The phylogeny of Clematis, section Viorna, was characterized in this study using molecular data. Two nuclear introns were sequenced for a variety of taxa: actin and nitrate reductase. Actin intron sequence data yielded very little phylogenetic information. Some basal clades were resolved, but there were very few well supported relationships between species of the Viorna section in both the neighbor joining and maximum parsimony analyses. Nitrate reductase intron sequence data was slightly more variable. The number of well supported relationships in both the neighbor joining and maximum parsimony analyses for nitrate reductase was greater, but still not sufficient to yield an informative tree. Two possible explanations for the lack of variation are that these species have not evolved many differences in these intron sequences or that common alleles are flowing between the species. Hybrid analysis using the actin intron was inconclusive because the experimentally generated hybrid possessed an allele that neither parents tested had. More sampling from multiple individuals from both parent and multiple hybrid individuals is necessary to answer this question. The hybrid specimen tested was homozygous for the nitrate reductase intron marker, and both parents also possessed the allele. This did not directly support or refute the use of these markers for tracking the hybridization history within Clematis.

Traditionally, autosomal genes, when expressed, are assumed to express both alleles equally. Exceptions to this tenet include genes for which a specific genotypic polymorphism controls expression level, as well as genes for which monoallelic expression is epigenetically dictated. In this work, we discovered and characterized allele-specific gene expression in a variety of tissues using multiple techniques. We used cell lines and fresh tissue from reciprocal crosses of F1 heterozygous mice and the homozygous parental strains. We relied on a variety of high-throughput genomic techniques including RNA-Seq and DNA SNP-arrays to examine multiple types of allele-specific expression. We searched for novel examples of random autosomal monoallelic expression (RMAE) by using DNA SNP-arrays and cDNA from lymphoblast and fibroblast clonal cell lines of heterozygous mice. We found that of the approximately 1,350 autosomal genes we assessed, over 10% showed evidence of RMAE in at least one cell type. This allele-specific expression was stable over long periods in cell culture and encompassed a variety of gene types, some of which also exhibit RMAE in human clonal lines. Additionally, for a subset of RMAE genes, there seemed to be preferential inactivation of one allele; this monoallelic expression was still considered random, as from clone to clone the gene could be expressed either monoallelically or biallelically. In a second set of experiments we developed an analysis pipeline to examine RNASeq data for allele-specific expression. Using a pilot data set, we assessed the murine liver for parent-of-origin monoallelic expression, examining both known and candidate novel imprinted genes. We observed imprinted monoallelic expression in the adult liver for some, but not all, imprinted genes that have been reported in studies of embryonic tissue. The results suggest that there are few, if any, novel imprinted genes to be discovered in the mouse liver. This pilot data set also allowed examination of the genetic basis of allele specific gene expression. In keeping with recent reports, we found evidence for genetically based allele-specific expression, ranging from mild to greater than 4-fold allelic imbalance. We examined the extent to which this allelic imbalance correlated with total expression levels in the parental strains.

Human Mendelian disease in Saudi Arabia is both significant and challenging. Next-generation sequencing (NGS) has resulted in important discoveries of the genetic variants responsible for inherited disease. However, the success of clinical genomics using NGS requires accurate and consistent identification of rare genome variants. Rarity is one very important criterion for pathogenicity. Here we describe a model to detect variants by analyzing allele frequencies of a Saudi population. This work will enhance the opportunity to improve variant calling workflow to gain robust frequency estimates in order to better detect rare and unusual variants which are frequently associated with inherited disease.

