Dissertations / Theses on the topic 'Chromosomal rearrangements'

Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. Using an inducible system that arrests replication forks at a specific locus in fission yeast, chromosomal rearrangement was investigated. In this system, replication restart requires homologous recombination. However, it occurs at the expense of gross chromosomal rearrangements that occur by either faulty template usage at restart or after the correctly restarted fork U-turns at inverted repeats. Both these mechanisms of chromosomal rearrangement generate acentric and reciprocal dicentric chromosomes. The work in this thesis analyses the timing of replication restart and appearance of chromosomal rearrangements in a single cell cycle after induction of fork stalling. This research also identifies the recombination-dependent intermediates corresponding to the two pathways of rearrangements. Moreover, the DNA integrity checkpoint responses after replication fork arrest, homologous recombination dependent replication restart, and the accumulation of GCRs are investigated.

To better understand the evolutionary dynamics of chromosomal inversions, a physical map for an Asian malaria vector, Anopheles stephensi, was created and compared with the maps of the major African malaria vectors A. gambiae and A. funestus No interchromosomal transposition was observed between A. gambiae and A. stephensi. Several cases of euchromatin and heterochromatin transitions weridentified between A. gambiae and A. stephensi. The study of paracentric inversions between lineages in Anopheles mosquitoes demonstrated that X chromosome has the fastest rate of inversion fixations and highest density of repetitive elements. Among the autosomes, 2R evolved faster than other autosomes. The slowly evolved autosomes have more M/SARs than rapidly evolving arms. Breakpoint regions are enriched with repetitive elements. The study revealed that fixed inversions are distributed nonrandomly and breakpoint clustering is common in lineages of A. gambiae and A. stephensi. The parallel association between the density of inversion fixations and polymorphisms suggests that polymorphic inversions can be fixed during evolution. To understand the direction of evolution in A. gambiae complex, the ancestral status of fixed inversions for this complex was identified. The presence of the 2La inversion in outgroups, A. stephensi and A. nili, confirmed the ancestral status of the 2La inversion. The presences of breakpoint structure of the 2Ro inversion in outgroup species, A. stephensi, indicated that the 2Ro is ancestral arrangement. The presence of SINE elements at the breakpoints of the 2R+p in A. gambiae PEST strain suggested that the 2R+p is a derived arrangement. Therefore, the carrier of 2Rop inversions, A. merus, was considered closest to the ancestral species. We have developed a new protocol for laser microdissection and whole genome amplification of polytene chromosomal fragments to obtain DNA for sequencing and assembly. The chromosomal regions spanning both breakpoints of the 2La in A. arabiensis and A. merus were laser microdissected from the polytene chromosomes. Subsequently, DNA samples were amplified using Illustra GenomePhi V2 DNA and Whole-pool amplification methods for obtaining amplicons. Successful amplification of our target DNA was confirmed by PCR with specific primers followed by Sanger sequencing.
Ph. D.

In this thesis I report three related studies that utilise state-of-the-art technologies to investigate germline and somatic chromosomal rearrangements in humans. Firstly, 16 patients with cytogenetically detectable deletions of 3p25-p26 were analysed with high density single nucleotide polymorphism (SNP) microarrays; Affymetrix 250K SNP microarrays (n=14) and Affymetrix SNP6.0 (n=2). Assuming complete penetrance, a critical region for congenitalheart disease (CHD) susceptibility gene was refined to approximately 200 kb and a candidate critical region for mental retardation was mapped to ~1 Mb interval containing SRGAP3. Secondly, I used SNP microarray and molecular cytogenetic studies to characterize chromosome 11p15 in 8 patients with the imprinting disorder Beckwith-Wiedemann syndrome (BWS). In addition to characterising 11p duplications in three patients, the breakpoints in two patients with balanced rearrangements were mapped to two distinct regions. Thirdly, I used high resolution SNP arrays (Affymetrix 250K Sty1 and 6.0 arrays) to identify copy number changes in renal cell carcinoma (RCC) primary tumours (n=81) and cell lines (n=23). Copy number changes most frequently involved large segments (>10Mb) and loss of 3p and gain of 5q were the most common copy number changes. A comparison of copy number changes in RCC cell lines and inherited and sporadic primary tumours was made.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and comprises a group of heterogeneous malignancies accounting for approximately 10% of all solid tumours in children under 15 years of age. It is a member of the small round blue cell tumours (SRBCT) family. Two main subtypes of RMS are recognised: embryonal (ERMS) and alveolar (ARMS). As is the case for many members of the SRBCT group, ARMS is characterised in 80% of cases by a specific chromosomal rearrangement [t(2;13)(q35;q14) or t(1;13)(p36;q14)] that gives rise to a fusion gene [PAX3/FOXO1A or PAX7/FOXO1A]. The fusion genes are believed to function as aberrant transcription factors. Characterisation of aberrant fusion proteins in human cancer has provided novel diagnostic and prognostic tools, and in some cases novel therapeutic strategies. To date no known cytogenetic abnormality characteristic of ERMS has been identified. This thesis reports the molecular cytogenetic investigation of nine ERMS cell lines (RD, 7763, CT10, RH36, YM, HX170, CCA, JR1, RUCH2) in an attempt to identify a consistent chromosomal aberration for ERMS, the most prevalent RMS subtype. Composite karyotypes of four cell lines (CCA, JR1, RUCH2 and RUCH3) were constructed following the application of an in house molecular cytogenetic screening protocol. A panel of nine ERMS cell line karyotypes was subsequently analysed from which chromosome 15 was revealed to be one of the most frequently rearranged chromosomes in ERMS. Detailed physical mapping of all breakpoints containing chromosome 15 in these nine cell lines suggested a number of genes potentially disrupted; but did not identify a consistent chromosomal aberration. A number of reciprocal chromosomal translocations were identified in the nine ERMS cell lines, and these were investigated in detail. A t(2;15)(q36;q11) in HX170 was noted to result in the disruption of PAX3, and may lead to the formation of a novel fusion gene in this cell line.

Natural environments expose organisms to multifarious selective pressures involving numerous aspects of the overall phenotype, therefore eliciting a response from multiple correlated loci. It has been hypothesized that chromosomal rearrangements can play a role in facilitating local adaptation by establishing new linkage relationships and modifying the recombination patterns between the different chromosomal forms, allowing coordinated adaptation of several loci. The central aim of the work presented here is to test this hypothesis using Drosophila americana as a model system. This species segregates several inversions and an X-4 centromeric fusion which makes it an excellent model to study the role of chromosomal rearrangements on local adaptation. This hypothesis was tested using several approaches. The geographic distribution of the chromosomal rearrangements was determined through sampling of wild populations from a broad geographic range. It was found that several of the chromosomal rearrangements exhibit clinal variation. Furthermore, many of these are found in high linkage disequilibrium. The X-4 fusion is highly associated with inversions on the X and 4th chromosome. Also, two inversions on chromosome 5 are in strong negative linkage disequilibrium. The sequence variation associated with rearrangements of the X was studied using inbred lines. The results show that the inversion and the fusion strongly influence variation on this chromosome. Regions of significant population differentiation and linkage with the rearrangements are found interspersed with loci showing neutral variation indicating that in spite of recombination, allelic associations are maintained on this chromosome. The analysis was also extended to flies directly collected from the wild sampled from a region encompassing a large part of the species' range. Loci throughout chromosome X and 4 were genotyped. Sites in high linkage disequilibrium with the rearrangements and with other assayed sites were found in close proximity with sites that did not show this pattern. In conclusion, the clinal distribution of chromosomal rearrangements and associated genetic variation in conjunction with the detection of islands of linkage disequilibrium among the rearrangements and loci on both chromosomes indicate that chromosomal rearrangements are facilitating local adaptation in D. americana.

The Selection during Niche AdaPtation (SNAP) hypothesis aims to explain how the gene order in bacterial chromosomes can change as the result of bacteria adapting to a new environment. It starts with a duplication of a chromosomal segment that includes some genes providing a fitness advantage. The duplication of these genes is preserved by positive selection. However, the rest of the duplicated segment accumulates mutations, including deletions. This results in a rearranged gene order. In this work, we develop a method to identify SNAP in bacterial chromosomes. The method was tested in Salmonella and Bartonella genomes. First, each gene was assigned an orthologous group (OG). For each genus, single-copy panorthologs (SCPos), the OGs that were present in most of the genomes as one copy, were targeted. If these SCPos were present twice or more in a genome, they were used to build duplicated regions within said genome. The resulting regions were visualized and their possible compatibility with the SNAP hypothesis was discussed. Even though the method proved to be effective on Bartonella genomes, it was less efficient on Salmonella. In addition, no strong evidence of SNAP was detected in Salmonella genomes.

Aberrant chromosome structures can promote tumors in the early stages of carcinogenesis and lead to tumor cells becoming resistant to chemotherapy, for example by changing in drug metabolism. Dicentric (containing two centromeres) and acentric (containing no centromeres) chromosomes are two abnormal chromosome structures that consider as precursors of a variety of gross chromosomal rearrangements (GCRs) generated by subsequent recombination events [1-7]. However, the mechanism of the dicentric and acentric palindromic chromosome formation and their subsequent metabolism is difficult to directly visualise. The previous results from our lab shows that replication forks stalled at a specific replication termination sequence (RTS1) can result in the formation of the dicentric and acentric palindromic chromosomes in the fission yeast Schizosaccharomyces pombe [48-52]. However, the formation of acentric and dicentric chromosomes results in a significant visability loss, due to instability and miss-segregation of the chromosomes in the yeast cells. Thus, their fate is difficult or impossible to follow. To resolve this problem, a non-essential mini-chromosome (Ch16) was developed as a novel model system in this project. The behaviour of rearranged chromosome in vivo and their subsequent fate have been visualised by integrating the lac operator (lacO) and tetracycline operator (tetO) arrays with auxotropic makers, adjacent to the RTSI locus on Ch16. The results reveal imbalanced segregation of a dicentric chromosome and subsequently undergoes a breakage event. An acentric chromosome appears to be decoupled or lost rapidly from the nucleus.

