Thesis--University of Oklahoma. / Includes bibliographical references.

A endometriose é uma doença ginecológica benigna comum, mas agressiva, caracterizada pela presença de tecido endometrial ectópico. A teoria mais aceita para explicá-la é a teoria de Sampson, na qual o tecido endometrial descamado durante a menstruação sofre refluxo através das tubas uterinas, adere e se prolifera em sítios ectópicos da cavidade peritoneal. Por outro lado, apenas o refluxo tubário não é capaz de estabelecer a doença e vários estudos sugerem uma etiologia multidimensional incluindo fatores hereditários, hormonais e imunológicos. Várias metodologias têm sido propostas com o objetivo de identificar genes candidatos para a endometriose. A hibridação genômica comparativa (CGH) é uma técnica que permite que o genoma inteiro seja analisado em um só experimento, sem a necessidade de cromossomos metafásicos obtidos por cultura celular. Nossa proposta foi avaliar, por CGH, amostras de endometriomas ovarianos e de tecido endometrial eutópico de dez pacientes com diagnóstico firmado de endometriose, para screening do genoma. No grupo eutópico, 6/10 amostras apresentaram alterações caracterizadas por perdas ou ganhos de regiões cromossômicas e no grupo ectópico foram encontradas alterações em 7/10 casos. A presença de perdas e ganhos de regiões cromossômicas no endométrio eutópico, histologicamente normal, de mulheres com endometriose ovariana, pode ser considerada como alteração primária ao desenvolvimento da doença. A metodologia de CGH permitiu a detecção das regiões cromossômicas 11q12.3-q13.1, 17p11.1-p12 e 17q25.3-qter como regiões críticas, direcionando investigações futuras para identificação de genes associados à endometriose. / Endometriosis is a common benign gynecological disease, very aggressive, characterized by the presence of ectopic endometrial tissue. The most accepted theory to explain it is Sampson\'s implantation theory, which says that the endometrial tissue exfoliated during menstruation undergoes reflux through the uterine tubes, adheres and proliferates in ectopic sites of the peritoneal cavity. On the other hand, only reflux is not enough to the establishment of the disease and a number of studies suggest a multidimensional etiology including hereditary, hormonal and immunological factors. Several methodologies have been proposed for the identification of candidate genes for endometriosis. The comparative genomic hybridization (CGH) is a versatile technique that allows the entire genome to be analyzed in only one experiment without the necessity of metaphasic chromosomes from the sample, excluding the cell culture. We aimed to evaluate, by CGH, ovarian endometriomas and eutopic endometrial tissue samples from 10 patients with confirmed diagnosis of endometriosis, for a genomic screening. In the eutopic group, 6/10 samples presented genomic imbalances and 7/10 cases showed alterations in the ectopic group. The presence of losses and gains of chromosomic regions in the histologically normal eutopic endometrium from women with ovarian endometriosis can be considered as a primary alteration in the development of the disease. The CGH methodology allowed the detection of chromosomic regions 11q12.3-q13.1, 17p11.1-p12 and 17q25.3-qter as critical regions, leading to future investigations for the identification of genes associated to endometriosis.

Orientadores: Luciana Bolsoni Lourenço, Carmen Silvia Busin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:08:20Z (GMT). No. of bitstreams: 1 Gatto_KalebPretto_M.pdf: 33988822 bytes, checksum: 7e3f3565dac9a540ea7c353f51068d4f (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

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<p>Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. </p><p>In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis.</p><p>In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.</p>

Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis. In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.

