The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies. IDH1 mutation was associated with MGMT promoter methylation when partially methylated tumours were grouped with methylated tumours. Grade II and grade III tumour comparison analysis revealed 14 novel significant miRNAs with differential expression. A miRNA signature was shown for histological subtypes, oligoastrocytoma and anaplastic oligoastrocytoma, following the miRNA expression analysis in grade II and grade III tumors based on histology. Oligoastrocytoma presented a more similar profile to oligodendroglioma, but anaplastic oligoastrocytoma was more similar to anaplastic astrocytoma. Five novel miRNAs were identified in grade III tumours, when comparing IDH1 mutant and IDH1 wild type tumours. Analysis of paired samples of primary/recurrent tumours revealed that additional genomic changes may promote tumour progression. For each of the pair, the two samples were genomically different and in each case, the reccurent tumours had more copy number aberrations than the corresponding primary tumours. Cell cultures derived from the tumour biopsies were not representative of the low grade glioma in vivo, which was evident from the differences identified in the miRNA expression and copy number changes in the paired samples. IDH1 mutation present in tumour biopsies was not maintained in their respective cell cultures. These findings give an insight into the molecular mechanisms involved in the tumourigenesis of low grade glioma and also tumour progression.

Mosaicism is represented by two or more chromosomally different cell lines in an individual. Mosaics are most often caused by chromosome malsegregation during mitosis, resulting in the gain or loss of chromosomes, known as aneuploidy, but structural aberrations can also occur in mosaic form. The problem is the limitation of detection with standart cytogenetic methods. The present study was carried out to compare the efficiency of FISH, array CGH and cytogenetic techniques in detection of mosaicism. In the practical part the results of 45 patients with mosaicisms of aneuplody of gonosomes (26 patients) and mosaicisms of autosomes (19 patients) were compared. The data show that we have different peripheral blood karyotype and FISH results in 23 of 37 patients (62%). There was a case of failure of detection of the mosaicism on the karyotype and the FISH method revealed a abnormal cell lines with a percentage of less than 5%. The array CGH method confirmed the karyotype and FISH results in 10 out of 12 patients (83%) in peripheral blood tests. The work also dealt with artificially made mosaics. From the results, it is obvious that the FISH method has a more accurate percentage of mosaic capture compared to the karyotype. The results indicate that using the techniques in parallel allow in clinical...