Recent theoretical developments have led to a renewed interest in the potential role of chromosomal rearrangements in speciation. Australian morabine grasshoppers (genus Vandiemenella, viatica species group) provide an excellent study system to test this potential role, because they show extensive chromosomal variation: 12 chromosomal races/species with parapatric distributions. The research in this thesis involves the application of molecular genetic analyses to examine patterns of gene introgression among chromosomal races of Vandiemenella at three different spatial scales: local-scale hybrid zone analysis, island-scale phylogeography, and continental-scale phylogeography. The aims of these multi-scale analyses are to investigate whether chromosomal races represent genetically distinct taxa with limited gene flow, and to infer the historical biogeography of Vandiemenella and evolutionary origins of their parapatric distributions. Karyotype and 11 nuclear markers revealed a remarkably narrow hybrid zone with substantial linkage disequilibrium and strong deficits of heterozygotes between the chromosome races P24(XY) and viatica17 on Kangaroo Island, suggesting that the zone is maintained by a balance between dispersal and selection against hybrids (tension zone). Selection that maintains the stable hybrid zone is unlikely to be operating only on loci linked to rearranged chromosomes. Island-scale and continental-scale phylogeography using multiple nuclear markers indicated that Vandiemenella chromosome races/species generally represent genetically distinct taxa with reduced gene flow between them. In contrast, analyses of a mitochondrial gene showed the presence of distinctive and geographically localised phylogroups that do not correspond with the distribution of the Vandiemenella taxa. These discordant population genetic patterns are likely to result from introgressive hybridization between the taxa and range expansions and contractions. Overall, our molecular analyses favour the allopatric mode of diversification for the evolution of Vandiemenella and do not support the stasipatric speciation model of White (1978). Patterns of genetic differentiation between the chromosomal races analysed at three different spatial scales show dynamic responses of the grasshoppers to past climatic fluctuations, leading to opportunities for long-term isolation and allopatric fixation of new chromosome variants and molecular mutations at many loci. Further analyses are necessary to assess potential roles of chromosomal rearrangements in facilitating diversification in Vandiemenella by reducing recombination within the rearranged chromosome segments.

Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A família Loricariidae é uma das mais diversificadas da ordem Siluriformes, com espécies distribuídas entre sete subfamílias: Hypoptopomatinae, Loricariinae, Hypostominae, Neoplecostominae, Lithogeninae, Delturinae e Ancistrinae. As espécies do gênero Ancistrus Kner, 1854, pertencem à subfamília Ancistrinae, têm mostrado grande variação cariotípica, além de características interessantes do ponto de vista citogenético como a presença de cromossomos sexuais e polimorfismos cromossômicos. Desta forma o objetivo do presente trabalho foi caracterizar os cromossomos de quatro espécies do gênero Ancistrus (Ancistrus sp. 1 “cupim”, Ancistrus sp. 2 “cupim”, Ancistrus sp. “mutuca” e Ancistrus sp. “soberbo”) pertencentes à bacia do Paraguai utilizando técnicas de citogenética clássica e molecular para melhor compreensão da evolução cariotípica dessas espécies. As espécies estudadas apresentaram número diploide variando de 2n=42 a 2n=54, NOR localizadas em regiões pericentroméricas e terminais, além de heteromorfismo de tamanho dessas regiões (NOR) em um dos homólogos nas quatro espécies estudadas. O bandamento C mostrou presença de pouca heterocromatina com exceção das espécies Ancistrus sp. 2 “cupim” e Ancistrus sp. “soberbo” que apresentaram dois blocos grandes de heterocromatina em um par de cromossomos, tanto nos machos quanto nas fêmeas. Um exemplar fêmea da espécie Ancistrus sp. 1 “cupim” também apresentou um bloco grande de heterocromatina em um dos homólogos do par 7, sendo esses resultados indicativos de provável relação entre esses blocos de heterocromatina e a diferenciação de cromossomos sexuais. Os resultados obtidos pela técnica de FISH utilizando sondas de DNAr 18S e 5S mostraram que o DNAr 18S está localizado na mesma região da NOR, o DNAr 5S está distribuído em quatro e cinco pares cromossômicos e o double FISH não mostrou co-localização desses genes. No entanto as espécies Ancistrus sp. 2 “cupim” e Ancistrus sp. “soberbo” mostraram variação nos resultados com marcações de DNAr em blocos de heterocromatina. O uso de sonda telomérica mostrou marcações nos telômeros dos cromossomos das quatro espécies estudadas e marcação pericentromérica em um par de cromossomos da espécie Ancistrus sp. 2 “cupim”. Nossos resultados evidenciam possíveis rearranjos cromossômicos do tipo fusão cêntrica, contribuindo com a redução do número diploide e inversões pericêntricas e paracêntricas resultando na localização dos sítios de DNAr.
The Loricariidae family is one of the most diversified of the Siluriformes order, with species distributed in seven subfamilies: Hypoptopomatinae, Loricariinae, Hypostominae, Neoplecostominae, Lithogeninae, Delturinae and Ancistrinae. The species of the genus Ancistrus Kner, 1854, belong to the subfamily Ancistrinae, have shown great karyotype variation, and interesting features of the cytogenetic point of view as the presence of sex chromosomes and chromosome polymorphisms. Thus the aim of this study was to characterize the chromosomes of four species of Ancistrus genus (Ancistrus sp. 1 "cupim", Ancistrus sp. 2 "cupim", Ancistrus sp. "mutuca" and Ancistrus sp. "soberbo") belonging to Paraguay basin using techniques of classical and molecular cytogenetics to better understand the karyotype evolution of these species. The species showed diploid number ranging from 2n = 42 to 2n = 54, NOR located in pericentomeric and terminal regions, and these regions size heteromorphism (NOR) in one of the homologous in the four species. The C-banding showed the presence of few heterochromatin with the exception of species Ancistrus sp. 2 "cupim" and Ancistrus sp. "soberbo" that had two large blocks of heterochromatin in a pair of chromosomes in both males and females. An exemplary female of the species Ancistrus sp. 1 "cupim" also presented a large block of heterochromatin in one of the pair of 7 homologous, and these results indicating probable relationship between these heterochromatin blocks and differentiation of sex chromosomes. The results obtained by FISH technique using probes 18S rDNA and 5S showed that the 18S rDNA is located in the same region of NOR, the 5S rDNA is distributed in four and five chromosome pairs and double FISH showed colocalization of these genes. However of Ancistrus species sp. 2 "cupim" and Ancistrus sp. "soberbo" showed variation in results with rDNA markings in heterochromatin blocks. The use of telomeric probe showed markings on the telomeres of the chromosomes of four species studied and pericentromeric marking on a pair of chromosomes of the species Ancistrus sp. 2 "cupim". Our results indicate possible chromosomal rearrangements type fusion centric, contributing to the reduction of the diploid number and pericentric inversions and paracentric resulting in the location of rDNA sites.
FAPESP: 2015/05993-3