La porphyrie aiguë intermittente (PAI) est la plus fréquente des porphyries hépatiques aiguës. Elle est décrite comme une maladie autosomique dominante dont le trait génétique est estimé à 1/1675 en France avec une pénétrance faible et variable allant de 10% à 50% dans les familles connues de PAI. La PAI est due à des mutations réduisant le niveau d’activité de l’hydroxyméthylbilane-synthase (HMBS). Son déficit entraîne l’accumulation de précurseurs neurotoxiques responsables de la symptomatologie clinique. Dans le foie, la synthèse d’hème est contrôlée par l’enzyme ALA-Synthétase 1 (ALAS1) dont l’activité est régulée par un rétrocontrôle négatif par le produit final : l’hème. Le traitement consiste à freiner l’induction d’ALAS1 induit par la carence en hème, par l’administration d’hème exogène. Ce traitement de la crise aiguë est très efficace mais génère rapidement une dépendance physique avec apparition de crises récurrentes nécessitant l’administration chronique d’hème exogène. L’objectif principal de ce projet a été d’étudier les mécanismes physiopathologiques et génétiques liés à cette pathologie afin de traiter et conseiller au mieux les patients. Une partie du projet a consisté à explorer les facteurs génétiques modulateurs de la pénétrance de la maladie. Tout d’abord, une prévalence minimale du trait génétique dans la population générale a été estimée à 1/1299 permettant d’en déduire une pénétrance de l’ordre de 1% alors que celle dans les familles PAI suivies par le CFP est estimée à 22,9 %. Ensuite, concernant les facteurs pouvant expliquer cette différence, la présence d’une mutation type non-sens est plus fréquemment associée aux formes sévères et à une pénétrance plus élevée. De plus, les études de corrélation et d’héritabilité suggèrent plutôt une transmission de type oligogénique associée à des facteurs épigénétiques modulateurs de la pénétrance dont le facteur environnemental. Une autre partie a consisté à explorer les effets de l’administration d’hème exogène sur les patients et un modèle murin de PAI créé génétiquement. Chez l’homme, le traitement est associé à une augmentation des formes chroniques (1,7 % avant vs 7,5 % après l’introduction du celui-ci). Dans le modèle murin de PAI, les injections intrapéritonéales répétées induisent une augmentation paradoxale d’ALAS1 (3 fois), une augmentation de l’hème oxygénase 1 qui catabolise l’hème (HMOX1, 9 fois) ainsi que des voies de l’inflammation (analyse transcriptomique et protéomique hépatique) et une surcharge en fer. De plus, cette administration induit une altération des complexes de la chaine respiratoire mitochondriale responsable d’anomalies du métabolisme énergétique au niveau hépatique, cérébral et musculaire pouvant expliquer la symptomatologie neuroviscérale. En conclusion, ce travail a permis d’explorer les caractéristiques génétiques de la maladie (prévalence, pénétrance) en remettant en cause le mode de transmission autosomique dominant jusqu’ici admis, et d’explorer les mécanismes physiopathologiques associées à l’administration d’hème exogène faisant de cette thérapeutique un pharmakon / The biosynthesis of porphyrins is one of the most conserved pathways known. By associating different metals, porphyrins give rise to the "pigments of life". The formation of haem is accomplished by a sequence of eight dedicated enzymes encoded by different genes, some being active in ubiquitous as well as in erythroid isoforms. In humans, the genes for each of the haem synthetic enzymes may become the target of mutations that give rise to an impaired cellular enzyme activity called porphyrias. The acute porphyrias are characterized by attacks of neuropsychiatric symptoms, which may be due to a toxic surplus of the porphyrin precursor 5-aminolevulinic acid, or a consequence of a deficit of vital hemoproteins. Mutations of the gene encoding the third enzyme: hydroxymethylbilane synthase, are associated with the most frequent type of acute hepatic porphyria, acute intermittent porphyria. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. In the classical form of AIP, HMBS activity is about 50% lower than normal in all tissues. These levels of activity in basal conditions are not sufficiently low to cause symptoms. However, factors increasing hepatic heme demand, resulting in an upregulation of hepatic aminolevulinate synthase (ALAS1, the first enzyme of the heme biosynthesis pathway), precipitate acute attacks. The treatment of the attack of AIP consists to repress ALAS1 and restores metabolic equilibrium. But this treatment leads side effects and dependency. The pathophysiological mechanism of the disease is partially known and difficult to explore because there is not an AIP model or prediction model of porphyrogenicity. We aimed to obtain further insight into the pathophysiological mechanism of AIP and into the genetic (prevalence and penetrance) of AIP, and the contribution of genetic factors to the variable clinical expression of HMBS mutations.We first calculated the penetrance of HMBS mutations in AIP patients seen at the French reference center for porphyria: 22.9%. We then used the Exome Variant Server (EVS) to estimate the prevalence of deleterious HMBS mutations in the general population: 1/1299; and the penetrance of the AIP genetic trait in France: 1%. Finally, we investigated further the genetic factors underlying the penetrance of AIP by analyzing genotype/phenotype correlations, and the pattern of familial correlations for the symptoms of the acute crises of AIP. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the era in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance suggesting that AIP inheritance does not follow the classical autosomal dominant model. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles.On the other hand, we explored the effect of heme administration. In human, the introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. We show that repeated hemin infusions in mice trigger a high level heme oxygenase 1 response, induce a pro-oxidative iron accumulation, a complex pattern of liver inflammation with macrophage infiltration and an alteration of oxidative phosphorylation

