Summary: Breast cancer (BC) is the most frequent cancer type in female population of Europe. Approximately 5 - 10 % accounts for its hereditary form which is characterized by high penetrance, early onset, risen recurrence risk and development of other cancers. Mutational analyses of high risk patients identify a predisposing mutation in one of the most studied genes (BRCA1, BRCA2, TP53, ATM, CHEK2, NBS1, PALB2) only in less than one third of tested breast cancer patients. Lately, with the use of new methods of next-generation sequencing, a number of other susceptibility or candidate genes were characterized, but the incidence of their pathogenic alteration is often geographically different. A notable proportion of high risk patients from families with hereditary BC can represent carriers of population-specific, or private mutations. Most of the to date identified BC susceptibility genes codes for proteins involved in DNA repair, especially repair of double strand break DNA repair. Nevertheless the mutation analysis was conducted only on a small fraction of these DNA repair genes. We can expect that in the group of yet nontested genes coding for DNA repair proteins a rare, but clinically important genetic alterations predisposing to BC in affected families can be discovered. This work describes a...

Next-Generation Sequencing (NGS) technologies have been evolving at an unparalleled pace. The ability to generate millions of base pairs of data in a short time and at lower cost than previously has led to a dramatic expansion of technologies within the field. This dissertation discusses the development and validation of new methods for assessing genomic variation, dynamic changes in gene expression, high-accuracy sequencing, and analysis of recombination events. By reducing the cost of analyzing many samples for genetic divergence by genotyping the same region of the genome in multiple samples, researchers can pursue investigations on a larger scale. Next-RAD (Nextera fragmentation with Restriction-Associated Digestion) allows analysis of a uniform subset of loci between organisms for comparison of populations by genetic differences with reduced burdens of cost and data analysis. This method was applied to the Anopheles darlingi mosquito to identify three distinct species that were thought to be a uniform population. The lowering cost of large-scale sequencing investigations allows for massively parallel analysis of genomic function in a single assay. Regulation of gene expression in response to stress is a complex process which can only be understood by analyzing many pathways in tandem. A novel method is described which quantifies on a genome-wide scale the expression of millions of randomer tags driven by associated transcriptional enhancers. This method provides novel data in the form of high-resolution analysis of gene regulation. Aside from generating novel data types, another force behind development of new technologies is to improve data quality. One limitation of NGS is the inherent error rate. PELE-Seq (Paired End Low Error Sequencing) was developed to address this problem, by employing completely overlapping paired-end reads as well as a dual barcoding strategy to eliminate incorrect sequences resulting from final library amplification. This new tool improves data quality dramatically. Finally, the rapid expansion of tools necessitates the identification of new applications for these technologies. To this end, 10x Genomics Linked-Read sequencing was employed to identify recombination events in multiple species. The haplotype-resolved nature of the data generated from such assays has many promising applications.

Breast cancer is a clinically and molecularly heterogeneous disease displaying distinct therapeutic responses. Although recent studies have explored the genomic and transcriptomic landscapes of breast cancer, the epigenetic architecture has received less attention. To address this, an optimised Reduced Representation Bisulfite Sequencing protocol was performed on 1482 primary breast tumours (and 237 matched adjacent normal tissues). This constitutes the largest breast cancer methylome yet, and this thesis describes the bioinformatics and statistical analysis of this study. Noticeable epigenetic drift (both gain and loss of homogeneous DNA methylation patterns) was observed in breast tumours when compared to normal tissues, with markedly higher differences in late replicating genomic regions. The extent of epigenetic drift was also found to be highly heterogeneous between the breast tumours and was sharply correlated with the tumour’s mitotic index, indicating that epigenetic drift is largely a consequence of the accumulation of passive cell division related errors. A novel algorithm called DMARC (Directed Methylation Altered Regions in Cancer) was developed that utilised the tumour-specific drift rates to discriminate between methylation alterations attained as a consequence of stochastic cell division errors (background) and those reflecting a more instructive biological process (directed). Directed methylation alterations were significantly enriched for gene expression changes in breast cancer, compared to background alterations. Characterising these methylation aberrations with gene expression led to the identification of breast cancer subtype-specific epigenetic genes with consequences on transcription and prognosis. Cancer genes may be deregulated by multiple mechanisms. By integrating with existing copy number and gene expression profiles for these tumours, DNA methylation alterations were revealed as the predominant mechanism correlated with differentially expressed genes in breast cancer. The crucial role of DNA methylation as a mechanism to target the silencing of specific genes within copy number amplifications is also explored which led to the identification of a putative tumour suppressor gene, THSZ2. Finally, the first genome-wide assessment of epigenomic evolution in breast cancer is conducted. Both, the level of intratumoural heterogeneity, and the extent of epiallelic burden were found to be prognostic, and revealed an extraordinary distinction in the role of epiclonal dynamics in different breast cancer subtypes. Collectively, the results presented in this thesis have shed light on the somatic DNA methylation basis of inter-patient as well as intra-tumour heterogeneity in breast cancer. This complements our genetic knowledge of the disease, and will help move us towards tailoring treatments to the patient's molecular profile.

