Review Article: Genetic Polymorphism Studies and Insurgence of Human Genetic Diseases

Single nucleotides polymorphism is the biological variant that affects people the most frequently (SNPs). Due of the link to hereditary illnesses, Polymorphisms are significant for hereditary investigations. Throughout this article, researchers examined a specific subset of SNPs that alter the sequencing of the related enzyme. Researchers created a brand-new technique that, beginning with sequencing data, can determine if a novel phenotypic resulting from an SNP is connected to a genetic abnormality. The greatest prevalent sort of genomic variability throughout the human genome is represented by solitary nucleotides polymorphism (SNPs). Understanding whether human genetic variants are associated with Chromosomal and complicated disorders is probably among a more essential objectives of SNP research. Non coding SNPs (NSSNPs), which cause solitary point mutations in molecules, are the subject of intense attention

Lineage-coupled clonal capture identifies clonal evolution mechanisms and vulnerabilities of BRAF

Targeted cancer therapies have revolutionized treatment but their efficacies are limited by the development of resistance driven by clonal evolution within tumors. We developed CAPTURE , a single-cell barcoding approach to comprehensively trace clonal dynamics and capture live lineage-coupled resistant cells for in-depth multi-omics analysis and functional exploration. We demonstrate that heterogeneous clones, either preexisting or emerging from drug-tolerant persister cells, dominated resistance to vemurafenib in BRA

Duchenne muscular dystrophy : mutation profiling in view of the emerging gene-based therapies

Includes bibliographical references (leaves 97-115).Duchenne Muscular Dystrophy (DMD) is a lethal, X-linked, recessive muscle-wasting disorder affecting 1 in 3 500 live male births worldwide, for which only palliative care is available to date. Large exonic deletions or duplications are found in approximately 70% of DMD patients, for which diagnostic testing is available. The remaining 30% carry point mutations, which go largely undetected, as no testing is currently offered due to the great size of the DMD gene and the logistical challenges involved

STRUCTURAL INSTABILITY OF HUMAN RIBOSOMAL RNA GENE CLUSTERS

The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity

An Investigation of Mutational Signatures in the Evolution of Oesophageal adenocarcinoma

An Investigation of Mutational Signatures in the Evolution of Oesophageal adenocarcinoma Sujath Abbas Oesophageal adenocarcinoma (OAC) remains a public health challenge with dismal survival rates and increasing incidence. This PhD study aimed to investigate how mutational processes act across different stages of OAC development and in metastasis for better understanding of the influence of mutational forces during tumour formation. To identify these signatures in clinical samples this study also aimed to develop a cost-effective DNA sequencing method in clinical formalin fixed OAC samples. A large study cohort was assembled comprising of 161 Barrett’s, 777 OAC primary tumours and 59 metastatic samples. Mutational signature analysis revealed 14 distinct single base substitution (SBS) mutational signatures in these genomes, SBS17b/a were most prevalent and presented early in Barrett’s. Traces of BER (SBS30), MMR(SBS44) and colibactin associated signature (SBS41) were uncovered for the first time, as well as a platinum signature (SBS35). Mostly signatures increased in their proportions from Barrett’s to invasive tumours and further in metastasis. SBS17 showed strong bias towards untranscribed and lagging strands. Nucleosome periodicity patterns were similar across the stages and SNVs were enriched in the inward facing minor groove suggesting a common mutational process throughout the disease evolution. Evaluation of evolutionary bottlenecks uncover a distinct SBS17b shift, with a decrease sub-clonally in Barrett’s, OACs and metastasis and this was by far the most dominant signal during OAC evolution. Clinical risk factors including alcohol, smoking and NSAIDs were positively correlated with signature proportions. APOBEC and colibactin processes were informative for Barrett’s and OAC classification, suggesting a role in transformation, and the BER signature (SBS30) was most prognostic in our cohort. Given that signatures have the potential to be clinically informative, a novel cost-effective DNA sequencing method to extract mutational signatures from archival FFPE tissues was developed successfully. Computational simulations on pan-cancer WGS and an experimental confirmation of the method showed very good concordance and mirrored the WGS-derived signatures (cosine similarity >0.9%). It is hoped that this work will pave the way for further studies to understand how mutations are laid down and determine their clinical application

The Role of Topologically Associating Domains for Developmental Gene Regulation - ⁠ A Systematic Functional Analysis at the Sox9 and Shh Loci

