An optimization framework for unsupervised identification of rare copy number variation from SNP array data

A highly sensitive and configurable method for calling copy number variants from SNP array data is presented that can identify even rare CNV

Assessing the Significance of Conserved Genomic Aberrations Using High Resolution Genomic Microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA

GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers

We describe methods with enhanced power and specificity to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth. By separating SCNA profiles into underlying arm-level and focal alterations, we improve the estimation of background rates for each category. We additionally describe a probabilistic method for defining the boundaries of selected-for SCNA regions with user-defined confidence. Here we detail this revised computational approach, GISTIC2.0, and validate its performance in real and simulated datasets

Copy number variations (CNVs) in the assessment of stroke susceptibility in patients with sickle cell anemia

Orientador: Mônica Barbosa de MeloTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Embora a anemia falciforme (AF) resulte da homozigose de uma única mutação, na posição 6 do locus da ?-hemoglobina, fenotipicamente, é uma doença muito heterogênea, de modo que diferentes pacientes apresentam evoluções clínicas significativamente distintas. Praticamente todos os órgãos podem ser afetados pela oclusão vascular, merecendo destaque o Sistema Nervoso Central (SNC), onde são observados acidentes isquêmicos transitórios, infartos e hemorragia cerebral, os quais acometem aproximadamente 25% dos pacientes com AF. As complicações neurológicas são graves, podendo ser fatais em até 15% dos casos. A identificação precoce de pacientes com anemia falciforme suscetíveis ao Acidente Vascular Cerebral (AVC) poderia diminuir os riscos, possivelmente prevenir a recorrência de infartos e potencialmente reduzir sua incidência. Portanto, estudos que visem identificar novos grupos de risco para o desenvolvimento de AVC em pacientes com anemia falciforme seriam fundamentais para otimizar o controle clínico desta enfermidade, sendo uma dessas vias a abordagem molecular deste grupo de pacientes. Desta forma, propusemos a investigação da presença de variação no número de cópias alélicas ou "Copy Number Variation" (CNV), utilizando-se lâminas de alta densidade (microarrays), com o objetivo de identificar regiões do genoma, potencialmente envolvidas com o risco aumentado de AVC em pacientes com anemia falciforme. Este trabalho propôs ainda avaliar os aspectos populacionais relativos à população brasileira. É de grande interesse nos estudos de genética humana entender a manifestação de dado fenótipo em populações distintas, e se a associação de variantes em uma população pode ser extrapolada para outras. A anemia falciforme fornece um modelo nesse intento, passível de ser extrapolado para doenças complexas dado ser um grupo de origem bem conhecida, exemplo clássico de seleção balanceada em humanos, primeira doença a ter base molecular desvendada e ser de herança mendeliana. Observou-se marcante diferença na ancestralidade de indivíduos afetados por AF brasileiros em relação a norte-americanos, tanto no genoma geral, quanto no cromossomo 11. Chegou-se a uma listagem de regiões com variações no número de cópias potencialmente associadas ao AVC em ambas as populaçõesAbstract: Although sickle cell anemia results from homozygosity for a single mutation at position 6 of gamma-hemoglobin locus, this disease presents high heterogenity in phenotype, so that different patients may have significantly different clinical outcomes. Virtually all organs may be affected by vascular occlusion, with emphasis on the Central Nervous System (CNS), where are observed transient ischemic attacks, stroke and cerebral hemorrhage, which affect approximately 25% of patients with SCA. Neurological complications are serious and can be fatal in up to 15% of cases. Early identification of patients with sickle cell anemia, susceptible to stroke (CVA) could reduce the risk, possibly preventing the recurrence of heart attacks and potentially reduce their incidence. Therefore, studies aimed at identifying new risk groups for development of stroke in sickle cell patients would be essential to optimize the clinical management of this disease, one of these pathways the molecular approach of this group of patients. Thus, we propose to investigate the presence of copy number variation in allele or "Copy Number Variation" (CNV), using high density microarray in order to identify genomic regions potentially involved in the increased risk of stroke in sickle cell patients. The present work also proposes to assess the Brazilian population genetic aspects. It is highly desirable in human genetics to unveil the manifestation of a given phenotype in distinct populations, as well as whether an association identified on a specific population may hold to other populations. Sickle cell anemia provides a model for this purpose, and may be extrapolated to complex diseases given its well known geographical region, and for being a classic case of balanced selection in humans, the first disease to have its molecular mechanism unraveled, and a Mendelian trait. Ancestral components markedly differ in Brazilian SCA patients compared to the United States subjects on both the genomic landscape and the local ancestry on chromosome 11. Here we present a list of CNV events potentially associated to stroke on both populationsDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecular140967/2012-52012/06438-5FAPESPCNP