Per tal de poder entendre la dinàmica evolutiva dels genomes de mamífers és necessari estudiar l’estructura dels cromosomes i quins han estat els canvis que s’han donat tant a gran escala (en forma de reorganitzacions cromosòmiques) com a petita escala (en forma de mutacions d’un sol nucleòtid). Les reorganitzacions cromosòmiques han jugat un paper crucial en el procés evolutiu ja que els canvis a què han donat lloc (inversions, translocacions, fusions o fissions) han provocat canvis genòmics amb conseqüències per a la diferenciació de les espècies. Dins dels mamífers, a més, els rosegadors són el grup que presenta més diversitat d’espècies amb un ampli espectre de cariotips. En aquesta tesi, hem estudiat les reorganitzacions cromosòmiques al llarg de l’evolució dels rosegadors així com els factors que han condicionat la distribució genòmica de les reorganitzacions. Gràcies a la creixent disponibilitat de genomes seqüenciats, hem pogut comparar els genomes de sis espècies de rosegadors (incloent com a referència el del ratolí) i de sis outgrups corresponents a diferents taxons de mamífers (Primats, Carnivora, Artiodactyla i Perissodactyla). Hem identificat les regions d’homologia (o Homologous Synteny Blocks; HSBs) i les regions de disrupció de l’homologia (o Evolutionary Breakpoint Regions; EBRs) que s’han donat al llarg de l’evolució dels rosegadors. La localització de les EBRs ens ha permès, estudiar les característiques genòmiques de les regions d’inestabilitat que han donat lloc a les reorganitzacions cromosòmiques en el genoma de ratolí i que s’han donat al llarg de l’evolució dels rosegadors. Els nostres resultats mostren que les EBRs presenten una distribució no-homogènea i es caracteritzen per presentar un alt contingut gènic, taxes de recombinació meiòtica més baixes i una proporció més baixa de lamina associated domains (cLADs), comparant amb la resta del genoma de ratolí. D’altra banda, està descrita la tendència de poblacions naturals de ratolí domèstic occidental (Mus musculus domesticus) a presentar una gran diversitat de nombres diploides a causa de l’aparició de fusions Robertsonianes (Rb). De totes les poblacions estudiades fins al moment, n’hi ha una, localitzada a les províncies de Barcelona, Lleida i Girona, que presenta una estructura diferent a la resta ja que no existeix en ella una raça metacèntrica i les fusions Rb detectades es troben en un estat de polimorfisme: aquesta zona es coneix amb el nom de sistema Robertsonià de Barcelona. En segon lloc, donades les característiques úniques d’aquest sistema Robertsonià hem analitzat: (i) el paper que podien jugar els telòmers en l’aparició de les fusions Rb i (ii) l’efecte d’aquestes fusions i del gen Prdm9 sobre la recombinació meiòtica. En el nostre treball hem detectat que l’escurçament telomèric dels braços p dels cromosomes acrocèntrics podria ser un dels factors que explicarien l’aparició de les fusions Rb ja que afavoririen la seva interacció i la posterior fusió. Em vist que la presència de les fusions Rb provoca una baixada en la taxa de recombinació que es deu a una redistribució dels crossovers (o punts de recombinació homòloga) cap al telòmers en els cromosomes fusionats. Aquest fenomen es deu a un efecte d’interferència del centròmer que suprimeix la recombinació a la zona pericentromèrica. També hem analitzat la distribució allèlica del gen Prdm9 en el sistema Robertsonià de Barcelona, així com un possible efecte de la seqüència d’aquest gen sobre la taxa de recombinació. Donats els nostres resultats, proposem que l’efecte de supressió de la recombinació en els individus amb fusions Rb és a causa d’un factor mecànic (com es l’efecte d’interferència centromèrica) i d’un factor genètic (com es la seqüència al·lèlica de Prdm9). Aquests resultats aporten una informació esencial per entendre la dinàmica dels processos d’especiació cromosòmica en poblacions naturals.
In order to understand the evolutionary dynamics of mammalian genomes, is necessary to analyze chromosome configuration as well as the genomic changes that have occurred at a large-scale (in the form of chromosomal rearrangements) and at a micro-scale (in the form of nucleotide changes) within species. Chromosomal rearrangements (i.e., inversions, translocations, fusions or fissions) have played a crucial role during evolution as they have led to genomic changes with consequences for the species differentiation. Within mammals, rodents represent the most specious taxon with a wide spectrum of karyotypes. In this thesis, we have first analyzed the chromosomal reorganizations along rodents evolution together with the factors that have been involved in the distribution of chromosomal rearrangements. Taking advantage of the increasing number of available whole-genomes sequenced, we have compared the genomes of six rodent species (including the mouse genome as a reference) and six outgroup species corresponding to different mammalian taxa (Primates, Artiodactyla, Carnivora and Perissodactyla). We have identified genomic regions of homology (or homologous synteny blocks, HSBs) and the regions of synteny disruption (or Evolutionary Breakpoint regions, EBRs) among rodents. Moreover, the localization of EBRs has permitted us to analyze the genomic features that could be involved in the origin of chromosomal rearrangements. Our results showed that EBRs present a non-homogeneus distribution across the mouse genome. Additionally, EBRs are characterized by specific genomic features such as higher gene content, lower recombination rates and low proportion of lamina associated domains (cLADs) compared with the rest of the mouse genome. Secondly, it is known that the western house mouse (Mus musculus domesticus) natural populations present a wide variety of diploid numbers due to the presence of Robertsonian (Rb) fusions. Within all these populations analyzed, one of them, localized in the Barcelona, Lleida and Girona provinces, presents a specific structure, where no metacentric race has been described, being the Rb fusions found in a polymorphic state. This chromosomal polymorphism zone is known as The Barcelona Rb system. Giving the specific characteristics of this population, we have: (i) analyzed the role of telomeres in the occurrence of the Rb fusions and (ii) studied the effect of the Rb fusions and Prdm9 gene on meiotic recombination. We have detected that telomere shortening in acrocentric p-arms can be one of the factors that could explain the occurrence of Rb fusions by promoting the interaction between chromosomal ends and thus, to the fusion events. Moreover, we have observed that the presence of Rb fusions leads to a decrease in recombination rates due to a re-distribution of crossovers towards the telomeres in metacentric chromosomes. Furthermore, we have detected that this phenomenon is due to an interference effect of the centromere in metacentric chromosomes, which acts suppressing recombination within the pericentromeric regions. Additionally, we have also characterized the Prdm9 allelic distribution within the Barcelona Rb polymorphism system, as well as an effect of the Prdm9 sequence on recombination rates. Therefore, and in the light of our results, we propose that the effect of suppression of recombination on individuals with Rb fusions is due to a mechanicstic (by the centromeric interference effect) and genetic (the Prdm9 allelic sequence) factors. These results, together with the characterization of the genomic features that have been involved in the occurrence of evolutionary chromosomal rearrangements in rodents, would help us to understand the dynamics of chromosomal speciation along evolution and how chromosomal rearrangements occur in natural populations.

This thesis illustrates the work I have developed as a Ph.D. student in the computational genomics group lead by Dr. David Torrents at the Barcelona Supercomputing Center. The group’s expertise in the analysis of biological data and the detection of variants to gain more knowledge about the genetic and molecular implications of human diseases, such as cancer, has allowed me to learn and conduct my research. Focusing on the analysis of structural variation in cancer, I have been able to apply different methodologies for sequencing data, retrieving, filtering, and determining the mutational profile foreach ofthe studied samples.Moreover,I have characterized new patterns of genomic rearrangements related to transposase-derived genes and extrachromosomal circular DNA elements in cancer. Therefore, this thesis is centered in the study of the genomic variation and mechanisms associated with oncogenic processes together with the analysis of elements of the human genome that are not generally included in comprehensive cancer studies, such as circular DNA elements. In summary, starting with the introduction, I give an overview of the methodological aspects of the study of cancer through the impact of sequencing technologies, the biological and molecular causes and consequences of this disease, focusing on structural variation, and the description of the circular DNA genomic component and its known implications in cancer. Finally, I introduce neuroblastoma, an example of how structural variants and circular DNA drive tumorigenesis. Next, I present the results of this thesis in three blocks, all of which have in common the study of structural variation in cancer. Two of the blocks correspond to the PGBD5 and neuroblastoma publications, and one corresponds to the continuation of the PGBD5 analysis in ICGC-Pan-Cancer data. As an overview of the trajectory of this thesis, I started with my involvement in a project focused on analyzing the role of PGBD5 —a transposase-derived gene— as an oncogenic mutator with an associated mechanism for site-specific DNA rearrangements. In this study, we describe how the expression of this gene promotes cell transformation and the generation of recurrent rearrangements, presenting a conserved motif in cell lines and childhood tumors. As a logical continuation of this publication and thanks to the access of our group to ICGC-PCAWG data, we expanded the study of these characteristic PGBD5-motif-related rearrangements to different patients and tumor types. The following part of this thesis is focused on the analysis, description, and classification of the genomic somatic rearrangements in neuroblastoma. With the aim of better grouping the patients with different clinical outcomes, we searched for differential patterns of structural variants across the samples. From this analysis, we were able to describe a new phenomenon that connects circular DNA with different integration sites around the genome through complex rearrangement clusters providing evidence on how circular DNA can act as a driver of genomic remodeling in neuroblastoma. To finalize, I present the general discussion of the results and questions addressed in this work to, then, end up disclosing the final conclusions of this thesis.

Chromosomal rearrangements of the nucleoporin genes NUP214 and NUP98 are recurrent in aggressive cases of acute myeloid and lymphoid leukemias. NUP214 and NUP98 are components of the nuclear pore complex, a giant multiprotein structure that mediates nucleocytoplasmic shuttling. The two nucleoporins are enriched in phenylalanine-glycine (FG) repeats, which form the NPC permeability barrier and are essential for the interaction with nuclear transport receptors. NUP214 and NUP98 exhibit high affinity for the nuclear export receptor chromosomal region maintenance 1 (CRM1), which, alone, mediates the nuclear export of thousands of proteins and ribonucleoproteins. In the first part of this project, we report that the leukemogenic fusion proteins SET-NUP214 and DEK-NUP214 affect nucleocytoplasmic transport by perturbing the localization of essential nuclear transport factors, including endogenous nucleoporins and CRM1 nuclear export complexes. We further demonstrate that the two fusion proteins are sensitive to CRM1 inhibition and that targeted inhibition of nuclear export is sufficient to reduce the cell viability and proliferation of patient-derived cell lines with SET-NUP214 and DEK-NUP214 rearrangements. In the second part of the project, we used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Though distinct endogenous binding partners have been documented for NUP214 and NUP98 chimeras, their total interactome has not been fully disclosed. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. We further purpose that the two fusion proteins affect distinct cellular processes. According to our results, NUP98-HOXA9 likely perturbs Wnt, MAPK and estrogen receptor signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, SET-NUP214 appears to affect cellular metabolism, likely due to the interaction with mitochondrial proteins and metabolic regulators. Overall, this research project provided new data supporting that CRM1 might be a possible therapeutic target in NUP214-related leukemia and revealed new clues on the mechanistic actions of nucleoporin fusion proteins. Hence, our findings might be of particular relevance in the search of new druggable targets for the treatment of nucleoporin-related leukemia.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Neste trabalho, estudamos duas deleções cromossômicas detectadas em indivíduos com retardo mental associado a sinais clínicos. O objetivo foi determinar que mecanismos originaram esses rearranjos e como a perda ou quebra dos segmentos cromossômicos está relacionada com o fenótipo dos portadores. A caracterização das seqüências nos pontos de quebra e junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. Para isso, este trabalho aliou o estudo cromossômico por hibridação in situ fluorescente (FISH) à análise do DNA.
Structural chromosomal alterations related to clinical phenotypes bring the opportunity to identify gene mutations determining the pathologies, because the causative genes may have been disrupted by the breaks or may have an altered number of copies. The delimitation of the segments involved in the chromosomal rearrangements is necessary for these genotype-phenotype correlations. The characterization of breakpoint and junction sequences in these chromosome alterations enables the identification of mechanisms originating them, and evidence has been produced pointing to the participation of particular genomic sequences in their formation. In this work, we studied two chromosomal deletions in patients with syndromic mental retardation, combining chromosomal analysis by fluorescent in situ hybridization (FISH) to DNA analysis. Our aim was to determine the mechanisms that originated these aberrations and how they were involved with the clinical phenotypes.