Smith-Magenis syndrome (SMS) is a multiple congenital abnormalities intellectual disability syndrome that results from a deletion of chromosome 17p11.2 or mutation of the retinoic acid inducted one gene (RAI1). SMS is characterized by a multitude of phenotypic features including craniofacial defects, short stature, obesity, intellectual disability, self-abusive behavior, sleep disturbance and behavioral abnormalities. Interestingly, although SMS is a clearly defined syndrome with a known molecular change at its foundation, ~40% of all candidate cases sent to the Elsea lab for evaluation do not have a mutation or deletion of RAI1. We hypothesize that at least one other locus must be responsible for this Smith-Magenis-like (SMS-like) phenotype. To address this hypothesis, we first compiled a cohort of 52 subjects who had been referred to the Elsea lab for a clinical diagnosis of SMS. Once these individuals were confirmed to not have an RAI1 mutation or deletion, their phenotypes were compiled and statically analyzed to distinguish whether SMS and SMS-like cohorts are different in the prevalence of the core phenotypes of SMS such as, but not limited to, sleep disturbance, self-abusive behavior and developmental delay. SMS-like and SMS cohorts are not different in prevalence for these core features. Next, all SMS-like subjects were sent for whole genome array comparative genomic hybridization (aCGH) to identify duplications or deletions of each individual’s genome which contribute to the phenotype observed. We identified 6 pathogenic copy number variants (CNVs) in six individuals which contribute directly to the clinical phenotype, including two del(2)(q37). This study enabled us to draw relationships between SMS and other syndromes that had never been appreciated before and helped to identify pathways in which RAI1 may function. Using the data from our SMS-like study we were able to further characterize two known syndromes; Deletion 2q37 syndrome (brachydactyly mental retardation syndrome) and deletion 2q23 syndrome. With regard to deletion 2q37, syndrome we used genomic data from known and new deletion 2q37 subjects to refine the critical region to one gene: the histone deacetylase 4 gene (HDAC4). Using both clinical and molecular clues, we were able to identify one subject from our SMS-like cohort who has an insertion in HDAC4 which results in a premature stop codon. We conclude from this study that mutation of HDAC4 results in brachydactyly mental retardation syndrome. With regard to deletion 2q23 syndrome there were only five known cases in the published literature to which we were able to add two more. Using as similar approach to our del2q37 study we refined the critical region for this syndrome to one gene, the methyl binding domain 5 gene (MBD5). Using a molecular and clinical approach we were able to conclude that haploinsufficiency of MBD5 results in the core phenotypes seen in del2q23 syndrome including microcephaly, intellectual disabilities, severe speech impairment, and seizures. Using all the data generated from the three previous studies we set out to characterize the molecular function of RAI1. We hypothesize that RAI1 is a transcription factor that regulates gene expression of core genes involved in development, neurological function, and circadian rhythm. Using a ChIP-chip based approach we identified 257 transcripts we believe RAI1 regulates. Following up on these transcripts, using in vitro and in vivo methods, we have been able to conclude that RAI1 is a positive regulator of CLOCK, the master regulator of the central circadian cycle. Taken together, these studies have given us insight into the specific molecular changes that contribute to SMS and SMS-like syndromes. We have unveiled pathways and genes which are important to normal human development and behavior and identified novel functions of RAI1. These studies will provide the foundation for the future discovery of the pathways affected.

