As malformações cardíacas congênitas (MCCs) são as malformações mais comuns ao nascimento, representando uma importante causa de morbidade e mortalidade em recém-nascidos. Nos últimos anos, estudos utilizando testes citogenômicos têm permitido elucidar e compreender melhor as causas das MCCs. O objetivo geral desse estudo foi investigar a presença de CNVs em amostras de tecido obtidas post-mortem de portadores de malformações cardíacas congênitas sindrômicas; e os objetivos específicos consistiram em avaliar a frequência das CNVs, destacando as mais relevantes, comparar a presença de CNVs nos diferentes tecidos e realizar a correlação genótipo-fenótipo. Para isso, foram estudados um total de 52 casos de natimortos e recém-nascidos provenientes do Serviço de Verificação de Óbitos - FMUSP. Amostras de DNA extraídas da pele, diafragma e do coração foram avaliadas utilizando o kit AmpFlSTR® MiniFiler(TM) PCR Amplification (Life Technologies(TM), USA) e a técnica de Multiplex Ligation-dependent Probe Amplification (MLPA) com diferentes kits (MCR-Holland, Holanda). A técnica de FISH foi utilizada para a confirmação dos resultados obtidos em um dos casos estudados. Foram encontradas CNVs relevantes em 21 casos, incluindo trissomia do 18 (10 casos), trissomia do 21 (4 casos), trissomia do 13 (2 casos), trissomia do 16 (1 caso), monossomia do X em mosaico (1 caso), dup 4p16 (1 caso), dup 11q25 (1 caso) e del GATA4 éxon 6 (1 caso). A análise genômica se mostrou eficiente na investigação das bases genômicas e na caracterização das diferentes malformações em amostras post-mortem de portadores de MCC sindrômicas / Congenital heart defects (CHDs) are the most common birth defect and represent an important cause of morbidity and mortality in newborns. In recent years, studies using cytogenomic tests have enabled an improved understanding of the causes of CHD. The general objective of this study was to investigate the presence of CNVs in post-mortem tissue samples from patients with congenital syndromic cardiac malformations; and the specific objectives were to evaluate the frequency of CNVs, highlighting the most relevant ones, to compare the presence of CNVs in the different tissues and to perform the genotype-phenotype correlation. For this, a total of 52 stillbirth and newborn cases from the Death Verification Service (SVO), FMUSP were investigated. DNA samples from skin, diaphragm and heart tissues were evaluated using an AmpFlSTR® MiniFiler(TM) PCR Amplification Kit (Life Technologies(TM), California, USA) and Multiplex Ligation-dependent Probe Amplification (MLPA) with different kits (MCRHolland, Amsterdam, The Netherlands). FISH was used to confirm the results of one of the studied cases. The results showed relevant copy number variations (CNVs) in 21 cases, including trisomy 18 (10 cases), trisomy 21 (4 cases), trisomy 13 (2 cases), trisomy 16 (1 case), mosaic monosomy X (1 case), dup 4p16 (1 case), dup 11q25 (1 case) and del GATA4 exon 6 (1 case). Genomic analysis was found to efficiently identify the genomic basis of, and characterize, various malformations found in postmortem samples from syndromic CHD carriers

Accelerated Adaptation through Stimulated Copy Number Variation in Saccharomyces cerevisiae Ryan Matthew Hull Repetitive regions of the genome, such as the centromeres, telomeres and ribosomal DNA account for a large proportion of the genetic variation between individuals. Differences in the number of repeat sequences between individuals is termed copy number variation (CNV) and is rife across eukaryotic genomes. CNV is of clinical importance as it has been implicated in many human disorders, in particularly cancers where is has been associated with tumour growth and drug resistance. The copper-resistance gene CUP1 in Saccharomyces cerevisiae is one such CNV gene. CUP1 is transcribed from a copper inducible promoter and encodes a protein involved in copper detoxification. In this work I show that yeast can regulate their repeat levels of the CUP1 gene through a transcriptionally stimulated CNV mechanism, as a direct adaptation response to a hostile environment. I characterise the requirement of the epigenetic mark Histone H3 Lysine 56 acetylation (H3K56ac) for stimulated CNV and its limitation of only working at actively transcribed genes. Based upon my findings, I propose a model for how stimulated CNV is regulated in yeast and show how we can pharmacologically manipulate this mechanism using drugs, like nicotinamide and rapamycin, to stimulate and repress a cell's ability to adapt to its environment. I further show that the model is not limited to high-copy CUP1 repeat arrays, but is also applicable to low-copy systems. Finally, I show that the model extends to other genetic loci in response to different challenging environments, such as formaldehyde stimulation of the formaldehyde-resistance gene SFA1. To the best of our knowledge, this is the first example of any eukaryotic cell undergoing genome optimisation as a novel means to accelerate its adaptation in direct response to its environment. If conserved in higher eukaryotes, such a mechanism could have major implications in how we consider and treat disorders associated with changes in CNV.

