This dissertation examines the history of the creation and development of the concept of anticipation, a pattern of heredity found in several diseases (e.g. Huntington’s disease and myotonic dystrophy), in which an illness manifests itself earlier and often more severely in successive generations. It reconstructs major arguments in twentieth-century debates about anticipation and analyzes the relations between different research communities and schools of thought. Developments in cutting-edge medicine, biology, and genetics are analyzed; many of these developments were centered in Britain, but saw significant contributions by people working in France, Germany, Switzerland, the Netherlands and North America. Chapter one traces precursor notions in psychiatric and hereditarian thought from 1840 to the coining of the term ‘anticipation’ by the ophthalmologist Edward Nettleship in 1905. Key roles in the following chapters are played by several figures. Prior to World War II, these include: the neuropathologist F.W. Mott, whose advocacy during 1911- 1927 led to anticipation being called “Mott’s law”; the biometrician and eugenicist Karl Pearson, who opposed Mott on methodological and political grounds; and two politically and theoretically opposed Germans – Ernst Rüdin, a leading psychiatrist and eugenicist who came to reject anticipation, and Richard Goldschmidt, a geneticist who offered a peculiar Mendelian explanation. The British psychiatrist and human geneticist, Lionel Penrose, makes a first interwar appearance, but becomes crucial to the story after World War II due to his systematic dismissal of anticipation, which discredited the notion on orthodox Mendelian grounds. The final chapters highlight the contributions of Dutch neurologist Christiaan Höweler, whose 1980s work demonstrated a major hole in Penrose’s reasoning, and British geneticist Peter Harper, whose research helped demonstrate that expanding trinucleotide repeats accounted for the transgenerational worsening without contradicting Mendel and resurrected anticipation as scientifically legitimate. Reception of the concept of anticipation is traced across the century through the examination of textbooks used in different fields. This dissertation argues against established positions regarding the history of the concept, including claims that anticipation’s association with eugenics adequately explains the rejection of the notion after 1945. Rejected, in fact, by many eugenicists from 1912, anticipation was used by physicians until the 1960s.

The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions. Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819

Les répétitions de trinucléotides sont des séquences répétées en tandem pouvant subir, chez l'homme, de larges expansions à l'origine de nombreuses maladies génétiques. La dystrophie myotonique de type 1 (DM1) est due à l'expansion d'une répétition CTG en 3'UTR du gène DMPK. Les mécanismes d'instabilités des répétitions, peu connus, reposeraient sur leur capacité à former des structures secondaires constituant un obstacle aux mécanismes impliquant une synthèse d'ADN. Nous avons montré qu'une TALEN induisant une cassure double brin dans les répétitions CTG à l'origine de la DM1 insérées chez la levure Saccharomyces cerevisiae permettait de manière efficace et spécifique d'aboutir après réparation à leur contraction. Le mécanisme de réparation est dépendant uniquement de deux gènes, RAD50 et RAD52, suggérant la formation de structures aux extrémités de la DSB devant être retirées pour initier la réparation, suivis d'une réaction de SSA entre les répétitions aboutissant à leur contraction. L'efficacité et spécificité d'un système CRISPR-Cas9 à contracter ces répétitions chez la levure ont été comparées à la TALEN. L'induction de CRISPR-Cas9 n'aboutit pas à la contraction des répétitions mais à des réarrangements chromosomiques suggérant un manque de spécificité et un mécanisme de réparation différent de celui de la TALEN. Enfin, nous avons étudié si ces nucléases peuvent contracter ces répétitions CTG à des tailles non pathologiques dans des cellules de mammifères. L'induction de la TALEN dans des cellules de souris transgéniques DM1, puis dans des fibroblastes humains de patients DM1 montre des résultats préliminaires encourageant de contraction des répétitions. / Trinucleotides repeats are a specific class of microsatellites whose large expansions are responsible for many human neurological disorders. Myotonic dystrophy type 1 (DM1) is due to an expansion of CTG repeats in the 3’UTR of DMPK gene, which can reach thousands of repeats. Molecular mechanisms leading to these large expansions are poorly understood but in vitro studies have shown the capacity of these repeats to form secondary structures, which probably interfere with mechanisms involving DNA synthesis. We shown that a TALEN used to induce double-strand break (DSB) in DM1 CTG repeats integrated in the yeast Saccharomyces cerevisiae is specific and leads to highly efficient repeat contractions after repair. Mechanism involved in TALEN-induced DSB only depends of RAD50 and RAD52 genes, suggesting the formation of secondary structures at DSB ends that need to be removed for repair initiation, followed by an intramolecular recombinaison repair such as SSA between repeats leading to their contraction. We compared the efficiency and specificity of a CRISPR-Cas9 and the TALEN to contract CTG repeats in yeast. Surprisingly, CRISPR-Cas9 induction do not lead to repeat contraction but to chromosomal rearrangement, suggesting a lack of specificity and a different repair mechanism than with the TALEN. At last, we studied whether these nucleases could contract CTG repeats to a non-pathological length in mammalian cells. Finally, TALEN induction in DM1 transgenic mice cells, and in DM1 human fibroblasts show promising repeat contractions.