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<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

Magister Scientiae - MSc / The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South Africa; a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot® and Multiplex AS-PCR genotyping assays, and also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the Cape Metropolitan area. Two SNaPshot® Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific – PCR (MAS-PCR) genotyping system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan large numbers of samples for novel genetic variations. / South Africa

Studies on stationary phase cultures of microorganisms in the laboratory have helped us understand the genetic and physiological basis of adaptation in their natural habitats. Stasis or decline in bacterial populations due to nutrient depletion during stationary phase has been shown to lead to the selection of mutants that are able to survive better than their parent. This phenomenon, where a mutant exhibits relatively better growth than its immediate parent, under the conditions that prevailed during its appearance, is termed as Growth Advantage in Stationary Phase (GASP) and constitutes a general strategy to survive prolonged stationary phase. A mutation conferring growth advantage in one environment, however, can result in the loss of fitness in another environment, typifying the environment-specificity of the adaptation. Several GASP loci have been isolated over the last two decades and they have enhanced our understanding of the rapid evolution of microorganisms under nutrient starvation and the forces driving it. The results presented in this thesis detail the isolation and characterization of one such survivor from the culture of an E. coli strain ZK819 that was grown for 28 days without any additional nutrients. The strain ZK819 carries the rpoS819 allele that was isolated as the first GASP mutation resulting in attenuated activity of the stationary phase - specific sigma factor RpoS. Among the several possible mutations accumulating in the survivors over this long incubation period, one was identified to be within the hns gene encoding the global regulator H-NS. Expression of a majority of genes regulated by H-NS is environmentally modulated and their products are required under various stress conditions. The presence of the hns66 mutation was identified by the derepression of the bgl operon, one of the well-studied targets of H-NS mediated repression. The primary question being addressed in this study is whether this naturally isolated hns66 mutation has any role in the survival of its bearer during prolonged stationary phase. The hns66 mutation results in a partially active longer polypeptide due to a single nucleotide alteration within the stop codon of the H-NS ORF. Strains carrying the hns66 allele showed differential fitness under two different environments relative to the parent. In early stationary phase there is a strong disadvantage associated with the hns66 allele in the rpoS819 background, when competed against the parent, highlighted by the observation that there is a sharp fall in the number of mutants and a concomitant appearance of revertants that lead to the rapid displacement of the original mutant. The reversion is predominantly due to suppressor mutations within the ssrA locus encoding a tmRNA involved in the release of stalled ribosomes and degradation of aberrant proteins. Analysis of the suppression phenomenon revealed that the altered H-NS66 protein is a substrate for SsrA-mediated tagging for proteolysis. The H-NS66 protein is sub-optimally functional and can repress the bgl operon when its level is increased either by over-expression from a plasmid or a mutation in ssrA. Mutation in ssrA suppressed the strong growth disadvantage displayed by the original survivor in early stationary phase. The growth disadvantage of the hns66 survivor was also lost upon introduction of the wild type rpoS allele, indicating that the combined presence of the hns66 and rpoS819 alleles is detrimental in early stationary phase. In the presence of the rpoS+ allele, the hns66 mutant showed a modet GASP phenotype relative to the parent in early stationary phase. In contrast to the crash observed during early stationary phase, strains carryingthe hns66 allele fared better in late stationary phase conditions compared to the parent. When present in its original genetic context, the hns66 allele conferred growth advantage to the original survivor in late stationary phase conditions when competed against the parent grown for 21 days. The hns66 allele, when introduced in the parent strain ZK819 by transduction could still confer a strong GASP phenotype when competed against one-day old parent cells in medium derived from a 21 day old culture. These observations suggest that the hns66 allele enables the cells to scavenge for limited quantities of specific nutrients available in the aged medium. This is consistent with the observation that the advantage is seen when the mutant is in minority and is lost when the mutant is in majority. These studies highlight the fact that the conditions under stationary phase are constantly changing and to adapt under these rapidly changing conditions, modulation of an already existing function would be a preferred strategy than selection for a new function or the complete loss of a function. In this context, genetic modulation of a global regulator offers a better option due to the pleiotropic effects it may generate. This study adds hns to the list of genes, which upon mutation confer a GASP phenotype to cells. It also highlights that the age of the cells as well as the medium has an impact on the GASP phenotype. Elucidation of the mechanism of the H-NS mediated growth advantage and its relation to the status of the rpoS locus can contribute to our understanding of the dynamics of the stationary phase.