A perda auditiva é o defeito mais comum ao nascimento e cerca de 70 milhões de pessoas no mundo apresentam algum grau de perda auditiva. Além da alta incidência, as implicações da perda auditiva na linguagem, na cognição e no desenvolvimento emocional e social reforçam sua importância. No entanto, em grande parte dos pacientes, a causa da deficiência auditiva não é esclarecida. Nós usamos hibridação comparativa do genoma baseada em arrays (Array Comparative Genomic Hybridization aCGH) para investigar alterações no número de cópias de segmentos de DNA (Copy Number Variation CNV) em 31 indivíduos que apresentavam deficiência auditiva e sinais clínicos adicionais, mas que não puderam ser classificados em síndrome conhecida. A escolha de indivíduos sindrômicos se baseou no pressuposto de que, em média, apresentam alterações genômicas maiores e, portanto, mais provavelmente detectáveis com o uso de aCGH de 1 Mb, que era a plataforma disponível no início do projeto. CNVs não descrita em bancos de dados de indivíduos normais foram identificadas em oito pacientes, quatro delas ocorreram de novo enquanto as outras quatro foram herdadas de um genitor fenotipicamente normal. As alterações de novo definem segmentos cromossômicos que provavelmente contém genes relacionados à deficiência auditiva e sensíveis a dose, especificamente: 1q23.3-q25.2, 2q22q23, 6p25.3 e 11q13.2-q13.4. As alterações raras identificadas tanto nos pacientes quanto em um genitor normal poderiam ser um evento ao acaso, sem papel na deficiência auditiva; no entanto, a possibilidade de que essas alterações possam funcionar como fatores de predisposição não podem ser descartadas. Se considerarmos apenas as CNVs de novo como causativas dos fenótipos investigados, detectamos quatro pacientes portadores entre os 31 investigados (13%). Se considerarmos também as CNVs herdadas como possivelmente causativas, a taxa de desequilíbrios cromossômicos associados à surdez será de 26%. Esses resultados são provavelmente uma substimativa e esses números seriam possivelmente maiores com o uso de uma das plataformas de alta resolução disponíveis atualmente. Esses resultados, embora limitados, indicam que investigação por aCGH em pacientes com surdez sindrômica idiopática está entre os testes mais eficientes para detectar etiologia dos fenótipos, devendo ser incorporado à rotina no diagnóstico e aconselhamento genético. / Hearing loss is the most common congenital deficiency and about 70 million people worldwide present some degree of hearing impairment. In addition to its high incidence, hearing loss impacts language, cognition and social and emotional development. However, in a large proportion of patients, the cause of the hearing deficiency cannot be elucidated. We screened copy number changes by 1 Mb-array Comparative Genomic Hybridization (aCGH) in 31 individuals with syndromic hearing impairment whose clinical features were untypical for known disorders. The choice of evaluating syndromic rather than non-syndromic individuals was based on the assumption that they are more likely to carry larger genomic alterations which could be more easily detected by the comparatively low resolution 1 Mb aCCG, which was the available platform when this project started. Copy number changes (CNV) not documented in the database of normal individuals were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2- q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but also have a possible role as a predisposition factor. When only the de novo CNVs were considered causative for the disease phenotypes, our study revealed relevant copy number changes in 4 patients (13%). If we also count the rare CNVs that had been inherited as possibly causative, the frequency of chromosome imbalances associated with syndromic deafness in our sample becomes 26%. These figures are probably underestimates and will probably become larger when high resolution oligoarray platforms are applied. These results indicate that aCGH is an efficient tool for defining the etiology of syndromic deafness and its use in routine diagnosis of hearing impairment and for genetic counseling is highly recommended.