No description available.

Advancements in the field of biotechnology are expanding the scientific horizon and a promising era is envisioned with personalized medicine for improved health. The amount of genetic data is growing at an ever-escalating pace due to the availability of novel technologies that allow massively parallel sequencing and whole-genome genotyping, that are supported by the advancements in computer science and information technologies. As the amount of information stored in databases throughout the world is growing and our knowledge deepens, genetic signatures with significant importance are discovered. The surface of such a set in the data mining process may include causative- or marker single nucleotide polymorphisms (SNPs), revealing predisposition to disease, or gene expression signatures, profiling a pathological state. When targeting a reduced set of signatures in a large number of samples for diagnostic- or fine-mapping purposes, efficient interrogation and scoring require appropriate preparations. These needs are met by miniaturized and parallelized platforms that allow a low sample and template consumption. This doctoral thesis describes an attempt to tackle some of these challenges by the design and implementation of a novel assay denoted Trinucleotide Threading (TnT). The method permits multiplex amplification of a medium size set of specific loci and was adapted to genotyping, gene expression profiling and digital allelotyping. Utilizing a reduced number of nucleotides permits specific amplification of targeted loci while preventing the generation of spurious amplification products. This method was applied to genotype 96 individuals for 75 SNPs. In addition, the accuracy of genotyping from minute amounts of genomic DNA was confirmed. This procedure was performed using a robotic workstation running custom-made scripts and a software tool was implemented to facilitate the assay design. Furthermore, a statistical model was derived from the molecular principles of the genotyping assay and an Expectation-Maximization algorithm was chosen to automatically call the generated genotypes. The TnT approach was also adapted to profiling signature gene sets for the Swedish Human Protein Atlas Program. Here 18 protein epitope signature tags (PrESTs) were targeted in eight different cell lines employed in the program and the results demonstrated high concordance rates with real-time PCR approaches. Finally, an assay for digital estimation of allele frequencies in large cohorts was set up by combining the TnT approach with a second-generation sequencing system. Allelotyping was performed by targeting 147 polymorphic loci in a genomic pool of 462 individuals. Subsequent interrogation was carried out on a state-of-the-art massively parallelized Pyrosequencing instrument. The experiment generated more than 200,000 reads and with bioinformatic support, clonally amplified fragments and the corresponding sequence reads were converted to a precise set of allele frequencies. / QC 20100813

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

Cystic fibrosis and diseases associated with trinucleotide expansions are serious hereditary disease that serves in my Diploma thesis as role models suitable for teaching interesting topics of human genetics at secondary schools. By using appropriate methods of prenatal genetic treatment, it is possible to make a diagnosis of the developing fetus and to determine a corresponding prognosis in next prenatal and postnatal development in families at risk. The practical part of the thesis is devoted to the content analysis of biology schoolbooks for secondary schools and to the preparation of a prototype class of genetics at secondary schools. The aim of the content analysis of biology schoolbooks for secondary schools is the evaluation of various textbooks according to several preselected criteria. The aim of the presentation of the proposed prototype class was to determine, based on the responses obtained from the questionnaires, whether pupils of higher grades of secondary schools are interested in the subject matter of genetics, to find out what engaged their attention the most during the lessons or what they did not understand and what they considered as difficult. The lesson was conducted as a specialized seminar, the teaching method was a lecture. From the selected biology schoolbooks for...

