Tesis sobre el tema "Tandem duplication"

This study concerns a specific molecular genetic mutation, the mixed lineage leukaemia partial tandem duplication ( MLL PTD). MLL is a transcriptional regulator that known to be instrumental in both normal haematopoiesis and in leukaemogenesis. This study aimed to evaluate the prognostic importance of this abnormality through investigation of its influence on clinical outcome, its utility as a marker of minimal residual disease (MRD) and the downstream effects of its expression. A qualitative PCR assay was established and determined the frequency of MLL PTD to be 5.2% in de novo AML. MLL PTD was found to be a useful independent marker being associated with a greater risk of relapse and a reduction of relapse free survival and overall survival. A quantitative PCR (QPCR) assay was developed that reliably distinguished AML-related MLL PTDs from the low level of background MLL PTDs occurring normally. Despite a high rate of cytogenetic clonal evolution MLL PTD expression remained stabled between diagnosis and relapse, making it a suitable marker for MRD. Results also suggested the QPCR assay could determine onset of remission and prediction of relapse. Gene expression profiling was used to identify a gene signature unique to the MLL PTD and was found to be distinct from that associated with MLL translocation. Analysis of the gene signature also identified three candidate chemotherapy compounds predicted to antagonise the effects of the MLL PTD. The results of this study could prove instrumental in improving the treatment of MLL PTD patients.

Après un rappel des notions fondamentales de biologie moléculaire et plus particulièrement des duplications en tandem, la thèse présente un panorama des outils existants permettant de détecter des régions homologues à grande échelle, en se focalisant sur les méthodes de chaînage d'ancres. Le document introduit alors un formalisme général de modélisation basé sur les graphes. Une nouvelle méthode de chaînage, capable de produire un ensemble de chaînes de score optimal et satisfaisant des contraintes de cohérences assurant une interprétation aisée des résultats est formulée, en exploitant la théorie des flots de coût minimum. Cette méthode est évaluée sur des problèmes de détection de duplications segmentales chez les plantes puis intégrée dans un pipeline de détection de grande régions dupliquées en tandem directement à partir de la séquence génomique. Cet outil est évalué sur le génome de la plante modèle Arabidopsis thaliana et confronté à l'annotation du génome, montrant ses capacités à détecter des régions dupliquées impliquant des éléments non-codants
After a quick introduction to molecular biology and more specifically tandem duplications, the thesis presents an overview of existing tools for detecting large scale homologuous regions, witha focus on anchor chaining methods. The thesis introduces a new graph-based general modelling formalism. A new chaining method, which is able to produce an optimal set of chains that satisfies specific consistency constraints that aim at easier interpretation is described, using minimum cost flow theory. This method is evaluated on segmental duplications detection in plants and then integrated in a pipeline targeted at tandem duplication detection directly from DNA. This tool is evaluated on the Arabidopsis thaliana genome and compared to the annotation, showing that it is able to detect tandem duplicated regions involving non coding elements

Protein-coding genes evolve together with their genome and acquire changes, some of which affect the length of their protein products. This explains why equivalent proteins from different species can exhibit length differences. Variation in length of proteins during evolution arguably presents a large number of possibilities for improvement and innovation of protein structure and function. In order to contribute to an increased understanding of this process, we have studied variation caused by tandem domain duplications and insertions or deletions of intrinsically disordered residues. The study of two proteins, Nebulin and Filamin, together with a broader study of long repeat proteins (>10 domain repeats), began by confirming that tandem domains evolve by internal duplications. Next, we show that vertebrate Nebulins evolved by duplications of a seven-domain unit, yet the most recent duplications utilized different gene parts as duplication units. However, Filamin exhibits a checkered duplication pattern, indicating that duplications were followed by similarity erosions that were hindered at particular domains due to the presence of equivalent binding motifs. For long repeat proteins, we found that human segmental duplications are over-represented in long repeat genes. Additionally, domains that have formed long repeats achieved this primarily by duplications of two or more domains at a time. The study of homologous protein pairs from the well-characterized eukaryotes nematode, fruit fly and several fungi, demonstrated a link between variation in length and variation in the number of intrinsically disordered residues. Next, insertions and deletions (indels) estimated from HMM-HMM pairwise alignments showed that disordered residues are clearly more frequent among indel than non-indel residues. Additionally, a study of raw length differences showed that more than half of the variation in fungi proteins is composed of disordered residues. Finally, a model of indels and their immediate surroundings suggested that disordered indels occur in already disordered regions rather than in ordered regions.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 4: Manuscript.