Disease associated balanced chromosomal rearrangements (DBCRs) are large-scale alterations in normal genomic sequence, which occur without copy number change in a phenotypically abnormal individual. Complete ascertainment of published DBCRs was attempted via recursive searches of the literature. 775 cases were identified with 1672 breakpoints and 406 different phenotypes. Physical mapping of DBCR breakpoints has elucidated the genetic basis of Mendelian disorders in 29 cases and was the first indication of a subsequently verified disease locus in a further 30 cases. Two interesting DBCR cases, which had no cell-lines or fixed cell suspensions, were available for study: Case 1 had a t(2;12)(p25;q23.3) translocation associated with upper limb peromelia and lower limb phocomelia; Case 2 had a t(1;2)(q41;p25.3) associated with lethal bilateral renal adysplasia. In case 1, the 2p25 breakpoint was found to interrupt the ROCK2 gene, which, on the basis of the phenotype of null mice, was considered to be a poor candidate for the peromelia/phocomelia phenotype. The 12q23.3 breakpoint lay 0-25 kb from the 5’ end of the CMKLR1gene, which encodes a chemokine-like receptor, the sole ligand for which is encoded by the retinoic acid responsive gene, RARRES2. Site and stage-specific expression of both the receptor and the ligand in the developing limb bud suggested that CMKLR1 was a good candidate for a causative gene. A phenotypically similar case was screened for mutations in CMKLR1 and a candidate regulatory region, but none could be identified. A mouse model is being developed to further elucidate the developmental role of this gene. In case 2, the 2p25.3 breakpoint mapped to a gap in the genome sequence. No good candidate gene could be identified in the vicinity of this gap.