A doença de Alzheimer (DA) é a causa mais comum de demência na população, sendo responsável por cerca de 50 a 60% dos casos. Embora o diagnóstico clínico da doença na maioria das vezes seja acurado, a confirmação da DA só é feita post mortem através principalmente da caracterização dos dois tipos principais de lesões neurais: depósitos extracelulares de placas de β amiloide e emaranhados de proteína tau hiperfosforilada. Até o momento, o envolvimento de apenas quatro genes foi confirmado na etiologia da DA, três deles (APP, PSEN1 e PSEN2) associados à forma familial de herança mendeliana, que corresponde a um tipo raro e grave. No entanto, apesar de inúmeros trabalhos de associação genômica, (Genome wide association studies- GWAS) sugerirem uma possível participação de vários outros genes na suscetibilidade à manifestação da forma multifatorial da DA, o gene APOE, ainda é o único consistente e reproduzivelmente associado à doença. As descobertas derivadas dos GWAS investigando o papel de SNPs coletivamente explicam somente uma pequena porcentagem da variação herdada que contribui para o risco de desenvolver a DA. Atualmente, há novas abordagens para investigar a base genética do restante da variabilidade fenotípica herdada e que pode influenciar a suscetibilidade ao desenvolvimento de doenças complexas. O papel da variação do número de cópias de segmentos de DNA (Copy Number Variation - CNV) na genética de doenças complexas foi demonstrado por diversos estudos nos últimos anos e evidencia que desequilíbrios genômicos também podem contribuir significantemente para a resistência ou susceptibilidade a várias patologias. Outro aspecto que vem assumindo crescente importância é a análise de modificações epigenéticas que podem constituir um mecanismo molecular básico e contribuir diretamente para a patogênese da DA. Logo, este trabalho teve como objetivo principal investigar dois aspectos relacionados à DA: (1) a identificação de CNVs que podem estar contribuindo para o desenvolvimento da forma multifatorial da DA, usando a técnica de array-CGH, e (2) a análise de alterações do padrão global de metilação do DNA no córtex frontal de indivíduos com a forma multifatorial da DA, usando um microarranjo que interroga o status de metilação de 450.000 sítios CpGs. Em nossa investigação sobre desequilíbrios genômicos na DA, identificamos 6 CNVs raras com conteúdo gênico relevante para o fenótipo investigado. Dois indivíduos distintos do grupo DA apresentam microduplicações em genes que codificam diferentes subunidades do mesmo tipo de canal de Ca2+ dependente de voltagem, o tipo L. Além disso, dos outros genes selecionados como especialmente interessantes, 4 estão envolvidos em diferentes processos inflamatórios e 1 é responsável por codificar a enzima nicotinamida fosforibosiltransferase, participante importante da via de biossíntese da molécula nicotinamida adenina dinucleotídeo (NAD). A implicação de um possível envolvimento de mediadores da sinalização celular do Ca2+ e da via de biossíntese da NAD na etiologia da DA também foi reforçada pelos nossos resultados sobre o padrão de metilação do DNA na DA. Dois genes importantes para a homeostasia intracelular do Ca2+ e via de biossíntese da NAD apresentaram sítios CpGs diferenciamente metilados nos sujeitos com DA / Alzheimer\'s disease (AD) is the most common form of dementia in the population, corresponding to 50-60% of all cases. Although clinical diagnosis seems to be accurate, the definitive diagnosis of the disease can only be made by a post mortem neuropathological exam that certifies the presence of the two hallmarks of AD: the accumulation of extracellular senile plaques containing β-amyloid (Aβ) and the intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Four genes are known to be involved in the etiology of AD, three of them (APP, PSEN1 and PSEN2) are associated to the familial form of the disease, which show autosomal dominant inheritance and correspond to the more severe and rare type of AD. Despite many genome wide association studies (GWAS), APOE still remains the only unequivocal genetic risk factor associated to the multifactorial form of AD. The discoveries from GWAS using SNPs collectively explain only a small percentage of heritable variation that may contribute in AD risk. Currently, new approaches have been used to