Made available in DSpace on 2016-08-10T10:38:31Z (GMT). No. of bitstreams: 1 LUCIANA ALVES ANTONIO MACHADO.pdf: 708554 bytes, checksum: ee6ad7a8ea7c2a08efc5ed340a9ce84c (MD5) Previous issue date: 2011-09-23 / Hearing loss is the most common sensory deficit in humans, which causes a negative impact in the structure of language and oral communication skills. One out of every thousand children are born deaf or become deaf to varying degrees before language is acquired, thus developing a pre-lingual deafness. Knowing the molecular genetic basis of the auditory system would provide early intervention, development of new therapies, genetic counseling and more productive social integration of individuals with hearing loss. Genomic studies would produce important information to support the development of technical, technological, and scientific tools that would help the regional community to provide better health care to the deaf . Moreover, genomic studies would ensure a molecular epidemiological analysis that portrays the reality of better health disorders in the population of Goiás. The current study was designed to identify chromosome regions containing genomic imbalances potentially associated with the nonsyndromic sensorineural hearing loss using molecular cytogenetics. Peripheral blood samples were obtained from 31 patients from the Clinical School of Speech Pathology, Pontifical Catholic University of Goiás, with sensorioneural hearing loss of unknown etiology, with or without family history and without a concurrent syndrome. All subjects were submitted to audiological evaluation, followed by short-term cultures of peripheral blood T lymphocytes in order to obtain metaphase chromosomes for karyotyping. Chromosomal imbalances was assessed by CGH. In the studied group, 45.2% were male and 54.8% female, mean age was 19.5 years, while 54.8% reported family history of deafness, 9% reported lack of familial history, and 3.2% could not say. All participants had sensorioneural hearing loss varying from mild to severe degrees, of which 87.1% exhibited prelingual hearing loss and 12.9% post-lingual. The multifactorial nature of hearing loss was certainly evident. However, chromosomal imbalances played an important role in the genesis and development of the disorder. Thus, a better understanding of the roles of genetic factors in the mechanisms related to the processes of cell differentiation, contributes to better understand the genesis of health problems, especially in the field of hearing loss.1 Referencial Teórico / A perda auditiva é o déficit sensorial mais comum em humanos e gera alterações na estruturação da linguagem e na capacidade de comunicação oral. Uma em cada mil crianças nasce surda ou se tornará surda em diferentes graus antes que a linguagem seja adquirida, período pré lingual. O conhecimento dos fundamentos genéticos moleculares do sistema auditivo proporcionará intervenções precoces, desenvolvimento de novas terapias, facilitação do aconselhamento genético e maior integração do indivíduo, com perda auditiva, na sociedade. Estudos genômicos poderão solidificar a geração e desenvolvimento do conhecimento técnico científico e tecnológico na comunidade científica regional, além de garantir uma análise epidemiológico molecular que retrate melhor a realidade dos agravos da saúde na população em Goiás. O presente estudo teve como objetivo identificar regiões cromossômicas contendo imbalanços genômicos, potencialmente associados nas perdas auditivas sensorioneurais não sindrômicas utilizando abordagens de citogenética molecular. Para a participação na pesquisa, amostras de sangue periférico foram obtidas de 31 pacientes da Clínica Escola de Fonoaudiologia da Pontifícia Universidade Católica de Goiás, com perda auditiva sensorioneural sem etiologia definida, com ou sem antecedentes familiares e sem características sindrômicas. Todos os indivíduos foram submetidos à avaliação audiológica, seguidos de culturas de linfócitos T do sangue periférico em curto prazo, conforme os protocolos para a obtenção de cromossomos metafásicos para a avaliação cariotípica. A investigação dos imbalanços cromossômicos foi realizada por CGH, utilizando o kit comercial para hibridação genômica comparativa, seguindo as recomendações do fabricante. Do grupo amostral, 45,2% (14/31) eram do sexo masculino e 54,8% (17/31) do feminino, com idade média de 19,5 anos, sendo que 54,8% citaram antecedentes familiares, 41,9% afirmaram não haver antecedentes e 3,2% não souberam informar. Todos os participantes apresentavam Perda Auditiva sensorioneural variando o grau de leve a profundo, dos quais 87,1% dos indivíduos referiram perda auditiva pré lingual e 12,9% pós lingual. A natureza multifatorial da perda auditiva é certamente uma situação muito evidente. Contudo, os imbalanços cromossômicos assumem um papel importante na gênese e desenvolvimento da doença. Deste modo, o melhor entendimento dos papéis dos fatores genéticos nos mecanismos relacionados com os processos de diferenciação celular, vem contribuir no melhor entendimento da gênese dos agravos à saúde, em especial, na perda auditiva.