Este estudo teve como objetivo identificar mecanismos pelos quais rearranjos cromossômicos aparentemente equilibrados possam estar associados de maneira causal a determinados quadros clínicos. Para isso estudamos seis translocações cromossômicas aparentemente equilibradas, detectadas em pacientes com malformações congênitas, comprometimento neuropsicomotor ou déficit intelectual. Os pontos de quebra desses rearranjos foram mapeados por hibridação in situ fluorescente (FISH). A busca por microdeleções e duplicações genômicas foi realizada por a-CGH. Estudamos duas translocações esporádicas, t(7;17)(p.13;q24) e t(17;20)(q24.3;q11.2), nas quais os pontos de quebra no cromossomo 17 foram localizados, respectivamente, a 917-855 kb e 624-585 kb upstream ao gene SOX9, em segmentos sem genes mapeados. Ambos os portadores apresentavam alterações esqueléticas que indicaram o diagnóstico de displasia campomélica acampomélica. Não foram detectados desequilíbrios cromossômicos submicroscópicos por a-CGH. Essas translocações podem levar à expressão alterada do gene SOX9, ao afetar a região reguladora desse gene. Sequências dos outros cromossomos participantes da translocação, que foram aproximadas ao gene pelo rearranjo, também podem ter afetado sua expressão. O estudo dos rearranjos t(7;17) e t(17;20) forneceu informação para o entendimento da região reguladora do gene. As manifestações clínicas associadas à t(17;20) permitiram redefinir o limite distal do cluster distal de rearranjos do cromossomo 17 associados ao espectro de manifestações clínicas do SOX9. A presença de testículo no portador dessa translocação indicou um elemento conservado candidato a atuar como enhancer do SOX9, para o desenvolvimento do testículo. Duas outras translocações equilibradas estavam associadas a desequilíbrios submicroscópicos em cis aos pontos de quebra. Caracterizamos uma t(10;21)(p13;q22) esporádica associada a atraso do desenvolvimento neuropsicomotor, microcefalia e espasticidade generaliza. Os pontos de quebra dos cromossomos 10 e 21, foram mapeados, respectivamente, em segmentos de 440 kb e 172 kb. Três genes estão mapeados no segmento que contém o ponto de quebra do cromossomo 10 e três outros, no intervalo delimitado para o ponto de quebra no cromossomo 21. O gene CDNF, que pode ter sido interrompido pelo ponto de quebra do cromossomo 10, é altamente expresso no sistema nervoso. A análise por meio de a-CGH detectou quatro deleções no cromossomo 10 todas de novo, indicando a complexidade do rearranjo. Duas deleções estavam próximas ao ponto de quebra: uma deleção de 973 kb em 10p14 e uma outra de 1,15 Mb em 10p13, mapeadas a 3,27 Mb e 210 kb do ponto de quebra da translocação, respectivamente. Outras duas deleções no cromossomo 10 ocorreram no braço longo: uma deleção de 700 kb em 10q26.13 estaria a 110,10 Mb do ponto de quebra da translocação, mas não conseguimos mapeá-la por FISH; uma outra deleção de 1,66 Mb em 10q26.2-q26.3 foi mapeada a 114,68 Mb do ponto de quebra da translocação. Quatorze genes estão localizados nas regiões das microdeleções. Os genes GPR26, OPTN, CUGBP2 são altamente expressos no sistema nervoso e, assim como o CNDF, podem ser considerados candidatos ao efeito fenotípico. O modelo de chromothripsis, em que o rearranjo resulta de uma série de quebras na dupla fita do DNA, seguida de ligação aleatória dos fragmentos resultantes, pode explicar a formação da translocação t(10;21). Aplicando a-CGH no estudo de uma translocação t(X;22)(q22;q13) esporádica, detectamos duplicações de 490 kb e 570 kb, respectivamente, em 22q13 e Xq22. A análise por FISH revelou que as cópias adicionais desses segmentos estavam localizadas nos pontos de quebra dos cromossomos derivativos X (segmento duplicado de 22q13) e 22 (segmento duplicado de Xq22). Não há genes mapeados no segmento duplicado do cromossomo 22. Um dos 14 genes duplicados no cromossomo X é o PLP1 (proteolipid protein 1), cujas mutações de ponto e duplicações causam a doença de Pelizaeus- Merzbacher, caracterizada pela hipomielinização do sistema nervoso central e afetando quase que exclusivamente indivíduos do sexo masculino. O exame neurológico, incluindo ressonância magnética, mostrou que o quadro clínico da paciente é compatível com o da doença de Pelizaeus-Merzbacher. A análise do padrão de inativação do cromossomo X em linfócitos de sangue periférico da paciente, com base na metilação do gene AR e também citologicamente em metáfases, após incorporação de 5-BrdU, revelou que, na maioria das células, o cromossomo X normal está inativo. Esse padrão de inativação torna as células funcionalmente equilibradas quanto aos segmentos translocados. O PLP1, entretanto, tem uma cópia adicional no cromossomo 22, além das cópias localizadas nos cromossomos X e der(X). Portanto, duas cópias ativas do gene estão presentes nas células da portadora da t(X;22). O mecanismo de formação de rearranjos cromossômicos baseado em bolhas de replicação explicaria a formação de translocações com duplicação em ambos os pontos de quebra, como ocorreu nessa t(X;22). Estudamos também uma aparente t(2;22)(p14;q12) familial que cossegregava com quadro de atraso do desenvolvimento neuropsicomotor e dificuldade de aprendizado associados a dismorfismos craniofaciais e alterações de mãos. A identificação de duplicações e deleções submicroscópicas, por meio de a-CGH e sua validação por FISH revelaram que se tratava, na verdade, de rearranjo, complexo entre três cromossomos 2, 5 e 22: um segmento de 1,2 Mb de 2p14 inseriu-se no braço curto do cromossomo 5, um evento que pode ter causado a deleção de um segmento de 1,4 Mb em 5p15.1; no cromossomo derivativo der(22) um segmento adicional de 5q23.2- 23.3 inseriu-se no ponto de quebra. Todos os afetados da família eram portadores do der(2) e do der(22). No entanto, o der(5) não segregava com o quadro clínico e foi detectado em um individuo fenotipicamente normal da família. Todos os afetados eram portadores da duplicação de 6,6 Mb do braço longo do cromossomo 5 (5q23.2-23.3). Os 17 genes duplicados são candidatos para o quadro clínico, por aumento da dosagem de seus produtos. Outra alteração comum a todos os afetados foi a haploinsuficiência do gene SLC1A4 mapeado em 2p14 e altamente expresso no sistema nervoso. É interessante que a deleção em 2p14, consequente à ausência do der(5), está restrita aos dois afetados que aparentam tem maior déficit cognitivo. Além do SLC1A4 , quatro genes mapeados nesse segmento CEP68, RAB1A, ACTR2 e SPRED2 podem contribuir para a variabilidade clínica dos afetados. A translocação t(2;5;22) pode ter-se originado a partir de duas quebras no braço curto do cromossomo 2, duas no braço curto e duas outras no braço longo do cromossomo 5 e uma quebra no braço longo do cromossomo 22. As quebras teriam ocorrido simultaneamente em um único evento. Após reunião de extremidades quebradas, formaram-se os cromossomos derivativos. Investigamos por a-CGH uma t(2;16)(q35;q24.1) esporádica cujos pontos de quebra foram mapeados anteriormente por FISH; nenhum gene estava mapeado nos segmentos que continham esses pontos de quebra. Não detectamos desequilíbrios cromossômicos submicroscópicos. A paciente portadora da translocação t(2;16) tinha quatro dígitos nas duas mãos e hexadactilia nos pés. A cerca de 1 Mb do ponto de quebra do cromossomo 2 está mapeado o gene IHH, que atua no desenvolvimento dos membros. A translocação pode ter interrompido elemento regulador do IHH ou separado o gene de elemento(s) regulador(es), levando à alteração de sua expressão e ao fenótipo. Este estudo fornece evidência adicional da importância da busca de desequilíbrios cromossômicos submicroscópicos em associação com rearranjos aparentemente equilibrados. Em três das seis translocações estudadas - t(10;21), t(2;22), t(X;22) - foram detectados desequilíbrios cromossômicos submicroscópicos em cis aos pontos de quebra, que podem ser responsáveis pelas manifestações clínicas dos portadores. Este estudo ressalta ainda a importância da técnica de FISH na análise dos desequilíbrios cromossômicos detectados por array, permitindo determinar a relação entre as perdas ou ganhos de segmentos submicroscópicos e os rearranjos equilibrados. A caracterização de rearranjos equilibrados neste estudo também contribuiu para sugerir mecanismos para sua formação
This study aimed at identifying mechanisms that lead to phenotypic abnormalities in carriers of balanced chromosomal rearrangements. We studied six apparently balanced chromosomal translocations detected in patients with congenital malformations, intellectual impairment or neuropsychomotor delay. Breakpoint mapping of apparently balanced chromosomal rearrangements was performed by fluorescence in situ hybridization (FISH), and cryptic genomic imbalances were investigated by array comparative genomic hybridization (a-CGH). We studied two sporadic translocations, t(7;17) (p13;q24) and t(17;20) (q24.3,q11.2). The breakpoints were located on chromosome 17, respectively, 917-855 kb and 624-585 kb upstream the SOX9 gene. There are no genes mapped to these segments. Patients had skeletal abnormalities that led to the diagnosis of acampomelic campomelic dysplasia. No submicroscopic chromosomal imbalances were detected by a-CGH. These translocations can alter gene expression by directly disrupting regulatory elements or by a position effect. The translocation t(7;17) and (17;20) provided additional information regarding the regulatory region of SOX9. The clinical manifestations associated with the translocation t(17;20) allowed the redefining of the limits of the distal breakpoint cluster of rearrangements on chromosome 17, which are associated with SOX9-related disorders. A conserved element was identified as a candidate SOX9 enhancer for testis development. Two additional sporadic translocations were associated with submicroscopic imbalances in cis to the breakpoints: t(10;21) and t(X;22). The translocation t(10;21)(p13;q22) was present in a girl with delayed motor development, microcephaly and generalized spasticity. The breakpoints on chromosomes 10 and 21 were mapped to 440 kb and 172 kb segments, respectively. Among the genes mapped to these breakpoint regions, only CDNF on chromossome 10, is highly expressed in the nervous system. Four de novo deletions on chromosome 10 were identified by a-CGH, revealing the complexity of the rearrangement. Two deletions were located at the vicinity of the translocation breakpoint: a 973 kb deletion on 10p14 and a 1.15 Mb deletion on 10p13 located, respectively, 3.27 Mb and 210 kb distal to the translocation breakpoint. Two other deletions were detected on the long arm of chromosome 10: a 700 kb deletion on 10q26.13, located 110.10 Mb distal to the translocation breakpoint, which we could not mapped by FISH; and a 1.66 Mb deletion on 10q26.2-q26.3, located 114.68 Mb distal to the translocation breakpoint. Fourteen genes are mapped to the microdeletion regions. Among these genes, GPR26, OPTN, CUGBP2 are highly expressed in the nervous system and, together with CNDF, are candidates for having clinical effects. The chromothripsis model, in which rearrangements result from a series of simultaneous double-stranded breaks followed by random joining of chromosomal fragments, might explain the formation of this t(10,21) translocation. Applying a-CGH to the apparently balanced translocation t(X;22)(q22;q13) carried by a girl, we detected duplicated segments on 22q13 and Xq22, encompassing 490 kb and 570 kb, respectively. FISH analysis revealed that the additional copies were located to the breakpoints of the derivative X chromosome (22q13 duplicated segment) and of the derivative 22 chromosome (Xq22 duplicated segment). No genes are mapped to the duplicated segment of chromosome 22. One of the 14 duplicated genes on the X chromosome is PLP1 (proteolipid protein 1). PLP1 point mutations and duplications cause Pelizaeus-Merzbacher disease, characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. Neurological examination of the patient, including MRI showed that her clinical manifestations were compatible with Pelizaeus-Merzbacher disease. The pattern of X chromosome inactivation was determined in peripheral blood lymphocytes, based on the AR gene methylation, and cytologically, in metaphases spreads, after 5-BrdU incorporation, and showed that the normal X chromosome was the inactive one in the majority of cells. This pattern of X inactivation makes cells functionally balanced for the translocated segments. A copy of the PLP1 gene, however, is present on chromosome 22, in addition to the copies located on the chromosomes X and der(X). Thus, two active copies of the gene are present in the cells, irrespective of the X-inactivation pattern. A mechanism based on replication bubbles can explain the formation of translocations with duplication at the breakpoints, such as this t(X;22). An apparently balanced familial translocation t(2;22)(p13;q12.2) was detected in association with learning disability and craniofacial and hand dysmorphisms. The combination of a-CGH and FISH revealed that the rearrangement, identified by Gbanding as a two-break balanced translocation, was a more complex three-chromosome rearrangement: a segment from chromosome 2 was inserted into chromosome 5 short arm, an event that probably caused a 5p15.1 deletion; on chromosome 22 a segment from 5q23.2-23.3 was inserted into the breakpoint. Chromosomes der(2) and der(22) were present in all affected individuals. However, the der(5) did not segregate with the clinical phenotype, and was detected in a phenotypically normal individual. The 6.6 Mb duplication of the long arm of chromosome 5 was the imbalance common to all affected individuals. The 17 genes in this region are candidates for the clinical phenotypes through dosage effect. In addition, common to all affected individuals is the haploinsufficiency of SLC1A4, a gene highly expressed in the nervous system, which is encompassed by the deletion on chromosome 2. Interestingly, learning disabilities were more pronounced in those patients who also carried chromosome 2 deletion. CEP68, RAB1A, ACTR2 and SPRED2, mapped to this deleted segment, might contribute to the variability of the clinical phenotype in the family. The translocation t(2;5;22) might have originated from a series of simultaneously occurring brakes, two on the short arm of chromosome 2, four breaks on the short arm and two on the long arm of chromosome 5, and one break on the long arm of chromosome 22. We also investigated by a-CGH a sporadic translocation t(2;16)(q35;q24.1) whose carrier had hand and feet defects. Submicroscopic imbalances were not detected. Previously performed FISH delimited the breakpoints segments on chromosomes 2 and 16, which encompassed no genes. The IHH gene, which is involved in limb development, is located approximately 1 Mb upstream chromosome 2 breakpoint. Therefore, the translocation might have disrupted a regulatory element of IHH or, alternatively, separated the gene from a regulatory region, thus altering IHH expression. This study provides further evidence for the occurrence of submicroscopic chromosomal imbalances in association with apparently balanced rearrangements. In three out of six translocations - t(10,21), t(2;5;22), t(X;22) - cryptic duplications/deletions in cis to the breakpoints were detected, which might account for the clinical manifestations of the patients. This study also highlights the importance of FISH in the analysis of genomic imbalances detected by array in determining how losses and gains of submicroscopic segments relate to the rearranged chromosomes. The characterization of the balanced translocations in this study also contributed to suggest mechanisms for their formation