Sepsis is one of the leading causes for fatalities in the intensive care unit, and also one of the biggest health problems worldwide. It is a disease caused primarily by bacterial infections but can also be caused by viral or fungal infections. Since it is such a big health problem being associated with increased risk of sepsis, coupled with longer stays in the intensive care unit, the need for fast diagnosis and treatment is very important. Currently, culture is the leading diagnostic method for identification of bacteria, although other methods are currently being tested to improve identification time and decrease cost and workload. Next generation sequencing (NGS) has the capacity to output several million reads in a single experiment, making it very fast and relatively cheap compared to other older sequencing methods such as Sanger sequencing. The ability to analyze genes and even whole genomes, opens the possibilities to identify factors such as bacterial species, virulence genes and antibiotic resistance genes. The aim of this study was to find any possible correlations between 16 species of streptococci and clinical data in patients with suspected sepsis. Initial species identification was performed using MALDI-TOF before the samples were sequenced using NGS. Sequence files were then quality controlled and trimmed before being assembled. Following assembly, coverage was controlled for all assembled genomes before the downstream analysis started. Different tools such as 16S RNA species identification, multi locus sequence typing and antibiotic resistance finder were used, among other tools. The results were extremely mixed, with the overall quality of the data being of good quality, but the assembly and downstream analysis being worse. The most consistent species was S. pyogenes. No correlation between sepsis patients and relevant clinical data was found. The mixed quality of results from assembly and downstream analysis were most likely contributed to difficulties in culturing and sequencing of the streptococci. Finding ways to circumvent these problems would most likely aid in general sequencing of streptococcal species, and hopefully in clinical applications as well.

abstract: Rapid advancements in genomic technologies have increased our understanding of rare human disease. Generation of multiple types of biological data including genetic variation from genome or exome, expression from transcriptome, methylation patterns from epigenome, protein complexity from proteome and metabolite information from metabolome is feasible. "Omics" tools provide comprehensive view into biological mechanisms that impact disease trait and risk. In spite of available data types and ability to collect them simultaneously from patients, researchers still rely on their independent analysis. Combining information from multiple biological data can reduce missing information, increase confidence in single data findings, and provide a more complete view of genotype-phenotype correlations. Although rare disease genetics has been greatly improved by exome sequencing, a substantial portion of clinical patients remain undiagnosed. Multiple frameworks for integrative analysis of genomic and transcriptomic data are presented with focus on identifying functional genetic variations in patients with undiagnosed, rare childhood conditions. Direct quantitation of X inactivation ratio was developed from genomic and transcriptomic data using allele specific expression and segregation analysis to determine magnitude and inheritance mode of X inactivation. This approach was applied in two families revealing non-random X inactivation in female patients. Expression based analysis of X inactivation showed high correlation with standard clinical assay. These findings improved understanding of molecular mechanisms underlying X-linked disorders. In addition multivariate outlier analysis of gene and exon level data from RNA-seq using Mahalanobis distance, and its integration of distance scores with genomic data found genotype-phenotype correlations in variant prioritization process in 25 families. Mahalanobis distance scores revealed variants with large transcriptional impact in patients. In this dataset, frameshift variants were more likely result in outlier expression signatures than other types of functional variants. Integration of outlier estimates with genetic variants corroborated previously identified, presumed causal variants and highlighted new candidate in previously un-diagnosed case. Integrative genomic approaches in easily attainable tissue will facilitate the search for biomarkers that impact disease trait, uncover pharmacogenomics targets, provide novel insight into molecular underpinnings of un-characterized conditions, and help improve analytical approaches that use large datasets. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

