletions of Y chromosome, however, the causes of oligozoospermia remain unclear. The development of new research technologies has allowed the detection of changes at genomic levels, such as mutations and copy number variations (CNVs). This study aimed to perform a genomic characterization of patients with idiopathic oligozoospermia to determine whether there is a correlation between changes of copy number and losses of heterozygosity (LOHs) in relation to the phenotype of infertility. Eighteen patients were selected for the cases after rigorous clinical examination and investigation of their reproductive history. Patients with chromosomal abnormalities or microdeletions of the Y chromosome were excluded. Six proven fertile men comprised the control group. Genomic investigation of both groups was performed by microarray comparative genomic hybridization (aCGH) using 4X180K platform (Agilent, US) analysed by Nexus 8.0 software. Potential pathogenic changes were detected on Y chromosome, as well as on the X and autosome chromosomes. A gain in AZFc region involving only DAZ1 and DAZ4 genes was detected in nine patients and four controls, and was considered as benign. However, changes in AZFc region, that could be related to the oligozoospermia phenotype were detected in three patients. These changes included extensive duplications and deletions involving the four copies of the DAZ gene together with copy number changes affecting other genes. After comparing the selected regions with the literature and with different databases, we suggest that changes such as LOH affecting PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 and ODF3B genes may influence sperm production. Our analysis indicates that, ten out of the twelve miRNAs present in LOH regions could be involved in the infertility phenotype and could have target genes with functions related to spermatogenesis and human reproduction. Additional studies involving gene sequencing and expression analysis are needed to confirm the the correlation between the genotype and oligozoospermia phenotype.

Loss of heterozygosity (LOH) is detectable in many forms of cancer including leukaemia. It contributes to tumorigenesis through the loss of one of the two alleles of one tumor suppressor gene at a given locus, caused by deletion or uniparental disomy (UPD). UPD can only be the result of homologous recombination. Little is known about the mechanisms of UPD and what connection this aberration has with the outcome of this disease. In this study, 146 patients with primary AML were analysed using a novel technique based on single nucleotide polymorphisms (SNPs). Leukaemic cells and healthy T-cells from each patient were obtained using FACS-Vantage cell sorting. In cases with very few sorted cells whole genome preamplification was done. Genome-wide SNP analysis was carried out according to the standard GeneChip Mapping Assay protocol (Affymetrix, USA) using the Human Mapping 10K Arrays. Moreover, the impact of the FLT3-ITD mutation on the homologous recombination using pmHPRT-DRGFP /pCbASce vectors system and yHA2x assay was investigated. Of 146 patients with normal karyotype LOH was found in 30 cases. The potential LOH regions, were confirmed by microsatellite analysis of short tandem repeat (STR) markers. In 21 of these cases STR-analysis of T-cells, representing the corresponding tumor-free material, confirmed the regions of partial UPD. This aberration affected different chromosomes, but most commonly chr. 2, 6, 11, 21, 13, and 7, and covered between 11.5 and 88 Mb. Interestingly, in 6 LOH cases, long stretches of homozygosity present at the same positions as in the healthy cells and in the blasts were found. The impact of this phenomenon is unknown. Additionally, chromosome losses were detected in 3 patients classified with normal karyotype according to current methods. These 9 cases were not included in the UPD positive group. No differences were observed regarding any clinical factors including age, WBC-counts and sex. The FAB M1 subtype was observed in 47.6% of the UPD positive patients, compared to only 19.2% of the UPD negative patients (P=0.04, n=146). In addition, no correlation between FLT3-ITD, MLL-PTD and NPM1 mutations in the UPD patients was found, but the data indicate that patients with UPD have a higher rate of treatment failure. Moreover, in this study the relationship between UPD and gene aberrations was able to be confirmed. In some cases, UPD found on chromosomes 21, 19 and 11 was correlated with mutations in the RUNX1, CEBPA and WT genes, respectively. Furthermore, AML cases with and without UPD showed different but specific gene expression profiles, revealing different expression levels for genes involved in double strand break repairs. Furthermore, it was found that different mutations could be responsible for the increase in efficiency of HR, such as FLT3-ITD or BCR-ABL. Moreover, cells with a FLT3-ITD mutation (without wt expression) rapidly increased the HR efficiency compared with heterozygous (FLT3-ITD/wt) cells. Preliminary results showed that the high repair efficiency was mainly dependent on the translocation of RAD51. In conclusion, SNP array technology allow the identification and mapping of LOH in AML patients with normal karyotype. The obtained data also point out the necessity of analysing tumour-free material to confirm the somatic origin of the alteration. Furthermore, the available results indicate that compared to patients without UPD, patients with UPD have a higher relapse rate, which might be used as a prognostic marker in the future. Also, it could be hypothesized that downregulation of RAD51 (for example by FLT3 inhibition) might be beneficial DNA damage occurs through the genotoxic agent by reducing the relapse risk of AML.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