Estudos de citogenética comparativa são rotineiramente desenvolvidos a partir da comparação dos padrões de bandamentos cromossômicos. Contudo, quando se trata de espécies que apresentam genomas altamente rearranjados, como no caso das espécies de Akodon, ou que são muito divergentes, a comparação de cromossomos por padrões de bandas torna-se inadequada. Como consequência, a pintura cromossômica tem se tornado o método de escolha assertivo, já que permite comparação genômica no nível citogenético. Nessa tecnologia sondas de um cromossomo inteiro de uma determinada espécie são hibridadas in situ em cromossomos de outra espécie, detectando as regiões homólogas entre os genomas. No presente estudo comparamos os cariótipos altamente diferenciados de algumas espécies de Akodon por meio de pintura cromossômica recíproca com uso de sondas espécie-específicas, obtidas a partir de cromossomos separados por citometria de fluxo. Os resultados revelaram homologia completa entre os complementos de Akodon sp. n. (ASP), 2n=10, A. cursor (ACU), 2n=15, A. montensis (AMO), 2n=24 e A. paranaensis (APA), 2n=44 e evidenciaram com precisão muitos rearranjos cromossômicos entre os complementos das espécies. Rearranjos Robertsonianos e em tandem, inversões pericêntricas e/ou reposicionamento de centrômero, inversão paracêntrica, translocações, inserções e existência de sítios frágeis nos complementos foram observados. A pintura cromossômica empregando o conjunto de sondas de APA para 21 autossomos mais cromossomos X e Y evidenciou oito segmentos sintênicos compartilhados entre A. montensis, A. cursor e Akodon sp. n., cinco associações exclusivas para A. cursor e seis para Akodon sp. n. Foi também detectada homologia completa dos cromossomos X, com exceção da região heterocromática de ASP X, e até mesmo dos cromossomos Y que geralmente não apresenta sinal hibridação entre diferentes espécies de mamíferos. Esses dados indicam que essas espécies intimamente relacionadas passaram por um processo recente de intensa diferenciação autossômica, no qual se conservou a homologia total, por exceção de Akodon sp. n., entre os cromossomos sexuais. Pertencente a tribo Akodontini, Deltamys Thomas 1917 é um táxon pouco estudado e que é raramente capturado. Utilizando-se de caracteres morfológicos ou genéticos, alguns autores consideram Deltamys como um gênero pleno enquanto outros acreditaram que esse devesse ser referido como subgênero ou sinônimo de Akodon. A única espécie formalmente descrita é Deltamys kempi que apresenta cariótipo básico com 2n=37 nos machos e 2n=38 nas fêmeas, NF=38, com sistema de determinação de sexo do tipo X1X1X2X2: X1X2Y. Uma característica citogenética que distingue Deltamys de Akodon é a presença de um pequeno par metacêntrico marcador em Akodon. Um cariótipo com 2n=40 e NF=40; XX: XY foi relacionado ao gênero Akodon porém, como em Deltamys kempi , esse complemento não apresenta o pequeno par metacêntrico. As análises filogenéticas de máxima parcimônia e máxima verossimilhança baseadas em sequências do gene mitocondrial do citocromo b evidenciaram o monofiletismo dos espécimens de Akodon sp. 2n=40, e o monofiletismo de Deltamys kempi. Além disso, revelaram que Akodon sp. é grupo irmão de Deltamys kempi, estando mais relacionado a este último gênero do que às demais espécies de Akodon, sugerindo a inclusão dos espécimens portadores do cariótipo com 2n=40 em Deltamys. Dessa forma, o gênero Deltamys se mostrou mais diverso do que até então se tinha conhecimento, agrupando duas linhagens: Deltamys kempi , 2n=37-38 ; X1X1X2X2: X1X2Y e Deltamys sp. 2n=40, XX: XY, que apresentam entre sí uma marcante divergência genética que chega a 12, 1%. Um cariótipo com 2n=50, NF=48 foi descrito para espécimes de Thaptomys Thomas,1916, coletados em Una, estado da Bahia, Brasil, que são indistinguíveis morfologicamente dos Thaptomys nigrita que apresentam 2n=52, NF=52, encontrados em outras localidades brasileiras. Foi então proposto que esse novo cariótipo com 2n=50 pertencesse a uma espécie críptica de Thaptomys nigrita, uma vez que os rearranjos cromossômicos observados somados a distância geográfica pode representar uma barreira reprodutiva entre as duas formas. Com o intuito de se estabelecer as relações entre indivíduos de Thaptomys com os dois números diplóides, análises filogenéticas moleculares foram realizadas com o uso de dezoito sequências do gene mitocondrial do citocromo b provenientes de espécimes cariotipados coletados ao longo da distribuição geográfica do gênero. Dois clados principais, Nordeste (A) que agrupa espécimens com 2n=50 e Sudeste (B) que agrupa espécimes com 2n=52, foram recuperados por análises de máxima parcimônia e máxima verossimilhança. As relações filogenéticas intra-genéricas corroboram os distintos números diplóides, e mostram os cariótipos com 2n=50 e 2n=52 como clados irmãos entre si separados pela cladogênese basal de Thaptomys . No presente trabalho são apresentados estudos de filogenia molecular e citogenética para o gênero monotípico de roedor fossorial Blarinomys Thomas 1896. Foram realizadas análises de máxima parcimônia e máxima verossimilhança com base em sequências do gene mitocondrial citocromo b , para amostra de 11 exemplares de B. breviceps provenientes de nove localidades de quatro estados do território brasileiro. Todas as topologias recuperaram duas linhagens principais: um clado Nordeste (A) e outro Sudeste. O clado sudeste agrupou dois clados irmãos, B e C. A divergência de sequência entre os indivíduos variou de: 4,7- 8,0% entre os clados nordeste e sudeste; 4,3-5,7% entre os clados B e C; 6,1-8,0% entre os clados nordeste; e B, e 4,7-6,4% entres os clados nordeste e C. Dentro dos clados a divergência variou de 0- 4,2% no clado nordeste, foi de 0,7% no clado B, e variou de 0,1- 1,3% no clado C. Variação entre espécimes da mesma região geográfica foi de 0-1,3%. Os estudos de citogenética de cinco exemplares revelaram alta diversidade cariotípica com cinco números diplóides distintos: 2n=52 (48A+2Bs, XY), 2n=43 (37A+4Bs, XX), 2n=37 (34A+1B, XY), 2n=34 (32A, XX) e 2n=31 (27A+2Bs, XX) e mesmo número de braços autossômicos (NF=50), excluindo-se os cromossomos sexuais e os supernumerários. Foram observados polimorfismos decorrentes de rearranjos Robertsonianos, além de variação de 0 a 4 cromossomos Bs, que são heterogêneos quanto a morfologia, constituição de heterocromatina e presença de sinais teloméricos intersticiais (ITS). ITS também foram observados na região pericentromérica de alguns pares autossômicos com dois braços em três dos exemplares. Foi realizada pintura cromossômica com sonda do cromossomo X de Akodon cursor (ACU X). Nossos dados revelaram uma diversidade até então desconhecida para Blarinomys , mostrando duas linhagens distintas correspondentes a regiões na Mata Atlântica e um extraordinário polimorfismo cromossômico.
Traditionally comparative cytogenetic studies are based mainly on banding patterns. Nevertheless, when dealing with species with highly rearranged genomes, as in Akodon species, or with other highly divergent species, cytogenetic comparisons of banding patterns prove to be inadequate. Hence, comparative chromosome painting has become the method of choice for genome comparisons at the cytogenetic level, since it allows complete chromosome probes of a species to be hybridized in situ onto chromosomes of other species, detecting homologous genomic regions between them. In the present study, we have explored the highly rearranged complements of the Akodon species using reciprocal chromosome painting through species-specific chromosome probes obtained by chromosome sorting. The results revealed complete homology among the complements of Akodon sp. n. (ASP), 2n=10, A. cursor (ACU), 2n=15, A. montensis (AMO), 2n=24 and A. paranaensis (APA), 2n=44 and extensive chromosome rearrangements have been detected within the species with high precision. Robertsonian and tandem rearrangements, pericentric inversions and/or centromere repositioning, paracentric inversion, translocations, insertions and fragile sites were observed. The chromosome painting using the APA set of 21 autosomes plus X and Y exhibited eight syntenic segments that are shared with A. montensis, A. cursor and Akodon sp. n. plus five exclusive associations for A. cursor and six for Akodon sp. n. Chromosomes X, except for the heterochromatin region of ASP X, and even chromosome Y that often present no hybridization signal when hybridized between species of mammals, shared complete homology among the species. These data indicate that all those closely related species have experienced a recent intensive process of autosomic differentiation, in wich, there is still complete maintenance, except for chromosome X of Akodon sp. n., of the sex chromosomes homologies. Member of the tribe Akodontini, Deltamys Thomas 1917 is a poorly studied and rarely collected taxon. Based on morphological or genetic characters, some authors considered Deltamys as a full genus while others regarded it as subgenus or synonym of Akodon. The single described species, Deltamys kempi presents a basic karyotype with 2n=37 in males and 2n=38 in females, FN=38, and with sex determination system of the type X1X1X2X2: X1X2Y. A cytogenetic character that distinguishes Deltamys from Akodon is the presence of a small metacentric pair marker in Akodon. A karyotype with 2n=40 and FN=40; XX: XY was related to the genus Akodon, but as in Deltamys kempi, this complement does not present the small metacentric pair. Phylogenetic analyses of maximum parsimony and maximum likelihood based on sequences of the mitochondrial gene cytochrome b evidenced the monophyly of a clade grouping specimens of Akodon sp. 2n=40 and monophyly of a clade containing specimens of Deltamys kempi. Besides that, the analyses showed that Akodon sp. is the sistergroup of Deltamys kempi, thus more related to this genus than to other species of Akodon and suggesting the placement of specimens with 2n=40 Deltamys. The genus Deltamys is, thus, more diverse than previously thought, grouping two lineages: Deltamys kempi, 2n=37-38 ; X1X1X2X2: X1X2Y and Deltamys sp. 2n=40, XX: XY, with a marked genetic divergence of 12,1% between them. A karyotype with 2n=50, FN=48 has been described for specimens of Thaptomys Thomas, 1916 collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita with 2n=52, FN=52 found in other Brazilian localities. It has been hence proposed that this new karyotype with 2n=50 could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosome rearrangements observed along with the geographic distance could represent a reproductive barrier between both forms. Molecular phylogenetic analyses using the cytochrome b sequences of eighteen karyotyped specimens of Thaptomys were performed attempting to establish the relationships among the individuals along the geographic distribution of the genus. Two major clades, Northeastern (A) with specimens with 2n=50 and Southeastern (B) with specimens with 2n=52, were reconstructed by maximum parsimony (MP) and maximum likelihood (ML). The intra-generic relationships recovered by phylogenetic analyses corroborated the distinct diploid numbers. The 2n=50 and 2n=52 karyotypes appeared as monophyletic separated by the basal cladogenesis of the genus, sister-group to each other. We present molecular phylogenetic and cytogenetic data on the monotypic fossorial rodent genus Blarinomys . Maximum parsimony and maximum likelihood based on cytochrome b gene sequences were performed for a sample of 11 individuals from nine localities of four states of Eastern Brazil. All topologies recovered two main lineages: a Northeastern (A) and a Southeastern clade. The Southeastern grouped two sister-clades B and C. Sequence divergence between individuals ranged from 4.7-8.0% between northeastern and southeastern clades, from 4.3-5.7% between clades B and C, from 6.1-8.0% between clades northeastern and B, and from 4.7-6.4% between clades northeastern and C. Within the clades, divergence varied from 0- 4.2% in the northeastern clade, was 0.7% in the clade B, and varied from 0.1- 1.3% in clade C. Variation among specimens from the same geographic regions ranged from 0-1.3%. Cytogenetic studies of five individuals revealed high karyotypic diversity with five distinct diploid numbers: 2n=52 (48A+2Bs,XY) from state of Bahia, and 2n=43 (37A+4Bs,XX), 2n=37 (34A+1B,XY), 2n=34 (32A,XX), and 2n=31 (27A+2Bs,XX) from state of São Paulo; and same number of autosomic arms (FN=50) excluding sex chromosomes and supernumeraries. Polymorphisms are due to Robertsonian rearrangements, in addition to the variation from none to four B chromosomes, which are heterogeneous regarding morphology, heterochromatin constitution and presence of interstitial telomeric signals (ITS). ITSs were also observed in the pericentromeric regions of some biarmed autosomic pairs of three specimens. Our results revealed a high unknown diversity for Blarinomys , showing two distinct lineages corresponding to regions of the Atlantic Rainforest, besides an extraordinary chromosomal polymorphism.