Molecular gene expression and genome wide profiling in tamoxifen-resistant breast cancer.

PhDOestrogen receptor positive (ER+) breast cancers (BC) are heterogeneous in both their clinical behaviour and response to therapy. The ER and Progesterone (PgR) are currently the best predictors of response to the anti-oestrogen tamoxifen, yet up to 40% of ER+ breast cancer will relapse despite tamoxifen treatment. New prognostic biomarkers and further biological understanding of tamoxifen resistance (TR) are required. There has been an explosion of greater understanding since the arrival of cutting-edge gene and genomic profiling technology. The two major aims of this research are to develop stable gene signatures that are effective at distinguishing „prognostic‟ groups and, when tested directly for response to tamoxifen, a set of „predictive‟ markers. In order to establish cellular pathways responsible for TR, tissue at relapse while on tamoxifen is preferred. However, in practice, this is difficult to obtain. Hence, in this study, I have established TR derivatives of breast cancer cell lines, T47D and ZR75-1, and analysed their gene-expression by microarray. MAGEA2 and EGLN3 were 4.0 and 3.8 fold upregulated respectively in TR cell lines. For MAGEA2- and EGLN3-overexpressing lines, the proliferation and growth rates in tamoxifen-containing media were significantly higher (p-value <0.001 and p<0.05, respectively) than for control cells. I have investigated possible downstream targets for each protein which may contribute to the mechanism of resistance. Immunohistochemistry validation was performed on a cohort of 196 tamoxifen-treated primary breast tumour tissues: MAGEA2 and EGLN3 were found to be valuable predictive (Positive predictive value of 89%, and 85%, with high sensitivity 38% and 42% respectively) biomarkers for TR in primary breast tumours. In the human breast tumour arm of this study, 25 frozen samples with known response to tamoxifen were analysed on both SNP6.0 and expression EXON arrays. The integrated analysis suggested that 5 genes (OPCML, OR10G7, SNF1LK2, PALM and ZBTB-16) are good predictors of TR, with high negative predictor values (68%, 71%, 59% and 73% respectively for the last 4 genes). Significant regions of copy number variation (CNV) were identified at chromosomes 8q24, 17q21-22 and 11q23-25. The application of this high-resolution approach should lead to a better understanding of the roles of complex genetic alterations in TR

A Systems Biology Approach to Epigenetic Gene Regulation

The ability to control when, and how much of the genetic code is being expressed is the underlying principle behind gene regulation. Control of gene production is able to influence a cell's phenotype by determining which structural components of the cell's observable traits (shape, growth, and behavior) are made. In multicellular organism’s different cell types are able to arise from the same genetic code due to a difference in the patterns of genes being expressed. Essentially anywhere in the process of gene expression from transcription, RNA processing, translation, and post-translational modifications of the protein is subject to regulation. As transcription is the first step in the process of gene expression, it is the first level of regulation for influencing the cell phenotype. The actions of transcription factors, histone modifiers, and other proteins work together to influence RNA polymerase's ability to complete the process of transcription. The actions of transcription factors are able to influence transcription by controlling the ability of RNA polymerase to be recruited to the start of a protein coding region and histone modifiers can rearrange the histones of the chromatin causing entire regions of a chromosome to become exposed or sequestered. These transcriptional regulators are able to work in a combinatorial fashion with one another to either activate and/or repress wide repertoires of transcriptional targets. Mapping out a network of interactions between these transcriptional regulators in gene expression programs allows researchers to understand how each protein is able to influence the phenotype of the cell, and how mutations to any of these transcriptional regulators are able to drive the cell into a diseased state. In the case of cancer, changes in the mechanisms of gene regulation brought on by mutations to these transcriptional regulators may drive the cell's hyper proliferative state. With the creation of next generation sequencing researchers are now better able to define where regulation is taking place in the genome, and how much it is able to influence gene expression. This gives researchers the ability to build these gene regulatory networks and evaluate their impact on gene expression. The subsequent chapters of this dissertation are a reflection of my published work investigating the contribution of oncogenic processes to gene regulatory networks in cancer through the study of hyperactivating somatic mutation of a histone modifier, changes in transcription factor response element specificity, epigenetic regulation of transcription factor signaling, and a transcription factor coactivation network