investigate the genetic basis of the phenotypical variability inheritance that can influence the susceptibility of complex diseases. The important role of DNA copy number variation (CNV) has been demonstrated by several studies over the last years and shows that genomic imbalances may also significantly contribute to resistance or susceptibility to various complex diseases. Additionally, there is now increasing interest in exploring how epigenetic modifications, in particular DNA methylation, could influence complex diseases etiology. Thus, the major aim of this work were to investigate two aspects related to the multifactorial form of AD: (1) identification of rare CNVs, using array-CGH, that could contribute to the development of the disease, and (2) analysis of the DNA methylation pattern in frontal cortex of individuals with AD. In our study, we identified 6 rare CNVs with relevant gene content to the investigated phenotype. Two distinct subjects with AD from our casuistic presented microduplications in genes that encode different subunits of the same type of Ca2+ voltage channel, the L-type. Furthermore, among the other selected genes, four are involved in different inflammatory process and one encodes the nicotinamide phosphoribosyltransferase enzyme, important mediator of nicotinamide adenine dinucleotide (NAD) biosynthesis. The implication of a possible involvement of Ca2+ intracellular signaling mediators and NAD biosynthesis pathway in the etiology of AD was also reinforced by our analysis of DNA methylation pattern. Interestingly, two important genes, one to intracellular Ca2+ homeostasis and the other to NAD biosynthesis pathway presented CpGs sites differently methylated in the AD subjects

<p>Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the <i>NF2 </i>gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the <i>NF2</i> gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the <i>NF2</i> locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the <i>NF2 </i>locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the <i>GSTT1/CABIN1</i> locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the <i>CABIN1</i> gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.</p>

<p>Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral vestibular schwannomas (VS). Patients affected by a severe NF2 phenotype also presents with peripheral schwannomas, meningiomas and ependymomas. The closely related disorder schwannomatosis also displays multiple schwannomas, but never VS. Mutation screening of the <i>NF2</i> gene in the above mentioned tumors did not identify mutations in numerous of cases. We analyzed the DNA sequence covering the <i>NF2</i> locus in order to identify evolutionarily conserved non-genic sequences (CNGs) with unknown regulatory function (paper I). The aim was to analyze CNGs for mutations in DNA derived from patients affected by NF2 associated tumors. During mutation analysis of the coding part of <i>NF2</i> and within the CNGs defined in paper I, were mutations detected in 39% of sporadic meningiomas (paper II). Two candidate regions were identified on 22q using array-CGH. Methylation profiling did not identify methylation of the <i>NF2</i> promoter in these tumors. Sporadic schwannomas were profiled for CNV using a 22q genomic array in the search for putative gene(s) that in addition to <i>NF2</i> could be involved in the development of schwannoma and/or schwannomatosis (paper III). The predominant aberration identified was monosomy 22. Terminal and interstitial deletions encompassing the <i>NF2</i> gene were detected in tumor DNA and eight loci affected by CNV in constitutional DNA. Some of these CNVs are unlikely to be phenotypically neutral, considering their size and gene content. Two schwannomatosis candidate regions were identified on 22q using array-CGH (paper IV). These regions were further characterized by a PCR-product based array with higher resolution. Rearrangements of the immunoglobulin lambda (<i>IGL</i>) locus detected were restricted to schwannomatosis patients. In the second candidate region spanning <i>GSTT1</i> and <i>CABIN1</i> genes, was frequent copy number polymorphism at the <i>GSTT1</i> locus identified. We further describe missense mutations in the <i>CABIN1 </i>gene, making this gene a plausible candidate which may contribute to the pathogenesis of these disorders. </p>

Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the NF2 gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the NF2 gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the NF2 locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the NF2 locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the GSTT1/CABIN1 locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the CABIN1 gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral vestibular schwannomas (VS). Patients affected by a severe NF2 phenotype also presents with peripheral schwannomas, meningiomas and ependymomas. The closely related disorder schwannomatosis also displays multiple schwannomas, but never VS. Mutation screening of the NF2 gene in the above mentioned tumors did not identify mutations in numerous of cases. We analyzed the DNA sequence covering the NF2 locus in order to identify evolutionarily conserved non-genic sequences (CNGs) with unknown regulatory function (paper I). The aim was to analyze CNGs for mutations in DNA derived from patients affected by NF2 associated tumors. During mutation analysis of the coding part of NF2 and within the CNGs defined in paper I, were mutations detected in 39% of sporadic meningiomas (paper II). Two candidate regions were identified on 22q using array-CGH. Methylation profiling did not identify methylation of the NF2 promoter in these tumors. Sporadic schwannomas were profiled for CNV using a 22q genomic array in the search for putative gene(s) that in addition to NF2 could be involved in the development of schwannoma and/or schwannomatosis (paper III). The predominant aberration identified was monosomy 22. Terminal and interstitial deletions encompassing the NF2 gene were detected in tumor DNA and eight loci affected by CNV in constitutional DNA. Some of these CNVs are unlikely to be phenotypically neutral, considering their size and gene content. Two schwannomatosis candidate regions were identified on 22q using array-CGH (paper IV). These regions were further characterized by a PCR-product based array with higher resolution. Rearrangements of the immunoglobulin lambda (IGL) locus detected were restricted to schwannomatosis patients. In the second candidate region spanning GSTT1 and CABIN1 genes, was frequent copy number polymorphism at the GSTT1 locus identified. We further describe missense mutations in the CABIN1 gene, making this gene a plausible candidate which may contribute to the pathogenesis of these disorders.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

Copy-number variation (CNV) has been implicated in many complex diseases. It is of great interest to detect and locate such regions through genetic association testings. However, the association testings are complicated by the fact that CNVs usually span multiple markers and thus such markers are correlated to each other. To overcome the difficulty, it is desirable to pool information across the markers. In this thesis, we propose a kernel-based method for aggregation of marker-level tests, in which first we obtain a bunch of p-values through association tests for every marker and then the association test involving CNV is based on the statistic of p-values combinations. In addition, we explore several aspects of its implementation. Since p-values among markers are correlated, it is complicated to obtain the null distribution of test statistics for kernel-base aggregation of marker-level tests. To solve the problem, we develop two proper methods that are both demonstrated to preserve the family-wise error rate of the test procedure. They are permutation based and correlation base approaches. Many implementation aspects of kernel-based method are compared through the empirical power studies in a number of simulations constructed from real data involving a pharmacogenomic study of gemcitabine. In addition, more performance comparisons are shown between permutation-based and correlation-based approach. We also apply those two approaches to the real data. The main contribution of the dissertation is the development of marker-level association testing, a comparable and powerful approach to detect phenotype-associated CNVs. Furthermore, the approach is extended to high dimension setting with high efficiency.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.