Die ALS ist eine fortschreitende neurodegenerative Erkrankung, deren Symptome durch den Untergang der Motoneuronen bedingt sind. Neueste zytogenetische Untersuchungen zeigen ein vermehrtes Auftreten konstitutioneller Chromosomenveränderunge bei ALS-Patienten. Dies lässt eine Verbindung zwischen dem Ausbruch der Erkrankung und der auffälligen Zytogenetik vermuten. Das Auftreten spontaner Chromosomenveränderungen als Zeichen einer chromosomalen Instabilität wurde in der vorliegenden Arbeit an ALS-Patienten untersucht. METHODE: Neben der Karyotypisierung, der Bestimmung der SCE-Rate und der Bruchrate nach Behandlung mit Bleomycin kam die Fluoreszenz in situ Hybridisierung zum Einsatz. Die Untersuchungen wurden an Patienten mit sporadischer ALS (45), an Kontrollpersonen (38) und Verwandten (9) durchgeführt. ERGEBNISSE: Die Karyotypisierung ergab bei den Patienten eine spontane Translokationsrate von 0,02 Translokationen/Zelle (t/Z), bei den Kontrollen 0,04 t/Z. Weitere numerische oder strukturelle Auffälligkeiten waren nicht signifikant verschieden. Es wurden keine konstitutionellen Chromosomenaberrationen gefunden. Die Häufigkeit der Schwesterchromatidaustausche (SCE-Rate) bewegte sich mit 7-8 SCE/Z in der Patientengruppe innerhalb der Normwerte. Durch die Zugabe von Bleomycin in die Zellkultur stieg die Zahl der Chromatidbrüche von 0,0 auf 0,8 Brüche/Z an. Dabei zeigten die untersuchten Gruppen ähnliche Progredienzen in den Bruchraten. Mit der Fluoreszenz in situ Hybridisierung werden quantitative Aussagen über spontane Translokationsraten gemacht. Sie betrug in der Kontroll-und Patientengruppe 0,03 bis 0,04 t/Z. DISKUSSION: Die vorliegenden Ergebnisse liefern keinen Anhalt für eine chromosomale Instabilität als Risikofaktor für die Entstehung der sporadischen ALS. Indizien für eine chromosomale Instabilität wie erhöhte Bruchraten und SCEs als auch vermehrtes Auftreten somatischer Aberrationen konnten bei den ALS-Patienten nicht nachgewiesen werden. Über mögliche Auffälligkeiten in den Motoneuronen lässt sich allerdings mit den Untersuchungen an Blutlymphozyten keine hinreichend sichere Aussage machen.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which is characterized by the degeneration of motor neurons. Recently, a high rate of constitutional structural chromosomal rearrangements has been reported in apparently sporadic ALS patients. It remains questionable whether or not these genomic rearrangements are caused by a chromosomal instability involved in the pathogenesis of the disease. Therefore, we performed different cytogenetic studies on chromosomal instability. METHOD: We performed chromosome analyses from patients (N=45), control subjects (N=38), and relatives (N=9) after culturing blood lymphocytes. Conventional chromosome analysis after GTG-banding, chromosomal breakage test after Bleomycin treatment, the rate of sister chromatid exchange (SCE), and whole chromosome painting were used for these analyses. RESULTS: Neither karyotyping nor whole chromosome painting revealed higher levels of structural or numerical aberrations in lymphocytes of patients with sALS. After karyotyping we found 0.02 t/cell in patients and 0.04 t/cell in controls. Whole chromosome painting revealed 0.04 t/cell in patients and 0.03 t/cell in controls. The chromosomal breaks increased likewise after Bleomycin treatment in the control group and the patient group as well. Cell cultures without Bleomycin did not show any breaks while the highest Bleomycin concentration induced up to 0.08 breaks/cell. The SCE rate in patients which corresponds to the chromatid repair activity did not rise to a higher level than in the control individuals. Both groups were in the normal range of 7 to 8 SCE/cell. DISCUSSION: The pathomechanism of neurodegeneration in ALS patients is still unknown. We tried to find a cytogenetic correlative being a risk factor for the development of ALS. However, so far there is no clue for chromosomal instability being involved in the neurodegenerative process.

L’alternance de périodes glaciaires et interglaciaires durant les 20 derniers Ma a mené à des changements environnementaux répétés au niveau du plateau continental antarctique. C’est dans ce contexte que les téléostéens de la famille des Nototheniidae se sont adaptés et diversifiés à travers plusieurs vagues de radiations (dont les Trematominae), dominant l’Ichtyofaune australe. Parmi les Nototheniidae, le groupe « Trematomus » (genres Cryothenia, Pagothenia, Trematomus et Indonotothenia) est celui où l’on observe la plus grande diversité chromosomique, avec des nombres diploïdes de chromosomes allant de 24 à 58, impliquant de nombreux réarrangements ayant accompagné les spéciations. Nous avons cherché à caractériser ces remaniements chromosomiques. Avec un caryotype ancestral inféré de 2n = 48, une conservation des unités chromosomiques entre espèces, et une constance des tailles de génome, l’hypothèse de réarrangements structuraux sans polyploïdisation préalable est la plus probable. Afin de reconstruire l’histoire évolutive de ces événements, nous avons recherché les homologies chromosomiques interspécifiques. Ceci nous a permis de reconstituer les remaniements (majoritairement des fusions) que nous avons repositionnés sur la phylogénie résolue des « Trematomus ». Contrairement à ce qui a été publié pour le genre Notothenia, nos résultats suggèrent des acquisitions multiples et indépendantes. Les éléments transposables (ETs) peuvent être impliqués dans les remaniements chromosomiques par le biais de recombinaisons ectopiques. Ils participent alors à la diversification des lignées au cours de l’évolution. En raison de leur régulation épigénétique, leur mobilisation massive peut être induite en cas de variations environnementales importantes. Nous nous sommes intéressés à trois super-familles d’ETs (DIRS, Gypsy and Copia) dans ces génomes. Les DIRS1 ont montré des patrons d’insertions en points chauds dans les régions centromériques et péricentromériques. Etant donné leur mode de transposition décrit et leur propension à s’insérer dans des copies préexistantes, nous proposons un rôle des éléments DIRS1 comme facilitateurs des fusions observées lors de la diversification des « Trematomus »
In the last 20 My, multiple glacial-interglacial cycles led to strong and repeated environmental changes on the Antarctic continental shelf. In this changing environment, nototheniid fishes diversified through several rounds of species radiation (one of which within Trematominae), and now constitute the dominant group in Antarctic teleosts. Among Nototheniidae, the group « Trematomus » (genera Cryothenia, Pagothenia, Trematomus and Indonotothenia) exhibits the highest chromosomal diversity, with diploid chromosome numbers ranging between 24 and 58, involving many rearrangements probably linked to speciation. We characterized the nature of these chromosomal repatternings. With an inferred ancestral state of 2n = 48 acrocentric chromosomes, a conserved number of chromosomal structural units, and a constancy of the genomes sizes we measured; the hypothesis of structural modifications is favored rather than a whole genome duplication associated to drastic reductions. In order to reconstruct an evolutionary scenario of such chromosomal rearrangements accompanying the trematomine diversification, we identified interspecific chromosomal homologies. This allowed us to reconstruct the rearrangements events (mostly centric and tandem fusions). We plotted them on a phylogeny we reconstructed based on our own ddRAD-seq data. Contrary to what was reported for the Notothenia, our results are in favor of independent acquisitions. Transposable elements (TEs) can lead to chromosomal rearrangements through ectopic recombination events, hinting at a role as drivers of specific-lineage diversification. Moreover, due to their epigenetic regulation, TEs can be mobilized when thermic changes occur. We focused on three retrotransposon superfamilies (DIRS, Gypsy and Copia) in nototheniid genomes. The DIRS1 showed unexpected accumulation patterns of insertion in the centromeric and pericentromeric regions. Given the mechanism of DIRS1 transposition and their tendency to sometimes insert on pre-existing copies (homing), we suggest a role of DIRS1 elements as facilitators of the fusions that occurred during the trematomine radiation