Infertilidade conjugal é uma doença do sistema reprodutivo que acomete cerca de 20% dos casais e na qual o fator masculino responde por metade desses casos. A infertilidade masculina é um fenótipo complexo que abrange diferentes fatores. Os fatores genéticos envolvidos variam desde mutações pontuais, microdeleções no cromossomo Y, até alterações cromossômicas, como a Síndrome de Klinefelter. Mesmo após avaliação clínicolaboratorial detalhada, metade dos pacientes permanece sem a identificação de um fator causal, caracterizando a infertilidade idiopática. Nesse grupo, observamos com maior frequência os pacientes com falha espermatogênica primária, que clinicamente apresentam oligozoospermia grave ou azoospermia não obstrutiva (ANO) e, no qual, preponderam fatores genéticos ainda desconhecidos. Para auxiliar na compreensão de possíveis alterações genômicas, sejam as variantes de número de cópias (CNVs) ou as regiões de perda de heterozigosidade (LOHs), envolvidas com infertilidade masculina idiopática, 16 pacientes com ANO e 6 controles foram investigados pela técnica de hibridação genômica comparativa (aCGH) utilizando a plataforma 4x180 CGH+SNP Agilent® com análise dos dados pelo software Nexus 8.0. Não foram observadas diferenças significativas tanto no número, como no tamanho das alterações genômicas em ambos os grupos. Foram descritas 18 novas alterações genômicas com efeito sobre a produção espermática, distribuídas na forma de 12 ganhos, 3 perdas e 3 LOHs. Os ganhos mais significativos para o fenótipo azoospermia não obstrutiva foram descritos em 7q36.3, 17q21.33, Xq21.1 e Yp11.2. Nessas regiões, os genes com maior impacto sobre o fenótipo foram, respectivamente, SHH, COL1A1, COX7B e LINC00279. Ganhos envolvendo a sub-banda Yq11.223 e contendo cópias dos genes DAZ1 e DAZ4 foram considerados benignos. As três perdas detectadas em 2q31.1, 3p21.1-21.31 e 15q11.2, contendo, respectivamente, os genes DLX1, CACNA2D2 e representantes da família de receptores olfatórios foram consideradas relevantes. A análise das LOHs em fenótipos complexos é escassa e desafiadora. No presente trabalho, foram descritas 3 dessas alterações, localizadas em 1p31.1, 7q21.1 e 12q21.1-21.2 e compartilhadas por mais de um indivíduo infértil. A descrição dessas alterações genômicas contribui para a compreensão de mecanismos complexos e ainda pouco estudados, que resultam em azoospermia não obstrutiva decorrente da falha espermatogênica primária. / Infertility is a disease of the reproductive system that affects about 20% of all couples, with half of the cases being related to the male factor. Male infertility is a complex phenotype associated with an interaction of different factors. The genetic factors involved may range from point mutations, microdeletions on the Y chromosome to chromosomal changes such as Klinefelter syndrome. Even after detailed clinical-laboratory evaluation, the etiology may remain unknown in approximately half of the patients, and, in such cases, the infertility can be classified as idiopathic. This group of patients more frequently present with primary spermatogenic failure, with severe oligozoospermia or non-obstructive azoospermia (NOA). Nevertheless, the underlying genetic factors are still largely unknown. In order to better understand the potential genomic changes involved with idiopathic male infertility, sixteen patients with NOA and 6 controls were investigated in this study. Copy number variants (CNVs) and regions of loss of heterozygosity (LOHs) were assessed by array comparative genomic hybridization technique (aCGH), using the Agilent® 4x180 CGH + SNP platform. Data analyses was performed using Nexus 8.0 software. No significant differences between the groups were observed in relation to either the number or the size of the genomic changes. Eighteen new genomic alterations were described that were associated with sperm production (12 gains, 3 losses and 3 LOHs). The most important gains for the nonobstructive azoospermia phenotype were observed in 7q36.3, 17q21.33, Xq21.1 and Yp11.2. In these regions, the genes related to greatest impact on the phenotype were SHH, COL1A1, COX7B and LINC00279, respectively. Gains involving the Yq11.223 sub-band and containing copies of the DAZ1 and DAZ4 genes were considered benign. All 3 losses detected in 2q31.1, 3p21.1-21.31 and 15q11.2, containing, respectively, the DLX1, CACNA2D2 genes and representatives of the olfactory receptor family were considered relevant. Analysis of LOHs in complex phenotypes such as male infertility has been infrequently reported and is challenging. In the present study, three significants LOHs were found (1p31.1, 7q21.1 and 12q21.1-21.2) and were identified in more than one infertile individual. The description of these genomic alterations contributes to a better understanding of this complex and poorly explored mechanisms that results in non-obstructive azoospermia due to primary spermatogenic failure.