Precise spatiotemporal gene expression during embryonic developmental is controlled by cis- regulatory elements (CREs) such as enhancers and promoters. Their physical chromatin proximity is correlated with active transcription and thought to be restricted to topologically associated domains (TADs) that help establish interactions between CREs and limit inappropriate contacts. Accordingly, TADs frequently overlap with gene regulatory landscapes, in which are contained diverse enhancers that transmit their activity across the domain towards their target promoter. Large structural variants reorganizing TADs were shown to cause gene misexpression and disease thereby linking gene regulation to chromatin structure. Recently, several studies revealed controversial results questioning the importance of TADs for transcriptional control. Acute depletion of CTCF and other architectural proteins in vitro led to loss of TAD structures with surprisingly modest effects on gene expression. However, the cytotoxicity of such depletion assays hindered analysis of more complex gene regulatory scenarios and their effect during development. This study specifically addresses the connection between TADs and developmental gene regulation through two projects using the murine limb as a model system. First, we took advantage of the Sox9/Kcnj2-locus that is subdivided into two adjacent TADs with distinct expression patterns of Sox9 and Kcnj2. The systematic deletion of individual CTCF binding sites at the TAD boundary and within the TAD resulted in gradual fusion of the neighboring domains without major effects on gene expression. TAD rearrangement by TAD-spanning inversions and repositioning of the boundary, however, redirected the regulatory activity and resulted in pathogenic gene misexpression. Thus, TAD structures may not be essential for developmental gene regulation, yet CTCF-dependent rearrangement of TADs can lead to the redirection of enhancer–promoter contacts and gene misexpression. In the second project, we studied how enhancer position relative to its TAD influences the function of an individual enhancer at the Shh-locus. Therefore, we repositioned the Shh-limb enhancer ZRS to five alternative locations inside and outside of its TAD. As expected, the enhancer lost all function in the positions outside of the Shh-TAD. Interestingly, the new positions inside the TAD also displayed decreased enhancer activity, albeit to varying degrees. Further analysis suggests that CTCF likely functions in some positions as a facilitator of enhancer-promoter contacts, while insulating short-range contacts in others. Ultimately, the ZRS is only able to ectopically activate some genes if repositioned to novel TADs, displaying strong enhancer-promoter selectivity. In summary, the results demonstrate that TADs provide robustness and precision to gene regulation, guiding enhancer-promoter interaction without being essential. The findings in this work build a basis for future studies aiming to understand enhancer-promoter interaction and can help in contextualizing potential disease-causing mutations disrupting TADs

Single molecule MATAC-seq reveals key determinants for DNA replication efficiency