Le radon est un carcinogène pulmonaire avéré chez l'homme à fortes doses mais l'existence d'un risque de cancer à faibles doses n'est pas établie car les approches épidémiologiques se heurtent, à ce niveau de doses, à de nombreux facteurs confondants. Ces données soulignent la nécessité de disposer d'un modèle d'étude des tumeurs pulmonaires chez l'animal. <br />Le but de ce travail de thèse est de caractériser, sur le plan moléculaire, une série de tumeurs pulmonaires radon-induites chez le rat qui comprend des adénocarcinomes (AC), des carcinomes épidermoïdes (Sq) et des carcinomes adénosquameux (ASq) formés des composantes cellulaires AC et Sq. Une analyse cytogénétique globale de ces tumeurs par la technique de CGH a permis de définir des déséquilibres récurrents de régions chromosomiques, sur lesquelles ont été ciblés des gènes candidats potentiellement impliqués dans le développement des tumeurs, comme p16Ink4a, p19Arf, Rb1, K-Ras ou c-Myc. Une analyse plus précise des voies p16Ink4a/Cdk4/Rb1 et p19Arf/Mdm2/Tp53 a été menée et a permis de mettre en évidence une inactivation fréquente de la voie Rb1, par la perte d'expression de la protéine p16Ink4a, indiquant que cette voie joue un rôle majeur dans le développement de ces tumeurs pulmonaires. Enfin, une analyse transcriptomique comparative des trois types de tumeurs induites a révélé que les tumeurs complexes ASq ont un profil d'expression génique qui reflète leur nature composite mais qu'elles présentent également des spécificités. <br />L'ensemble de ce travail à mis à jour des caractéristiques moléculaires communes aux tumeurs pulmonaires murines et humaines qui encouragent à poursuivre l'étude des spécificités des tumeurs pulmonaires radio-induites chez le rat pour aider à une meilleure caractérisation de ces tumeurs chez l'homme.

<p>Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the <i>PDGFRA</i> gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called <i>Sequence Allocator</i>, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.</p>

<p>Chromosomal abnormalities are defined as changes in the chromosome structure and fall in one of two categories. The first category is numerical alterations while the second category consists of structural abnormalities. Structural chromosomal abnormalities do not always interrupt genes in order to cause disease. They can also affect gene expression by separating a gene and its promoter element from distant regulatory elements. We have used characterisation of structural chromosomal abnormalities to identify the genetic bases for several congenital disorders.</p><p>In papers I-III, we have applied molecular characterisation of chromosomal translocations in order to identify candidate genes involved in mental retardation, hypospadias and anal malformation and premature ovarian failure. In paper I, we localised the chromosome X translocation breakpoint in a t(X;15) to be in the immediate proximity of the gene <i>ZDHHC15 </i>in a patient with severe mental retardation. Subsequent experiments revealed loss of <i>ZDHHC15</i> transcription in the patient which suggests this gene to be involved in the aetiology of the patient’s phenotype. In paper II, we show that a balanced translocation between chromosomes 6 and 17 in a patient with urogential malformation disrupts 2 genes, one at each translocation breakpoint. We also identified a fusion-gene as a result of the translocation. Our hypethesis is that the translocation together with its molecular consequences is important for the phenotype in the patient. Similarly, in paper III, we have used molecular characterisation of the breakpoints in a balanced translocation between chromosomes X and 11 in order to localise candidate genes in ovarian function. Our results indicate a number of genes affected by the translocation. In paper IV, we have used array-based comparative genomic hybridisation (array-CGH) in order to investigate a cohort of autistic sib-pairs for submicroscopic chromosomal alterations. We have identified several novel duplications and one novel deletion with strong association with autism.</p>

<p>Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. </p><p>In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions.</p><p>In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD.</p><p>In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype.</p>

Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the PDGFRA gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called Sequence Allocator, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.

Chromosomal abnormalities are defined as changes in the chromosome structure and fall in one of two categories. The first category is numerical alterations while the second category consists of structural abnormalities. Structural chromosomal abnormalities do not always interrupt genes in order to cause disease. They can also affect gene expression by separating a gene and its promoter element from distant regulatory elements. We have used characterisation of structural chromosomal abnormalities to identify the genetic bases for several congenital disorders. In papers I-III, we have applied molecular characterisation of chromosomal translocations in order to identify candidate genes involved in mental retardation, hypospadias and anal malformation and premature ovarian failure. In paper I, we localised the chromosome X translocation breakpoint in a t(X;15) to be in the immediate proximity of the gene ZDHHC15 in a patient with severe mental retardation. Subsequent experiments revealed loss of ZDHHC15 transcription in the patient which suggests this gene to be involved in the aetiology of the patient’s phenotype. In paper II, we show that a balanced translocation between chromosomes 6 and 17 in a patient with urogential malformation disrupts 2 genes, one at each translocation breakpoint. We also identified a fusion-gene as a result of the translocation. Our hypethesis is that the translocation together with its molecular consequences is important for the phenotype in the patient. Similarly, in paper III, we have used molecular characterisation of the breakpoints in a balanced translocation between chromosomes X and 11 in order to localise candidate genes in ovarian function. Our results indicate a number of genes affected by the translocation. In paper IV, we have used array-based comparative genomic hybridisation (array-CGH) in order to investigate a cohort of autistic sib-pairs for submicroscopic chromosomal alterations. We have identified several novel duplications and one novel deletion with strong association with autism.