A Síndrome de Rubinstein-Taybi (RTSs) é uma doença rara de herança autossômica dominante, caracterizada por dismorfismos craniofaciais, polegares e háluces alargados, deficiência intelectual e de crescimento. RTSs tem sido associada com mutações no gene CREBBP (CBP) e mutações menos frequentes no gene EP300 que foram descritas em oito indivíduos. CBP e p300 possuem alta homologia e são extremamente importantes em várias vias de sinalização, principalmente como coativadores de transcrição e na acetilação das histonas. Nosso estudo baseou-se na análise de alterações moleculares por sequenciamento direto do CBP, FISH e array-CGH em 20 pacientes com RTSs. Dos 20 pacientes avaliados por sequenciamento direto foram identificadas oito alterações moleculares, dentre estas, seis são alterações moleculares novas as quais não foram descritas na literatura, são elas: i) duas deleções (p.M747fs STOP830 e p.G1011fs STOP1021) ii) duas alterações do tipo nonsense (p.Arg1341X, p.Arg1498X) iii) três do tipo missense (p.Arg1907Trp, p.Leu604Pro e p.His1291Arg). Também identificamos um polimorfismo de único nucleotídeo (SNP) (rs115594471/ c.5874CT). Dois pacientes apresentaram deleção do gene CBP em um dos alelos, identificado pelo método array-CGH. Outro, apresentou uma translocação aparentemente equilibrada t(2;16), cuja análise subsequente com FISH revelou uma quebra na região do CBP. Neste trabalho, a taxa de detecção de alteração molecular no CBP por sequenciamento direto foi de 40% (08/20). Porém, a taxa de detecção das alterações moleculares no CBP foi de 55% (11/20), considerando a combinação das diferentes técnicas utilizadas (FISH, sequenciamento direto e array-CGH). Não houve correlação genótipo-fenótipo, exceto por uma maior frequência da presença de epicanto nos pacientes com alteração no CBP. Os resultados obtidos neste trabalho servem como o diagnóstico molecular para os pacientes com RTSs atendidos no Ambulatório do Laboratório de investigação Médica 001 (ALIM 001) do Instituto da Criança - FMUSP, contribuindo para uma melhor orientação médica, como também para realização do aconselhamento genético às famílias / Rubinstein-Taybi syndrome (RTSs) is a rare autosomal dominant disease characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency. RTS has been associated with CREBBP (CBP) gene mutations and less frequently with mutations in EP300 gene, which have been reported in eight individuals. CBP and p300 have high homology and are extremely important in many signaling pathways especially as transcriptional coactivators and histone acetylation. Our study was based on the alteration analysis by direct sequencing of the CBP, by FISH and array-CGH in 20 RTSs patients. We identified eight molecular alterations in 20 RTSs patients evaluated by direct sequencing: i) two deletions (p.M747fs STOP830 and p.G1011fs STOP1021) ii) two nonsense alterations (p.Arg1341X and p.Arg1498X) iii) Three missense alteration (p.Arg1907Trp, p.Leu604Pro and p.His1291Arg). Single-nucleotide polymorphism were also identified (rs115594471 / c.5874CT), and six of these are new molecular alterations, not described in literature. Two RTSs patients studied had CBP gene deletion in one allele, identified by array-CGH method. Other patient, presented with apparent balanced translocation t(2;16) in which the subsequent analysis using FISH, showed a break in region of CBP. In this work, the rate of detection of molecular alteration in CBP by direct sequencing in RTSs patient was 40.0% (08/20). However, the rate of detection of molecular alteration in CBP was 55.0% (11/20), considering the combination of different techniques (FISH, direct sequencing and array-CGH. No significant correlation could be established in this study between the different types of mutations and genotype-phenotype of RTSs patients, except a higher frequency of the presence of epicanthus in the RTS patients with alteration in the CBP. The results of this study serve as a molecular diagnosis for RTSs patients treated at the Ambulatory of the Medical Investigation Laboratory 001 (ALIM 001) of the Instituto da Criança - FMUSP, and this contributes to better clinical management, such as making an appropriate genetic counseling for families