abstract: Obesity and its underlying insulin resistance are caused by environmental and genetic factors. DNA methylation provides a mechanism by which environmental factors can regulate transcriptional activity. The overall goal of the work herein was to (1) identify alterations in DNA methylation in human skeletal muscle with obesity and its underlying insulin resistance, (2) to determine if these changes in methylation can be altered through weight-loss induced by bariatric surgery, and (3) to identify DNA methylation biomarkers in whole blood that can be used as a surrogate for skeletal muscle. Assessment of DNA methylation was performed on human skeletal muscle and blood using reduced representation bisulfite sequencing (RRBS) for high-throughput identification and pyrosequencing for site-specific confirmation. Sorbin and SH3 homology domain 3 (SORBS3) was identified in skeletal muscle to be increased in methylation (+5.0 to +24.4 %) in the promoter and 5’untranslated region (UTR) in the obese participants (n= 10) compared to lean (n=12), and this finding corresponded with a decrease in gene expression (fold change: -1.9, P=0.0001). Furthermore, SORBS3 was demonstrated in a separate cohort of morbidly obese participants (n=7) undergoing weight-loss induced by surgery, to decrease in methylation (-5.6 to -24.2%) and increase in gene expression (fold change: +1.7; P=0.05) post-surgery. Moreover, SORBS3 promoter methylation was demonstrated in vitro to inhibit transcriptional activity (P=0.000003). The methylation and transcriptional changes for SORBS3 were significantly (P≤0.05) correlated with obesity measures and fasting insulin levels. SORBS3 was not identified in the blood methylation analysis of lean (n=10) and obese (n=10) participants suggesting that it is a muscle specific marker. However, solute carrier family 19 member 1 (SLC19A1) was identified in blood and skeletal muscle to have decreased 5’UTR methylation in obese participants, and this was significantly (P≤0.05) predicted by insulin sensitivity. These findings suggest SLC19A1 as a potential blood-based biomarker for obese, insulin resistant states. The collective findings of SORBS3 DNA methylation and gene expression present an exciting novel target in skeletal muscle for further understanding obesity and its underlying insulin resistance. Moreover, the dynamic changes to SORBS3 in response to metabolic improvements and weight-loss induced by surgery. / Dissertation/Thesis / Appendix A / Appendix B / Appendix C / Appendix D / Appendix G / Doctoral Dissertation Biology 2017

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response system and can occur when the immune system over- or under- reacts to an infection. Klebsiella spp. has been found to be one of the leading causes of sepsis, and the increasing occurrence of antibiotic resistance observed has become a major concern in clinical care. To study the genome and increase knowledge of the biodiversity of K. pneumoniae, K. variicola, and K. oxytoca, bacterial isolates were collected from blood, urine, nasopharynx, and wounds of patients with suspected sepsis. Next-generation sequencing was performed, and the presence of antibiotic resistance genes and plasmids were studied. Furthermore, a prediction of traits for each phylogroup was performed and the results from whole-genome sequencing were compared to phenotypic results. Among the K. pneumoniae isolates obtained, almost half had been misidentified by standard phenotypic methods and were found to be K. variicola, K. quasipneumoniae, and K. quasivariicola. A significant difference in the number of antibiotic resistance genes were observed between K. pneumoniae and K. variicola compared to K. oxytoca, however no significant difference was observed between K. pneumoniae and K. variicola, suggesting the underestimated pathogenicity of K. variicola. A genetic agreement was observed between the type of beta-lactamase harboured and presence or absence of nitrogen-fixation genes to the phylogroup, providing a way of species identification. Further studies should be conducted on the pathogenicity and virulence of K. variicola and K. quasipneumoniae to avoid misidentification, find organism-specific treatments, and narrow down the antibiotic prescription. / Biodiversitet vid Sepsis

abstract: Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS. / Dissertation/Thesis / Ph.D. Chemistry 2014