Hydrothermal vents (HV) are deep-sea ecosystems where hypoxia/anoxia is one of the major constraints. Respiratory pigments play a key function in the adaptation to hypoxia. HV polynoids are abundant, diverse and widely distributed and are the only ones to possess hemoglobins (Hb), and these are unique among annelids. The goal of this work was to know their evolutionary history. There are 2 types of Hb in HV polynoids: 1- and 4-domains. While they are extracellular, they are more related to intracellular globins that to annelid extracellular ones, indicating a distinct origin. We studied the evolutionary history of the 4-domain gene Hb and found that originated from the tandem duplication of a myoglobin-like ancestral. Both globin types possess residues in the heme pocket that are responsible for the high oxygen affinity in other organisms' Hb. But the adaptive value of these Hb probably resides in their expression at high levels. Plus, some amino acid sites were shown to be under positive selection (PS). Some are located in the heme pocket where they might affect functional properties. Others are in areas where they can affect interactions between subunits and domains.

La compréhension de la dynamique des réarrangements génomiques est importante en phylogénie. La phylogénie est l'étude de l'évolution des espèces. Un but majeur est d'établir les relations d'évolution au sein d'un groupe d'espèces, pour déterminer la topologie de l'arbre d'évolution formé par ce groupe et des ancêtres communs à certains sous-ensembles. Pour ce faire, il est naturellement très utile de disposer d'un moyen d'évaluer les distances évolutionnaires relatives entre des espèces, ou encore d'être capable d'inférer à un groupe d'espèces le génome d'un ancêtre commun à celles-ci. Ce travail de thèse, dans la lignée d'autres travaux, consiste à élaborer de tels moyens, ici dans des cas particuliers où les génomes possèdent des gènes en multiples copies, ce qui complique les choses. Plusieurs hypotèses explicatives de la présence de duplications ont été considérées, des formules de distance ainsi que des algorithmes de calcul de scénarios ont été élaborés, accompagnés de preuves de complexité.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Härtill 4 uppsatser.

In acute myeloid leukaemia (AML), further prognostic determinants are required in addition to cytogenetics to predict patients at increased risk of relapse. Recent studies have indicated an internal tandem duplication (ITD) in the FLT3 gene may adversely affect clinical outcome. The studies reported in this thesis evaluated the impact of a FLT3/ITD on outcome in 854 patients treated in UK MRC AML trials. A FLT3/ITD was present in 27% patients and was associated with leucocytosis and a high percentage of bone marrow blast cells. With respect to clinical outcome it predicted for increased relapse risk (RR), adverse disease-free survival (DFS), event-free survival (EFS) and overall survival (OS). In multivariate analysis, presence of a mutation was the most significant prognostic factor predicting for RR and DFS. In order to evaluate whether FLT3 mutations can be used as markers of minimal residual disease, paired presentation and relapse samples were studied. Twenty four patients were wild type FLT3 at diagnosis and 4 acquired a FLT3 mutation at relapse. Of 20 patients positive at diagnosis, 5 who were all originally ITD+, had no detectable mutation at relapse. Furthermore, another patient had a completely different ITD at relapse which could not be detected in the presentation sample. These results suggest that, at least in some patients, FLT3 mutations are secondary events in leukaemogenesis, are unstable and thus should be used cautiously for the detection of minimal residual disease. Given the high frequency of activating FLT3 mutations in patients with AML, FLT3 and its downstream pathway are attractive targets for directed inhibition. Several promising tyrosine kinase inhibitors have recently been identified and in vitro data reported here suggest that FLT3 inhibitors may have a role either as monotherapy or in combination with conventional cytotoxic agents in the treatment of AML.

Despite being found in all presently sequenced genomes, the evolution of tandemly repeated sequences has only just begun to be understood. We can represent the duplication history of tandemly repeated sequences with duplication trees. Most phylogenetic techniques need to be modified to be used on duplication trees. Due to gene loss, it is not always possible to reconstruct the duplication history of a tandemly repeated sequence. This thesis addresses this problem by providing a polynomial-time locally optimal algorithm to reconstruct the duplication history of a tandemly repeated sequence in the presence of gene loss. Supertree methods cannot be directly applied to duplication trees. A polynomial-time algorithm that takes a forest of ordered phylogenies and looks for a super duplication tree is presented. If such a super duplication tree is found then the algorithm constructs the super duplication tree. However, the algorithm does not always find a super duplication tree when one exists. The SPR topological rearrangement in its current form cannot be used on duplication trees. The necessary modifications are made to an agreement forest so that the SPR operation can be used on duplication trees. This operation is called the duplication rooted subtree prune and regraft operation (DrSPR). The size of the DrSPR neighbourhood is calculated for simple duplication trees and the tree shapes that maximize and minimize this are given.