Fortschritte in der molekulargenetischen Charakterisierung von Tumorerkrankungen haben in den letzten Jahren die klinische Praxis zunehmend beeinflusst. Das Ziel dieser Arbeit war die Untersuchung von astrozytären Tumoren der WHO-Grade II bis IV und ihre Subtypisierung anhand der gefundenen chromosomalen Aberrationen. Ferner sollte der Einfluss der gefundenen Aberrationen auf klinische Parameter wie das Gesamtüberleben oder die rezidivfreie Zeit untersucht werden. Dazu wurden paraffinfixierte Proben von insgesamt 184 primären astrozytären Tumoren (28 low-grade Astrozytome, 6 low-grade Oligoastrozytome, 50 anaplastische Astrozytome, 4 anaplastische Oligoastrozytome, 96 Glioblastoma multiforme) mit der Comparativen Genomischen Hybridisierung (CGH) untersucht. Häufige Aberrationen in allen Malignitätsgruppen stellten chromosomale Zugewinne auf Chromosom 7 sowie Verluste von Chromosom 10 und 9p dar. High-grade Astrozytome zeigten ferner häufig Zugewinne von Chromosom 19 und 20 sowie Verluste von 13q, 14q und 15q. WHO-Grad-II-Astrozytome wiesen häufig Zugewinne auf Chromosom 8 sowie Verluste von Chromosom 4q und 6q auf. Eine kürzeres Gesamtüberleben zeigten high-grade Gliome mit Verlusten von Chromosom 10q und Zugewinnen auf 7p. In Glioblastomen verursachten zusätzlich Zugewinne auf 7q sowie Verluste von 14q, in anaplastischen Astrozytomen zusätzlich Verluste von 10p ein verringertes Gesamtüberleben. WHO-Grad-II-Astrozytome zeigten bei Verlusten von 3p ein schlechteres Gesamtüberleben. Chromosomale Aberrationen, die zu einem verlängerten Gesamtüberleben führten, waren Verluste von 1p und Zugewinne von 10p in WHO-Grad-III-Tumoren. Die rezidivfreie Zeit wurde in high-grade Gliomen durch Zugewinne auf 7p und Verluste von 10p verringert. Eine Verkürzung der rezidivfreien Zeit in Glioblastomen zeigten außerdem Tumoren mit Verlusten von 7q, 10q und 14q. In anaplastischen Astrozytomen führten Verluste von 1p und 19q sowie Zugewinne auf 8q und 10p, in WHO-Grad-II-Astrozytomen Verluste von Chromosom 6 zu einer verlängerten Zeit bis zum Rezidiv. Die Anfertigung onkogenetischer Baummodelle stellte verschiedene genetische Wege der Tumorgenese dar. Ein Cluster war gekennzeichnet durch einen Verlust von 6q, ein weiterer wurde initialisiert durch den Verlust von 13q, der dritte durch den Verlust von 9p. Der vierte Cluster wurde charakterisiert durch Zugewinne auf Chromosom 7 und Verluste von Chromosom 10, während der fünfte Cluster Zugewinne auf 8q sowie Verluste von 4q aufwies.