The stochastic nature of origin activation results in significant variability in the way genome replication is carried out from cell to cell. The reason for the diversity in efficiency and timing of individual origins has remained an unresolved issue for a long time. Cell-to-cell variability has been demonstrated to play a crucial role in cellular plasticity and cancer in mammalian cells. Although population-based methods have provided valuable insight into biological processes, it is necessary to use single molecule techniques to uncover events that are hidden by the population average. Many biological processes, such as DNA replication, transcription, and gene expression, are closely linked to the local chromatin structure. In yeast, although DNA replication origins have conserved DNA sequences, they display remarkable differences in timing and efficiency. Some origins initiate replication earlier during S-phase or more frequently than others, resulting in a high degree of heterogeneity among the cells in a population, with no two cells having the exact same replication profile. Our hypothesis is that the local nucleosomal structure may affect the DNA replication profile of individual origins. To explore this relationship, we have developed Methylation Accessibility of Targeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility maps of specific genomic locations after targeted purification in their native chromatin context. Our analysis of selected early-efficient (EE) and late-inefficient (LI) replication origins in Saccharomyces cerevisiae using MATAC-Seq revealed significant cell-to-cell heterogeneity in their chromatin states. Disrupting the INO80 or ISW2 chromatin remodeling complexes led to changes at individual nucleosomal positions that correspond to changes in replication efficiency. Our results show that a chromatin state with a narrow size of accessible origin DNA in combination with well-positioned surrounding nucleosomes and an open +2 linker region was a strong predictor for efficient origin activation. MATAC-Seq provides a single-molecule assay for chromatin accessibility that reveals the large spectrum of alternative chromatin states that coexist at a given locus, which was previously masked in population-based experiments. This provides a mechanistic basis for origin activation heterogeneity that occurs during DNA replication in eukaryotic cells. As a result, our single-molecule assay for chromatin accessibility will be ideal for defining single-molecule heterogeneity across many biological processes, such as transcription, replication, or DNA repair in vitro and ex vivo.Die stochastische Natur der Aktivierung von Replikationsursprüngen führt zu einer signifikanten Variabilität in der Art und Weise, wie die DNA Replikation von Zelle zu Zelle durchgeführt wird. Der Grund für die Diversität in Effizienz und Zeitpunkt der individuellen Aktivierung von Ursprüngen blieb lange ein ungelöstes Problem. Es wurde gezeigt, dass die Zell-zu-Zell-Variabilität eine entscheidende Rolle bei der zellulären Plastizität und Krebs in Säugetierzellen spielt. Obwohl populationsbasierte Methoden wertvolle Einblicke in biologische Prozesse geliefert haben, ist es notwendig, Einzelmolekültechniken zu verwenden, um Ereignisse aufzudecken, die durch das Durchschnittsverhalten aller Moleküle verborgen sind. Viele biologische Prozesse wie DNA-Replikation, Transkription und Genexpression sind eng mit der lokalen Chromatinstruktur verbunden. Obwohl die DNA-Replikationsursprünge in Hefe konservierte DNA-Sequenzen aufweisen, zeigen sie bemerkenswerte Unterschiede im Zeitpunkt und Effizienz der Replikation. Einige Ursprünge initiieren die Replikation früher während der S-Phase oder häufiger als andere, was zu einem hohen Grad an Heterogenität zwischen den Zellen in einer Population führt, wobei keine zwei Zellen das exakt gleiche Replikationsprofil aufweisen. Unsere Hypothese ist, dass die lokale nukleosomale Struktur das DNA-Replikationsprofil beeinflussen kann. Um diese Beziehung zu untersuchen, haben wir Methylation Accessibility of Targeted Chromatin Domain Sequencing (MATAC-Seq) entwickelt, um Einzelmolekül-Chromatin-Zugänglichkeitskarten spezifischer genomischer Orte nach gezielter Reinigung in ihrem nativen Chromatin-Kontext zu bestimmen. Unsere Analyse ausgewählter früh-effizient (EE) und spät-ineffizient (LI) feuernde Replikationsursprünge in Saccharomyces cerevisiae mit MATAC-Seq ergab eine signifikante Zell-zu-Zell-Heterogenität in ihren Chromatinzuständen. Die genetische Deletion der INO80- oder ISW2-Chromatin-Remodeling Komplexe führte zu Veränderungen an einzelnen nukleosomalen Positionen, die mit Veränderungen der Replikationseffizienz korrespondierten. Unsere Ergebnisse zeigten, dass ein Chromatinzustand mit einem engen Fenster an zugänglicher Replikationsursprungs-DNA in Kombination mit gut positionierten umgebenden Nukleosomen und einer offenen +2-Linkerregion ein starker Prädiktor für eine effiziente Ursprungsaktivierung war. MATAC-Seq bietet einen Einzelmolekül-Assay für die Zugänglichkeit von Chromatin, der das große Spektrum alternativer Chromatinzustände aufzeigt, die an einem bestimmten genomischen Lokus koexistieren, der zuvor in populationsbasierten Experimenten maskiert war. Dies liefert eine mechanistische Grundlage für die Heterogenität der Ursprungsaktivierung, die während der DNA-Replikation in eukaryotischen Zellen auftritt. Infolgedessen ist unser Einzelmolekül-Assay 5 für Chromatin-Zugänglichkeit ideal für die Definition der Einzelmolekül-Heterogenität über viele biologische Prozesse hinweg, wie z. B. Transkription, Replikation oder DNA-Reparatur in vitro und ex vivo