Tandemly arrayed genes (TAG) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. We identified all the TAGs in the genomes of human, chimp, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG gene orientations and intergenic distances, and TAG gene functions. TAGs account for about 14-17% of all the genomic genes and nearly one third of all the duplicated genes in the four genomes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests while others are enriched with TAG deserts. The majority of TAGs are of size two for all genomes, similar to the previous findings in C. elegans, A. thaliana, and O. sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes with parallel orientation in the genome. The analysis of GO function indicate that genes with receptor or binding activities are significantly over-represented by TAGs. Simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. It is noteworthy to mention that gene family sizes are significantly correlated with the extent of tandem duplication, suggesting that tandem duplication is a preferred form of duplication, especially in large families. There has not been any systematic study of TAG genes' expression patterns in the genome. Taking advantage of recent large-scale microarray data, we were able to study expression divergence of some of the TAGs of size two in human and mouse for which the expression data is available and examine the effect of sequence divergence, gene orientation, and physical proximity on the divergence of gene expression patterns. Our results show that there is a weak negative correlation between sequence divergence and expression similarity between the two members of a TAG, and also a weak negative correlation between physical proximity of two genes and their expression similarity. No significant relationship was detected between gene orientation and expression similarity. Moreover, we compared the expression breadth of upstream and downstream duplicate copies and found that downstream duplicate does not show significantly narrower expression breadth. We also compared TAG gene pairs with their neighboring non-TAG pairs for both physical proximity and expression similarity. Our results show that TAG gene pairs do not show any distinct differences in the two aspects from their neighboring gene pairs, suggesting that sufficient divergence has occurred to these duplicated genes during evolution and their original similarity conferred by duplication has decayed to a level that is comparable to their surrounding regions.
Master of Science

A central topic of evolutionary biology is the inference of phylogeny, i. e., the evolutionary history of species. A powerful tool for the inference of such phylogenetic relationships is the arrangement of the genes in mitochondrial genomes. The rationale is that these gene arrangements are subject to different types of mutations in the course of evolution. Hence, a high similarity in the gene arrangement between two species indicates a close evolutionary relation. Metazoan mitochondrial gene arrangements are particularly well suited for such phylogenetic studies as they are available for a wide range of species, their gene content is almost invariant, and usually free of duplicates. With these properties gene arrangements of mitochondrial genomes are modeled by permutations in which each element represents a gene, i. e., a specific genetic sequence. The mutations that shape the gene arrangement of genomes are then represented by operations that rearrange elements in permutations, so-called genome rearrangements, and thereby bridge the gap between evolutionary biology and optimization. Many problems of phylogeny inference can be formulated as challenging combinatorial optimization problems which makes this research area especially interesting for computer scientists. The most prominent examples of such optimization problems are the sorting problem and the distance problem. While the sorting problem requires a minimum length sequence of rearrangements that transforms one given permutation into another given permutation, i. e., it aims for a hypothetical scenario of gene order evolution, the distance problem intends to determine only the length of such a sequence. This minimum length is called distance and used as a (dis)similarity measure quantifying the evolutionary relatedness. Most evolutionary changes occurring in gene arrangements of mitochondrial genomes can be explained by the tandem duplication random loss (TDRL) genome rearrangement model. A TDRL consists of a duplication of a consecutive set of genes in tandem followed by a random loss of one copy of each duplicated gene. In spite of the importance of the TDRL genome rearrangement in mitochondrial evolution, its combinatorial properties have rarely been studied. In addition, models of genome rearrangements which include all types of rearrangement that are relevant for mitochondrial genomes, i. e., inversions, transpositions, inverse transpositions, and TDRLs, while admitting computational tractability are rare. Nevertheless, especially for metazoan gene arrangements the TDRL rearrangement should be considered for the reconstruction of phylogeny. Realizing that a better understanding of the TDRL model is indispensable for the study of mitochondrial gene arrangements, the central theme of this thesis is to broaden the horizon of TDRL genome rearrangements with respect to mitochondrial genome evolution. For this purpose, this thesis provides combinatorial properties of the TDRL model and its variants as well as efficient methods for a plausible reconstruction of rearrangement scenarios between gene arrangements. The methods that are proposed consider all types of genome rearrangements that predominately occur during mitochondrial evolution. More precisely, the main points contained in this thesis are as follows: The distance problem and the sorting problem for the TDRL model are further examined in respect to circular permutations, a formal concept that reflects the circular structure of mitochondrial genomes. As a result, a closed formula for the distance is provided. Recently, evidence for a variant of the TDRL rearrangement model in which the duplicated set of genes is additionally inverted have been found. Initiating the algorithmic study of this new rearrangement model on a certain type of permutations, a closed formula solving the distance problem is proposed as well as a quasilinear time algorithm that solves the corresponding sorting problem. The assumption that only one type of genome rearrangement has occurred during the evolution of certain gene arrangements is most likely unrealistic, e. g., at least three types of rearrangements on top of the TDRL rearrangement have to be considered for the evolution metazoan mitochondrial genomes. Therefore, three different biologically motivated constraints are taken into account in this thesis in order to produce plausible evolutionary rearrangement scenarios. The first constraint is extending the considered set of genome rearrangements to the model that covers all four common types of mitochondrial genome rearrangements. For this 4-type model a sharp lower bound and several close additive upper bounds on the distance are developed. As a byproduct, a polynomial-time approximation algorithm for the corresponding sorting problem is provided that guarantees the computation of pairwise rearrangement scenarios that deviate from a minimum length scenario by at most two rearrangement operations. The second biologically motivated constraint is the relative frequency of the different types of rearrangements occurring during the evolution. The frequency is modeled by employing a weighting scheme on the 4-type model in which every rearrangement is weighted with respect to its type. The resulting NP-hard sorting problem is then solved by means of a polynomial size integer linear program. The third biologically motivated constraint that has been taken into account is that certain subsets of genes are often found in close proximity in the gene arrangements of many different species. This observation is reflected by demanding rearrangement scenarios to preserve certain groups of genes which are modeled by common intervals of permutations. In order to solve the sorting problem that considers all three types of biologically motivated constraints, the exact dynamic programming algorithm CREx2 is proposed. CREx2 has a linear runtime for a large class of problem instances. Otherwise, two versions of the CREx2 are provided: The first version provides exact solutions but has an exponential runtime in the worst case and the second version provides approximated solutions efficiently. CREx2 is evaluated by an empirical study for simulated artificial and real biological mitochondrial gene arrangements.