Examination And Characterization Of Cancer Risk Variants

Genome-wide association studies (GWAS) have been a useful tool in identifying numerous genetic loci that associate with increased risk for numerous cancers. However, as most of the identified risk variants are found in non-coding regions of the genome, the field has been slow in moving beyond identifying the risk variants to functionally determining the mechanism for cancer predisposition. The ultimate goal of genome-wide association studies is to identify and understand the role of these loci disease etiology to ultimately enable more effective screening and therapeutic treatments. Framed by better understanding GWAS results, my dissertation has 3 main aspects, where I: 1) developed a computational approach to characterize GWAS results using publically-available epigenomic databases, 2) identified novel germline susceptibility loci for myeloproliferative neoplasms, and 3) examined molecular mechanisms by which a prostate cancer single-nucleotide polymorphism may increase risk

Molecular classification of uterine leiomyomas by genome-wide methods

Uterine leiomyomas, often called fibroids, are highly common tumors arising from smooth muscle cells of the myometrium. Whereas cancers have the ability to metastasize, leiomyomas are benign tumors that grow only locally. Nevertheless, leiomyomas frequently cause a variety of health complications, including abdominal pain, abnormal menstrual bleeding, and impaired fertility. Leiomyomas are the leading indication for hysterectomy worldwide, and pose a significant socio-economic impact. Despite their major public health impact, this disease attracts relatively little research. Epidemiological and molecular studies have indicated that, in the etiology of leiomyomas, genetic factors play a central role. Early cytogenetic studies revealed that approximately half of all leiomyomas display non-random chromosomal abnormalities such as high mobility group AT-hook 2 (HMGA2) gene translocations. Furthermore, family-based linkage studies revealed that germline mutations in the fumarate hydratase (FH) gene result in high penetrance susceptibility to uterine leiomyomas. Sporadic leiomyomas, however, rarely harbor FH mutations and the majority lack chromosomal abnormalities, suggesting that some driver genes remain undiscovered. Recent advances in sequencing technologies have made it possible to examine tumor genomes on a previously unprecedented scale. The aim of this thesis was to characterize the molecular underpinnings of uterine leiomyomas by the use of genome-wide methods such as massively parallel sequencing technology and gene expression microarrays. Using exome sequencing, we discovered that 71% of leiomyomas display localized mutations in the mediator complex subunit 12 (MED12) gene, making it their most commonly mutated gene. Furthermore, with whole-genome sequencing, we discovered that a subset of leiomyomas display highly complex chromosomal rearrangements, ones previously undetectable by conventional cytogenetic techniques. These rearrangements closely resembled chromothripsis, a phenomenon in which one or a few chromosomes are shattered into multiple pieces and randomly stitched together in a single event. We also found these events to have occurred multiple times, and some had resulted in genetic changes with a selective value, such as collagen type IV alpha 5 chain and collagen type IV alpha 6 chain (COL4A5-COL4A6) deletions. Patients affected by leiomyomas frequently harbor multiple distinct tumor nodules. Whereas the majority of studies have proposed that each leiomyoma arises independently, we found some leiomyomas to display identical chromosomal abnormalities, suggesting a common clonal origin. Whole-genome sequencing of clonally related leiomyomas revealed intratumor genetic heterogeneity suggestive of a branching model of tumor growth. Furthermore, we also discovered DEP domain containing 5 (DEPDC5) as