L’amélioration génétique de la luzerne (Medicago sativa), une légumineuse fourragère autotétraploïde, pourrait bénéficier de l’allele mining. Cette méthode consiste à rechercher, dans la diversité naturelle, des allèles ayant potentiellement un effet sur le phénotype. Pour évaluer l’intérêt de cette stratégie qui exploite la diversité naturelle, cinq gènes impliqués dans des caractères sélectionnés ont été retenus : CAD1 et CCoaOMT (digestibilité), CONSTANS-like (rendement fourrager), NHX1 (tolérance à la salinité) et WXP1 (tolérance à la sécheresse). La diversité de ces gènes a été étudiée en séquençant 387 génotypes cultivés et 20 génotypes sauvages. L’analyse des données confirme la présence d’un goulot d’étranglement durant la domestication et la sélection de la luzerne. CONSTANS-like et WXP1 révèlent de nombreux variants alors que CAD1, CCoaOMT et NHX1 sont très peu variables. Des variants ayant un effet potentiel sur le phénotype ont été identifiés dans les zones de la séquence protéique qui sont conservées au sein des Faboideae. L’impact sur le phénotype de deux variants du gène CONSTANS-like a été étudié : Constans-634, à l’origine d’un codon stop prématuré et Constans-4111, situé dans une région conservée du gène. Pour cela des croisements ont été réalisés afin d’obtenir une descendance avec toutes les doses possibles (AAAA, AAAB, AABB, ABBB and BBBB) pour chaque mutation étudiée. Des marqueurs KASPar ont été développés afin de déterminer les doses de mutations chez les descendants. Aucun génotype homozygote muté pour Constans-634 n’a été détecté parmi les 1505 descendants. Ce variant a induit une floraison plus précoce de trois jours pour les génotypes portant trois doses d’allèle muté. Le variant Constans-4111 a induit un effet additif sur la hauteur de tige. Les génotypes homozygotes de type sauvage étaient en moyenne 11,8 cm plus petits que les génotypes homozygotes portant trois ou quatre doses du variant. L’intégration de la stratégie allele mining dans les schémas de sélection des espèces végétales autotétraploïdes hétérozygotes a été discutée. / Lucerne (Medicago sativa) is an autotetraploid forage legume, whose breeding could beneficiate from allele mining. This strategy is based on the natural diversity and consists in seeking alleles with a potential effect on the phenotype. The interest of this approach was evaluated by studying five genes of agronomic interest: CAD1 and CCoaOMT (digestibility), CONSTANS-like (forage yield), NHX1 (salt tolerance) and WXP1 (drought tolerance). The diversity of these five genes was evaluated by sequencing 387 genotypes of cultivated accessions and 20 genotypes of wild accessions. The results confirmed a bottleneck during lucerne domestication and selection. CONSTANS-like and WXP1 were very variable whereas CAD1, CCoaOMT and NHX1 contained very few variants. Variants with a potential strong impact on the phenotype were identified in conserved parts of protein sequence within the Faboideae. The impact on phenotype was studied for two mutations of the CONSTANS-like gene: constans-634, causing a premature stop codon and constans-4111, located in a conserved region of the gene. Genotypes carrying one to three doses of the mutations (AAAB, AABB and ABBB) were polycrossed in order to obtain offsprings with every allele combination (AAAA, AAAB, AABB, ABBB and BBBB). KASPar markers were developed to determine the mutation doses in offspring progeny. No homozygous genotype was found for constans-634 in the 1505 offspring progeny. This mutation induced a premature flowering of three days for the genotypes carrying three doses of the mutation. The mutation constans-4111 induced an additive effect on stem height and the homozygous genotypes without the variant where on average 11.8 cm shorter than homozygous genotypes carrying three or four doses of the variant. The application of allele mining strategy in plant schemes of heterozygous autotetraploid species was discussed.