Deficiência intelectual (DI), associada ou não a outras alterações congênitas, é a razão mais frequente de procura por aconselhamento genético pelas famílias. Até alguns anos atrás, a realização de cariótipo, triagem para doenças metabólicas e fra(x) elucidavam apenas ∼40% dos casos de pacientes com DI idiopática. Com o surgimento de arrays genômicos, as causas moleculares por trás de outros ∼20% dos quadros de DI foram elucidadas; porém, mesmo com esse avanço, muitos pacientes ainda permanecem sem causa molecular clara que justifique o fenótipo. O sequenciamento do exoma (WES) é hoje um dos recursos disponíveis para o diagnóstico e possível elucidação das causas genéticas por trás da deficiência intelectual idiopática, abrindo caminho também à identificação de novos genes. O presente trabalho realizou o sequenciamento de exoma de 8 probandos que tinham em comum a deficiência intelectual esporádica, acompanhada ou não de outros sinais clínicos, e de seus genitores não afetados (trios). Esses pacientes foram previamente triados para a síndrome do X frágil, e submetidos a exame de array CGH para investigação de perdas e ganhos de segmentos cromossômicos, ambos com resultados negativos. O objetivo desse estudo foi detectar alterações e possivelmente novos genes associados com a DI, usando pipelines de padrões de herança mendeliano. Treze alterações em 9 genes foram detectadas por sequenciamento de exoma e confirmadas por sequenciamento Sanger: 8 mutações bialélicas em genes recessivos (TBC1D24, ADAMTSL2, NALCN, VPS13B), uma ligada ao X (MID1), e 4 alterações de novo (RYR2, GABBR2, CDK13, DDX3X); 5 dessas alterações ainda não haviam sido descritas nos bancos de dados consultados, caracterizando mutações novas. Dos 8 trios, em 5 identificamos alterações moleculares provavelmente responsáveis pelos quadros apresentados; dois desses casos foram em genes recessivos (mutações homozigotas ou em heterozigose composta) e potencialmente teriam sido detectados mesmo se apenas os probandos houvessem sido sequenciados. Para as alterações em heterozigose, porém, a avaliação dos genitores e constatação de status de novo da mutação foram importantes para avaliar o impacto da variante. Esse trabalho resultou em uma taxa de diagnóstico de 62,5%; mesmo considerando o pequeno tamanho da amostra, esse valor está bem acima dos 15-30% relatados na literatura quando essa metodologia é utilizada para o estudo de casos esporádicos de DI. Em dois casos, mutações foram identificadas em genes que só foram descritos como mutados recentemente e que ainda não são considerados genes de deficiência intelectual no OMIM: o gene CDK13 foi descrito como mutado em pacientes de uma única coorte com malformação cardíaca congênita (sindrômica ou não), porém sua contribuição para coortes de DI ainda não foi investigada. O gene GABBR2, identificado mutado em heterozigose em um dos nossos pacientes, já havia sido considerado um candidato potencial para DI, mas apenas 2 trabalhos detectaram mutações nesse gene entre pacientes com DI e epilepsia. Os resultados aqui apresentados substanciam o papel desses genes como implicados na DI sindrômica de herança autossômica dominante, e devem contribuir para serem considerados genes OMIM de deficiência intelectual / Intellectual disability (ID), associated or not with other congenital abnormalities, is the most frequent reason for families to seek genetic counseling. Until some years ago, karyotyping, metabolic disease and FRAXA screening elucidated only ∼40% of patients with idiopathic ID. Importantly, with the introduction of genomic arrays, the molecular cause behind a further ∼20% of ID cases was determined; however, despite this improvement, many patients are still not provided with a clear molecular explanation and cause for their phenotype. Nowadays, whole exome sequencing (WES) is one of the methods available for diagnosis and a further means of possible elucidation of the genetic causes of idiopathic intellectual disability; in many cases this method also allows identification of genes that have not been previously related to ID. In the present project, we sequenced the exome (WES) of 8 sporadic patients that all had ID, with or without other clinical signs, and their unaffected parents (trios); these patients had been previously screened for fragile X syndrome and for losses and gains of chromosomal segments by array CGH, both with negative results. The objective of this study was to detect mutations and possibly new genes associated with ID, using pipelines for Mendelian inheritance patterns. Thirteen mutations in 9candidate genes were detected by exome sequencing and confirmed by Sanger sequencing, among them 8 biallelic mutations in autossomal recessive genes (TBC1D24, ADAMTSL2, NALCN, VPS13B), one mutation in an X-linked gene (MID1), and 4 de novo alterations (RYR2, GABBR2, CDK13, DDX3X); 5 of these mutations had not been described in the databases consulted characterizing new variants. Of the 8 trios, we obtained a probable diagnosis of the molecular alteration responsible for the presented phenotypes in 5. Two of these cases were in recessive genes (homozygous mutations or compound heterozygous), and the mutations would probably have been detected even if only the probands had been sequenced. However, for the heterozygous mutations, the assessment of the parents and the confirmation of the de novo status of the mutation was important to evaluate the impact of the variant. This work resulted in a diagnosis rate of 62.5%; even considering the small sample size, this value is well above the average of 15-30% reported in the literature when the methodology used for the study of ID sporadic cases is considered. In two cases, mutations were detected in genes only recently described as mutated and which are not considered yet as OMIM ID genes. The CDK13 gene had already been described as mutated in a single cohort of patients with syndromic congenital heart defects, but its contribution to ID cohorts has not been established. The GABBR2 gene, where a heterozygous mutation was identified in the patient, had already been considered a potential candidate for ID; there are only 2 studies that detected mutations in this gene among patients with ID and epilepsy. This contribution may pave the way to establishing GABBR2 and CDK13 as causations of ID and acceptance by OMIM