a novel tumor suppressor gene, acting as a secondary driver gene in a subset of leiomyomas. Our integrative analyses demonstrated that specific genetic defects were the major determinants of expression changes in leiomyomas. Our observations indicate that at least four molecular subtypes exist: leiomyomas harboring a MED12 hotspot mutation, HMGA2 overexpression, FH inactivation, or COL4A5-COL4A6 deletion. We also detected subtype-specific expression differences in key tumorigenic pathways, including Wnt/β-catenin, Prolactin, IGF-1, and NRF2 signaling. Using genome-wide methods in this thesis work, we have discovered several novel molecular defects that underlie leiomyoma etiology. These studies emphasize the importance of stratification in leiomyoma research and offer a set of candidate biomarkers that may facilitate the molecular classification of uterine leiomyomas. Millions of women suffer from uterine leiomyomas, and the ability to classify each lesion should pave the way towards personalized treatments.Myom är godartade tumörer av glattmuskelceller som växer i livmodern. Kraftiga blödningar och smärtor är de vanligaste symptomen. En del kvinnor kan även bli infertila av myom. Hysterektomi är för tillfället den vanligaste och effektivaste operationen för myom. Trots deras kliniska och socioekonomiska konsekvenser, undersöks dessa tumörer relativt lite. Tidigare studier har visat att genetiska faktorer spelar en central roll i uppkomsten av myom. Undersökningar med hjälp av cytogenetik har visat att ungefär hälften av myom har kromosomavvikelser, såsom translokationer av genen high mobility group AT-hook 2 (HMGA2). Genetisk kopplingsanalys identifierade att ärftliga mutationer i genen fumarate hydratase (FH) är förknippade med en hög risk för myom. Sporadiska myom har dock sällan somatiska FH mutationer och majoriteten saknar kromosomavvikelser, vilket tyder på att en del gen-mutationer är oupptäckta. Framsteg inom sekvenseringsteknologin har gjort det möjligt att sekvensera människors hela genom mycket billigare och på en kortare tid. Syftet med denna avhandling var att karakterisera de molekylära faktorerna som bidrar till utveckling av myom med hjälp av massiv parallell sekvensering och mikromatriser. Med exom-sekvensering upptäckte vi att 71% av myom har specifika mutationer i genen mediator complex subunit 12 (MED12). Med genom-sekvensering upptäckte vi att en del myom har väldigt komplexa kromosomavvikelser ( chromothripsis ) som har uppstått på en och samma gång. En del av dessa kromosomavvikelser hade resulterat i specifika genetiska förändringar, såsom deletioner i generna collagen type IV alpha 5 chain och collagen type IV alpha 6 chain (COL4A5-COL4A6). Patienter som drabbas av myom har oftast flera myom i livmodern. Även om de flesta studier indikerar att varje myom uppstår självständig, så upptäckte vi att vissa myom kan ha identiska kromosomavvikelser, vilket tyder på att flera myom kan ha ett gemensamt ursprung. Dessutom upptäckte vi att genen DEP domain containing 5 (DEPDC5) har en sekundär roll i tillväxten av sådana myom. Vi upptäckte att specifika genetiska mutationer leder till unika genuttrycks mönster i myom. Våra observationer indikerar att åtminstone fyra molekylära underklasser av myom existerar: myom med mutationer i MED12, myom med överuttryck av HMGA2, myom med inaktivering av FH, och myom med deletioner i COL4A5 och COL4A6. Vi upptäckte att dessa underklasser har unika genuttrycks mönster i Wnt/β-catenin, prolaktin, IGF-1, och Nrf2 signalering. Med hjälp av dessa nya teknologier har vi nu upptäckt flera nya molekylära faktorer som bidrar till uppkomsten och tillväxten av myom. Dessa studier betonar betydelsen av att klassificera myom på molekyl-nivå, och vi identifierade potentiella biomarkörer som kan underlätta denna klassificering. Miljontals kvinnor drabbas av myom, och förmågan att klassificera myom kommer att förbättra utvecklingen av behandlingar mot denna vanliga sjukdom