[Français] Une fraction importante des génomes eucaryotes est constituée de Gènes Répétés en Tandem (GRT). Un mécanisme fondamental dans l’évolution des GRT est la recombinaison inégale durant la méiose, entrainant la duplication locale (en tandem) de segments chromosomiques contenant un ou plusieurs gènes adjacents. Différents algorithmes ont été proposés pour inférer une histoire de duplication en tandem pour un cluster de GRT. Cependant, leur utilisation est limitée dans la pratique, car ils ne tiennent pas compte d’autres événements évolutifs pourtant fréquents, comme les inversions, les duplications inversées et les délétions. Cette thèse propose différentes approches algorithmiques permettant d’intégrer ces événements dans le modèle de duplication en tandem classique. Nos contributions sont les suivantes: • Intégrer les inversions dans un modèle de duplication en tandem simple (duplication d’un gène à la fois) et proposer un algorithme exact permettant de calculer le nombre minimal d’inversions s’étant produites dans l’évolution d’un cluster de GRT. • Généraliser ce modèle pour l’étude d’un ensemble de clusters orthologues dans plusieurs espèces. • Proposer un algorithme permettant d’inférer l’histoire évolutive d’un cluster de GRT en tenant compte des duplications en tandem, duplications inversées, inversions et délétions de segments chromosomiques contenant un ou plusieurs gènes adjacents.
[English] Tandemly arrayed genes (TAGs) represent an important fraction of most genomes. A fundamental mechanism at the origin of TAG clusters is unequal crossing-over during meiosis, leading to the duplication of chromosomal segments containing one or many adjacent genes. Such duplications are called tandem duplications, as the duplicated segment is placed next to the original one on the chromosome. Different algorithms have been proposed to infer the tandem duplication history of a TAG cluster. However, their applicability is limited in practice since they do not take into account other frequent evolutionary events such as inversion, inverted duplication and deletion. In this thesis, we propose different algorithmic approaches allowing to integrate these evolutionary events in the original tandem duplication model of evolution. Our contributions are summarized as follows: • We integrate inversion events in a tandem duplication model restricted to single gene duplications, and we propose an exact algorithm allowing to compute the minimum number of inversions explaining the evolution of a TAG cluster. • We generalize this model to the study of orthologous TAG clusters in different species. • We propose an algorithm allowing to infer the evolutionary history of a TAG cluster through tandem duplication, inverted duplication, inversion and deletion of chromosomal segments containing one or many adjacent genes.