Microarrays in molecular profiling of cancer : focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.Biolääketieteessä mikrosiruilla tutkitaan samanaikaisesti tuhansia molekyylejä solu- tai kudosnäytteestä. Mikrosirut koostuvat kiinteällä alustalla, kuten mikroskooppilasilla, olevista tuhansista pienistä pisteistä. Jokainen piste voi sisältää esimerkiksi 25-60 emäksen pituisia oligonukleotidejä, jotka vastaavat tiettyä geeniä. Näin mikrosirujen avulla voidaan tutkia vaikkapa useiden geenien ilmentymistä näytteestä. Mikrosiruilla on paljon sovelluksia biolääketieteen alalla. Erityisesti siruja on käytetty syöpätutkimuksessa. Mikrosiruja geenien ilmentymisen määrittämiseen valmistetaan paikallisesti tutkimuslaboratoriossa tai ostetaan kaupallisilta valmistajilta. Kaupallisia valmistajia on useita. Monimuotoisuus asettaa haasteita tiedon keräämiselle eri sirutyypeiltä ja kerätyn tiedon vertaamiselle. Tässä väitöskirjatyössä verrattiin geenien ilmentymisen tuloksia kolmelta erityyppiseltä mikrosirulta. Joukossa oli kaksi kaupallista sekä yksi itsetehty siru. Vertailu osoitti, että kaupalliset sirut antoivat samankaltaisempia tuloksia, korrelaatio sirujen välillä 0.78-0.86, kuin itsetehty siru. Vaikka tulokset osoittivat, että eri siruilta voidaan saada vertailukelpoista tietoa, ei vertailu tulosten välillä ole suoraviivaista. Haasteena ovat mikrosirujen tekniset eroavaisuudet sekä tiedon saattaminen vertailukelpoiseen muotoon. Mikrosirujen kehittyessä tutkimustyökalusta kliinisiin sovelluksiin standardoinnilla - ja sitä kautta tulosten paremmalla vertautuvuudella - on tärkeä tehtävä. Työssä tutkittiin myös biologisen tiedon yhdistämistä eri mikrosirumenetelmistä pään ja kaulan alueen syövässä. Mittaamalla mikrosiruilla geenin kopiomäärän muutoksia DNA-tasolla ja yhdistämällä tämä tieto geenin ilmentymistason muutoksiin saatiin tietoa syövälle merkityksellisistä geeneistä. Erityisesti korkeasteiset kopioluvun muutokset vaikuttivat geenien ilmentymiseen, sillä 14-32% näillä alueilla sijaisevista geeneistä oli myös kohonnut ilmentymistaso. Tilastollisin menetelmin osoitettiin satoja geenejä, joilla kopioluvun ja ilmentymän muutos näyttävät olevan yhteydessä toisiinsa. Tässä joukossa oli uusia kohdegeenejä ennestään tunnetuille kromosomialueille, kuten FADD ja PPFIA1 geenit kromosomissa 11. Työssä tunnistetut geenit antavat hyvän pohjan jatkotutkimuksille, jotka voivat vuosien kuluessa johtaa edistysaskeliin pään ja kaulan alueen syövän hoidossa

Development of computational approaches for the analysis of bisulfite next-generation sequencing data

The scientific contribution of this thesis consists of three articles that have been published in Bioinformatics (Oxford Journals) and Nature Methods and the third article being under review at Leukemia (Nature Publishing Group), respectively. The implications of these articles for the field of computational epigenetics and future perspectives of this research area are discussed. The main challenge within the framework of this thesis was the development of a bioinformatics tool for bisulfite sequencing analysis. The article in Bioinformatics presents the bioinformatics tool B-SOLANA for the analysis of DNA methylation data generated by two-base encoding bisulfite sequencing on the SOLiD platform of Life Technologies. Additionally, benchmark analyses revealed that B-SOLANA exhibits a significantly higher sensitivity and specificity compared to other software approaches which were developed at the same time. The review article in Nature Methods summarizes challenges of bisulfite sequencing analysis as they appear on different high-throughput sequencing platforms. Especially primary analyses including the quality control and mapping of raw sequences are discussed. Furthermore, the article debates the effect of sequencing errors and contaminations on inferred DNA methylation levels and recommends the most appropriate way to analyze this type of data. This review is a helpful reference for the analysis of DNA methylation by high-throughput sequencing, a currently rapidly developing research area. The third article, which has been submitted to Leukemia, comprises the analysis of a DNA methylome of the DAUDI cell line at single base resolution. On the genetic level, this endemic Burkitt Lymphoma cell line is characterized by the presence of the hallmark IG-MYC translocation. Recent publications about this cell line suggested a high number of DNA methylation changes. However, until now only array-based studies were published, which have concentrated their focus on loci-specific DNA methylation patterns. We showed that the mechanisms of DNA methylation associated with transcriptional regulation in lymphomas go by far beyond the usually studied promoter methylation. Furthermore, we characterized the DNA methylome of the mitochondria and the Epstein-Barr virus, whereas upregulation of the latter has already been identified in DAUDI before. As the DAUDI cell line is used over decades in many laboratories throughout the world, the obtained methylome data prove valuable as a "reference epigenome" for future studies