<p>Pancreatic endocrine tumors (PETs) occur sporadically or in the familial multiple endocrine neoplasia type 1 (MEN1) syndrome, whereas midgut carcinoids are nonfamilial, malignant endocrine tumors of the intestine. For these tumor entities morphological criteria are of limited use for prognostic prediction and selection of treatment. Genetic characterization may give additional information of clinical use and reveal pathways involved in tumor development.</p><p>Molecular genetic alterations in sporadic and MEN1-associated PETs and midgut carcinoids were studied with LOH and mutational analysis. In addition, immunohistochemistry was used to clarify gene expression. Detected genetic aberrations were correlated to the disease course of individual patients.</p><p>Somatic mutations of the<i> MEN1</i> gene at chromosome <i>11q13</i> were detected in 1/3 of sporadic PETs<i>. </i>Moreover, LOH was found in 70% of the lesions. All tumors with somatic <i>MEN1</i> mutations displayed loss of the remaining allele showing that the <i>MEN1</i> gene is involved in development of sporadic PETs.</p><p> Sporadic and MEN1 PETs were analyzed for LOH at <i>3p,</i> <i>11q13</i> and <i>18q</i>. A relation of LOH at <i>11q13</i> and <i>3p</i> to malignancy was found for the sporadic tumors. None of the benign tumors (all of them insulinomas) had allelic loss at <i>3p</i> or <i>11q13</i>, versus 92 % (p<0.01) of the malignant tumors (including malignant insulinomas). 1/4 of both sporadic and MEN1 lesions displayed LOH at <i>18q</i>, without altered <i>Smad4/DPC4</i>.</p><p>Genome-wide LOH screening of MEN1 PETs revealed multiple allelic deletions without general correlation to tumor size or malignancy. All tumors displayed LOH at the <i>MEN1 </i>locus, and 30% on chromosomes 3, 6, 8, 10, 18 and 21. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. </p><p>A similar genome-wide LOH screening was performed on midgut carcinoids. Deletions at chromosome <i>18q</i> were found in 88% of the tumors indicating a potential tumor suppressor locus.</p>

Pancreatic endocrine tumors (PETs) occur sporadically or in the familial multiple endocrine neoplasia type 1 (MEN1) syndrome, whereas midgut carcinoids are nonfamilial, malignant endocrine tumors of the intestine. For these tumor entities morphological criteria are of limited use for prognostic prediction and selection of treatment. Genetic characterization may give additional information of clinical use and reveal pathways involved in tumor development. Molecular genetic alterations in sporadic and MEN1-associated PETs and midgut carcinoids were studied with LOH and mutational analysis. In addition, immunohistochemistry was used to clarify gene expression. Detected genetic aberrations were correlated to the disease course of individual patients. Somatic mutations of the MEN1 gene at chromosome 11q13 were detected in 1/3 of sporadic PETs. Moreover, LOH was found in 70% of the lesions. All tumors with somatic MEN1 mutations displayed loss of the remaining allele showing that the MEN1 gene is involved in development of sporadic PETs. Sporadic and MEN1 PETs were analyzed for LOH at 3p, 11q13 and 18q. A relation of LOH at 11q13 and 3p to malignancy was found for the sporadic tumors. None of the benign tumors (all of them insulinomas) had allelic loss at 3p or 11q13, versus 92 % (p&lt;0.01) of the malignant tumors (including malignant insulinomas). 1/4 of both sporadic and MEN1 lesions displayed LOH at 18q, without altered Smad4/DPC4. Genome-wide LOH screening of MEN1 PETs revealed multiple allelic deletions without general correlation to tumor size or malignancy. All tumors displayed LOH at the MEN1 locus, and 30% on chromosomes 3, 6, 8, 10, 18 and 21. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. A similar genome-wide LOH screening was performed on midgut carcinoids. Deletions at chromosome 18q were found in 88% of the tumors indicating a potential tumor suppressor locus.

<p>A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. </p><p>In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. </p><p>In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.</p>

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

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