A fisiopatologia das síndromes de falência da medula óssea (FMO) está relacionada a mecanismos adquiridos de destruição das células-tronco hematopoeiticas na medula ou a defeitos constitucionais em genes fundamentais para o reparo do DNA e manutenção dos telômeros. A anemia aplástica (AA), o protótipo das doenças de FMO, pode ter etiologia adquirida ou constitucional. A avaliação genética de pacientes com AA adquirida tem como objetivo a detecção de mutações somáticas que possam ser usadas como marcadores de resposta ao tratamento imunossupressor. Diferentemente, em pacientes com AA constitucional, a avaliação genética é fundamental para detecção de mutações etiológicas na doença do paciente, sendo essencial para o tratamento e seleção de doadores de medula óssea. Contudo, o papel das mutações constitucionais na fisiopatologia e modulação imunológica da AA adquirida ainda não é conhecido. Neste estudo, nós sequenciamos pacientes com AA de duas coortes independentes utilizando diferentes painéis de sequenciamento de genes alvos. A primeira coorte, composta por 13 pacientes com AA adquirida, foi sequenciada utilizando um painel com 165 genes relacionados à FMO, neoplasias hematológicas, reparo de DNA, manutenção dos telômeros e vias de resposta imune. A segunda coorte, composta por 59 pacientes investigados para doença constitucional, foi sequenciada com um painel de sequenciamento comercial com 49 genes relacionados à FMO hereditária. Foram identificadas alterações potencialmente patogênicas em três dos cinco pacientes com AA adquirida que não responderam à imunossupressão: dois pacientes com variantes em TERT e um com uma variante em DHX36. Não foram identificadas variantes funcionalmente relevantes nos pacientes que responderam ao tratamento imunossupressor. Em contraste, foram identificadas variantes potencialmente patogênicas em RTEL1 em 8 pacientes com AA constitucional. Variantes em RTEL1 foram associadas tanto ao encurtamento telomérico quanto à erosão excessiva do 3\' overhang, independentemente do comprimento dos telômeros. Desse modo, apenas a medida do comprimento dos telômeros não foi suficiente para identificar todos ospacientes com disfunções teloméricas. As plataformas de sequenciamento de nova geração diminuíram o custo e o tempo para a avaliação genética dos pacientes com FMO. Em nosso estudo, os pacientes com AA adquirida não apresentaram um padrão genético associado à sua resposta ao tratamento com imunossupressores, no entanto, o sequenciamento da coorte com suspeita de AA constitucional foi capaz de identificar o defeito genético associado à doença do paciente em 40% dos casos. O uso de dados clínicos, investigação familiar, análises in silico e testes funcionais foram essenciais para uma correta interpretação da patogenicidade de novas variantes identificadas por sequenciamento de nova geração. / The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. The genetic screening of patients with constitutional AA is performed to detect germline mutations that are etiologic in patients\' disease. That is critical for treatment decisions and to identify a donor for a bone marrow transplant. In acquired AA, the genetic screening has been used to detect somatic mutations that can predict patients\' outcomes after treatment, as the role of germline mutations in this disease is yet not clear. To investigate the role of germline variants in AA, we screened two independent cohorts with two different targeting sequencing panels; a first cohort composed by 13 patients with acquired AA that was screened using a panel with 165 genes related to BMF, hematologic malignancies, DNA repair, telomere maintenance, and immune response pathways. A second cohort composed of 59 patients suspected to have a constitutional disease screened by a commercial Inherited Bone Marrow Failure Sequencing panel. In our first cohort, while patients without functional relevant germline variants responded to immunosuppression treatment (n=8), three out of 5 nonresponder patients were identified with variants in telomere biology genes. We found patients carrying TERT and DHX36 variants. In our constitutional AA cohort, we identified 8 patients carrying variants in the RTEL1 gene, a helicase critical to telomere maintenance. RTEL1 variants associated with both patients\' overall telomere shortening and single-stranded 3\' overhang erosion independent of telomere length. Also, 3\' overhang erosion was associated with patients\' predisposition to clonal evolution. In this context, the variants identified in the helicases genes DHX36 and RTEL1 were both associated with patients\' normal telomere length and poor outcomes. Also, telomere length measurement alone was insufficient to identify all primary telomere defects. The platforms of next-generation sequencing decreased the cost and time for the genetic screening of patients with BMF. In our study, acquired AA patients did not display a clear genetic pattern associated with their immunosuppressive treatment response. In contrast, the sequencing of the cohort selected based on their suspicion to have an inherited diseaseidentified a molecular defect that might be pathogenic in up to 40% of patients, including the RTEL1 variants. Pathogenicity assessment of genetic variants requires a combination of clinical, in silico, and functional data required to avoid misinterpretation of common variants.