Les maladies à expansion de polyglutamine sont un ensemble de troubles neurodégénératives héréditaires se développant lorsqu’il y a répétitions de trinucléotides CAG dans les gènes causatifs au-delà d’un certain seuil. L’expansion des répétitions de trinucléotides CAG entraîne des désordres neurologiques héréditaires précoces, dont de multiples formes d’ataxie spinocérébelleuse (SCA). Parmi celles-ci, le type le plus commun et dominant est l’ataxie spinocérébelleuse de type 3 (SCA3), aussi connue sous le nom de la maladie de Machado-Joseph (MMJ). Ce dernier est un désordre neurologique progressif autosomique dominant. Le gène causatif de MMJ est ATXN3 (ATAXINE-3). Plusieurs études récentes suggèrent une association entre ce gène et la modulation du stress du réticulum endoplasmique (RE). Lors de ce travail de maîtrise, des souches transgéniques de C. elegans exprimant les formes sauvage et mutante du gène ATXN3 humain ont été générées. Les résultats suggèrent des phénotypes importants chez la souche transgénique mutante associés à la pathologie humaine: défaut de motilité, longévité réduite et profil neurodégénératif considérable. Ceci dit, ces résultats nous ont poussé à vouloir déterminer si l’utilisation des composés chimiques, connus en tant que modulateurs du stress du RE et possédant des rôles neuroprotecteurs, sont capables de restaurer les phénotypes notés. Les composés utilisés, c’est-à-dire le Bleu de Méthylène, le Salubrinal et le Guanabenz, ont démontré une capacité de corriger les phénotypes rapportés dans la souche transgénique mutante. De plus, ces composés ont aussi été en mesure de prévenir une augmentation du niveau du stress oxydatif et de la réponse au stress du RE exhibé chez les vers mutants. Par le développement de nouveaux modèles C. elegans pour la MMJ, où il y a expression du gène ATXN3 complet dans les motoneurones, il a été possible de trouver qu’une modulation chimique du stress du RE peut réduire considérablement la neurodégénérescence et par conséquent, être une possible nouvelle approche thérapeutique pour traiter cette pathologie. / Polyglutamine expansion diseases are a class of dominantly inherited neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders such as multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited spinocerebellar ataxia is the type 3 (SCA3) also known as Machado-Joseph disease (MJD), an autosomal dominant, progressive neurological disorder. The gene causing MJD is ATXN3 (ATAXIN-3): MJD is caused by an abnormal CAG trinucleotide repeat expansion in the ATXN3 gene. Several recent studies have shown that this gene is associated with endoplasmic reticulum (ER) stress. In this study, we generated transgenic C. elegans strains expressing wild type or mutant human ATXN3 genes and tested them for recovery of locomotor phenotype, lifespan and neurodegeneration phenotypes upon treatment with compounds known to modulate ER stress and having neuroprotective roles. We observed differences between both transgenic lines and found that the motility defects, the reduced lifespan and the neurodegeneration can be rescued by methylene blue, guanabenz and salubrinal. These compounds were also able to prevent the oxidative stress and the ER stress response induced by mutant transgenic worms. We introduce novel C. elegans models for MJD based on the expression of full-length ATXN3 in GABAergic motor neurons. Using these models we discovered that chemical modulation of the ER unfolded protein response reduced neurodegeneration and could be a new therapeutic approach for the treatment of MJD.