碩士
國立陽明大學
生物化學研究所
83
1993年Brockington在粒線體肌病和腦肌病患者的粒線體DNA(mtDNA)之D－loop區域發現了第一型序列重複突變，Poulton等，認為在所發現得序列重複突變可能是先產生類似在病所看到的序列重複突變種，的過程所產生的。最近又有第二型的序列重複突變被證實存在於正常老年人的肌肉組織中，而且可能與人類老化的過程有關。第一型序列重複突變亦可於老年人的組織中發現。因此，我們乃進一步研究在正常老化組織中mtDNA的D－loop區域所發生的序列重複突變，探討其發生的分子機轉。 在本研究中，我們在mtDNA的D－loop區域發現了另外九種序列重複突變，依序列重複突變發生的位置，接合位置的核甘酸序列以及在各組織之出現的頻率，我們將其歸為二類。第一類從近一個重股DNA合成的起始點開始產生重複，接合位置為polyC序列或12bp的正向重複序列(direct repeat)，在不同組織中各型序列重複突變出現的頻率不同，有相當高的組織特異性。而且在較老的組織中出現的頻率亦較高，可能與老化有關，其中第五型重複突變在較耗能的組織中出現的頻率較高，我們認為這一類序列重複突變可能是在重股DNA合成接近完成時，藉由DNA末端的poly C序列或正向重複序列與模板上其他位置的相同序列產生錯誤配對(mis－pairing)，再經由DNA polymerase重複合成該段DNA所造成。 第二類序列重複突變由核甘酸序列第16257或16191位置附近開始產生重複，這個位置與第一類一至四型開始產生重複的位置之序列相似，而開始被重複的位置，也和第一類的第一、二、三型一樣，為第567、493及455位置附近。因此，第455、493、567位置區域是序列重複突變的好發生部位;所以第二類與第一類序列重複突變的產生必有一些共同的因素，有可能是因為未合成完全的(incompletely replicated)7S DNA與模板DNA經由錯誤配對而產生；但因其接合位置之核甘酸序列與在各組織中出現頻率之趨勢與第一類相差甚多，所以也有可能經由其他約方式產生，因為其產生在特定的核甘酸位置，所以可能與DNA結構或與某些特定的蛋白質結合有關係；這一類mtDNA序列重複突變在年輕人的組織、血液、以及幾對的母子血液檢體均可測得，所以有可能會經由母系遺傳的方式傳給子代。 A heteroplasmic tandem duplication (type) with a size of about 260 bp in the D-loop of human mitochondial DNA (mtDNA) has recently been identified in the muscle of several patients with mitochondrial myopathy. An aging-associated type Ⅱ tandem duplicated mtDNA was also observed in the mtDNA of aging human muscle. In the study, we have tried to search for the other tandem duplications in the D-loop region of human mtDNA and analyzed the nucleotide sequences flanking the insertion sites with an aim to answer how the tandem duplications are generated. By use of several back-to-back primers, we have identified with PCR techniques another nine types of tandem duplications in the D-loop region of human mtDNA. These tandem duplications are classified into two groups according to the duplicated regions in the mtDNA, the nucleotide sequences in the junction sites and the frequency of occurrence in different tissues. Group I tandem duplications bear some important characteristics. First, the duplications start from the CSB Ⅱ and CSB Ⅲ region near the start site of the heavy-strand DNA synthesis. Second, the junction sites are localized in the poly C sequence or a 12 bp direct repeat sequence in the D-loop region. Third, they are tissue-specific and aging-associated. In addition, typeⅤ tandem duplication occurs much more frequently in the tissues of high-energy demand. On the basis of these observations, we propose that group I tandem duplications are caused by mis-pairing of directly repeated poly C runs or similar ones at the end of replication of the heavy strand mtDNA. In the group Ⅱ tandem duplications, the nucleotide sequence in the start sites of the duplicated regions and the positions of the start sites are similar to those of group Ⅰ tandem duplications. Thus, there are some common factors involved in the generation of groupⅠand group Ⅱ tandem duplications. Group Ⅱ tandem duplications are most likely caused by the mis-pairing of the incompletely replicated 7S DNA with template DNA. Moreover, there are some characteristics that differentiate group Ⅰ from group Ⅱ tandem duplications. Most notably, the nucleotide sequences flanking the junction sites and frequency of occurrence in different tissues were found to be highly variable. It may be related to a unique mtDNA structure or DNA-protein binding as judged by the junction sites of these duplications. Thus, the group Ⅱ tandem duplications are caused by a different mechanism different from that of group Ⅰ tandem duplications. Interestingly, the group Ⅱ tandem duplications were detectable in the tissues from very young subjects and the blood cells of several mothers and their children. This indicates that the group Ⅱ tandem duplications may be transmitted through maternal lineage.