L’alternance de périodes glaciaires et interglaciaires durant les 20 derniers Ma a mené à des changements environnementaux répétés au niveau du plateau continental antarctique. C’est dans ce contexte que les téléostéens de la famille des Nototheniidae se sont adaptés et diversifiés à travers plusieurs vagues de radiations (dont les Trematominae), dominant l’Ichtyofaune australe. Parmi les Nototheniidae, le groupe « Trematomus » (genres Cryothenia, Pagothenia, Trematomus et Indonotothenia) est celui où l’on observe la plus grande diversité chromosomique, avec des nombres diploïdes de chromosomes allant de 24 à 58, impliquant de nombreux réarrangements ayant accompagné les spéciations. Nous avons cherché à caractériser ces remaniements chromosomiques. Avec un caryotype ancestral inféré de 2n = 48, une conservation des unités chromosomiques entre espèces, et une constance des tailles de génome, l’hypothèse de réarrangements structuraux sans polyploïdisation préalable est la plus probable. Afin de reconstruire l’histoire évolutive de ces événements, nous avons recherché les homologies chromosomiques interspécifiques. Ceci nous a permis de reconstituer les remaniements (majoritairement des fusions) que nous avons repositionnés sur la phylogénie résolue des « Trematomus ». Contrairement à ce qui a été publié pour le genre Notothenia, nos résultats suggèrent des acquisitions multiples et indépendantes. Les éléments transposables (ETs) peuvent être impliqués dans les remaniements chromosomiques par le biais de recombinaisons ectopiques. Ils participent alors à la diversification des lignées au cours de l’évolution. En raison de leur régulation épigénétique, leur mobilisation massive peut être induite en cas de variations environnementales importantes. Nous nous sommes intéressés à trois super-familles d’ETs (DIRS, Gypsy and Copia) dans ces génomes. Les DIRS1 ont montré des patrons d’insertions en points chauds dans les régions centromériques et péricentromériques. Etant donné leur mode de transposition décrit et leur propension à s’insérer dans des copies préexistantes, nous proposons un rôle des éléments DIRS1 comme facilitateurs des fusions observées lors de la diversification des « Trematomus »
In the last 20 My, multiple glacial-interglacial cycles led to strong and repeated environmental changes on the Antarctic continental shelf. In this changing environment, nototheniid fishes diversified through several rounds of species radiation (one of which within Trematominae), and now constitute the dominant group in Antarctic teleosts. Among Nototheniidae, the group « Trematomus » (genera Cryothenia, Pagothenia, Trematomus and Indonotothenia) exhibits the highest chromosomal diversity, with diploid chromosome numbers ranging between 24 and 58, involving many rearrangements probably linked to speciation. We characterized the nature of these chromosomal repatternings. With an inferred ancestral state of 2n = 48 acrocentric chromosomes, a conserved number of chromosomal structural units, and a constancy of the genomes sizes we measured; the hypothesis of structural modifications is favored rather than a whole genome duplication associated to drastic reductions. In order to reconstruct an evolutionary scenario of such chromosomal rearrangements accompanying the trematomine diversification, we identified interspecific chromosomal homologies. This allowed us to reconstruct the rearrangements events (mostly centric and tandem fusions). We plotted them on a phylogeny we reconstructed based on our own ddRAD-seq data. Contrary to what was reported for the Notothenia, our results are in favor of independent acquisitions. Transposable elements (TEs) can lead to chromosomal rearrangements through ectopic recombination events, hinting at a role as drivers of specific-lineage diversification. Moreover, due to their epigenetic regulation, TEs can be mobilized when thermic changes occur. We focused on three retrotransposon superfamilies (DIRS, Gypsy and Copia) in nototheniid genomes. The DIRS1 showed unexpected accumulation patterns of insertion in the centromeric and pericentromeric regions. Given the mechanism of DIRS1 transposition and their tendency to sometimes insert on pre-existing copies (homing), we suggest a role of DIRS1 elements as facilitators of the fusions that occurred during the trematomine radiation

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Pimelodidae is a family of fishes of South America, and although several taxonomic and molecular studies have been conducted, the phylogenetic relationships among the genera are not still fully understood. In order to provide data to assist in the understanding of the relationships within this family, cytogenetic studies were performed in two species of Iheringichthys and seven species of Pimelodus from three river systems. The specimens were collected in the Piquiri River, Upper Paraná River basin; in the Iguaçu River, downstream to the Iguaçu Falls in the Middle Paraná River basin; in the Iguaçu River, Lower Iguaçu River basin and in the Ijuí River, Upper Uruguay River basin. The analysis showed the presence of 2n=56 chromosomes for all species, corroborating the hypothesis of this basal diploid number for the family. The AgNORs, confirmed by 18S rDNA-FISH, were localized in the terminal position on long arm of a chromosome pair for all analyzed species, which has been reported for all species of Pimelodidae and may indicate a basal trait for the family. The heterochromatin distribution pattern found herein is similar to those described for other Pimelodidae, and allowed us to differentiate most of the species, becoming an important marker. The location of 5S rDNA sequences in Iheringichthys species allowed their differentiation, and can be used as a taxonomic marker. In Pimelodus species, it was verified a variation in the number and position of 5S rDNA sites. In P. britskii and P. maculates, sites of 5S rDNA and 18S were found in synteny, which may indicate a derived condition for these species, considering that they are the only for pimelodids species till now studied that have this feature. The results of this study provided data that contribute to the knowledge of the evolutionary history of the species for Pimelodidae; establishing phylogenetic relationships and assisting in the identification of these species.
Pimelodidae é uma família de peixes da região Neotropical, e embora vários estudos taxonômicos e moleculares tenham sido realizados, as relações filogenéticas entre seus gêneros ainda não são totalmente compreendidas. Com o intuito de fornecer dados para auxiliar no entendimento das relações dentro desta família, foram realizados estudos citogenéticos em duas espécies de Iheringichthys e em sete espécies de Pimelodus de três sistemas hidrográficos. Os exemplares foram coletados no rio Piquiri, Bacia do Alto rio Paraná; no rio Iguaçu, jusante às Cataratas do Iguaçu na Bacia do Médio rio Paraná; no rio Iguaçu, Bacia do Baixo rio Iguaçu e no rio Ijuí, Bacia do Alto rio Uruguai. As análises mostraram a presença de 2n=56 cromossomos em todas as espécies, reforçando a hipótese de número diplóide basal para a família. As AgRONs, confirmadas pela FISH-DNAr 18S, foram localizadas na região terminal do braço longo de um par de cromossomos em todas as espécies estudadas, sendo que posição terminal desta região é observada em todas as espécies de Pimelodidae e pode indicar um caracter basal da família. O padrão de distribuição de heterocromatina encontrado é semelhante ao observado em outros Pimelodidae, e permitiu diferenciar a maioria das espécies, sendo um importante marcador. A localização das sequências de DNAr 5S nas espécies de Iheringichthys permitiu diferenciá-las, podendo ser utilizado como marcador taxonômico. Em Pimelodus, variação quanto ao número e posição de sítios do DNAr 5S foi observada. Em P. britskii e P. maculatus os sítios de DNAr 5S e 18S foram localizados em sintenia, o que pode indicar uma condição derivada para estas espécies, visto que são as únicas espécies de Pimelodidae que apresentam esta característica até o momento. Os resultados do presente estudo fornecem dados que contribuem para o conhecimento da história evolutiva das espécies de Pimelodidae, permitem estabelecer relações filogenéticas e auxiliam na identificação destas espécies.

El ratolí domèstic de l’Europa occidental (Mus musculus domesticus Schwarz i Schwarz 1943) mostra una predisposició particularment elevada vers l’esdeveniment i fixació de translocacions robertsonianes; un tipus de reorganització cromosòmica que comporta la fusió de cromosomes a nivell centromèric i, per tant, una disminució del número diploide. Com a resultat, aquesta subespècie de ratolí domèstic presenta una elevada diversitat cariotípica. L’acumulació de translocacions robertsonianes té el potencial de dificultar el flux genètic. Conseqüentment, aquestes reordenacions cromosòmiques són considerades potencials factors desencadenants d’especiació cromosòmica. A més, la restricció d’intercanvi genètic entre poblacions finalment podria conduir a la seva divergència morfològica. La present tesi doctoral pretén aprofundir en el paper que les translocacions robertsonianes podrien tenir en la covariació entre trets morfològics, la diversificació fenotípica d’estructures esquelètiques, així com en el creixement d’aquestes estructures al llarg de l’ontogènia postnatal inicial, en poblacions naturals del ratolí domèstic de l’Europa occidental. L’àrea d’estudi inclou el sistema robertsonià Barcelona de Mus musculus domesticus, caracteritzat per set cromosomes metacèntrics diferents amb una distribució clinal i constituït per poblacions metacèntriques amb números diploides d’entre 27 i 39 cromosomes, així com poblacions circumdants d’espècimens amb el cariotip estàndard de 40 cromosomes. La present tesi específicament es centra en anàlisis comparatius de covariació morfològica i variació fenotípica de la mandíbula i la caixa craniana en espècimens adults, i del patró de creixement mandibular en sèries ontogenètiques d’espècimens juvenils d’entre la segona i la vuitena setmana de vida postnatal. A més, el creixement mandibular és avaluat en una sèrie ontogenètica de la soca endogàmica clàssica de ratolí domèstic (Mus musculus) C57BL/6J, amb la intenció de contextualitzar les potencials diferències en creixement mandibular entre ratolins salvatges amb el cariotip estàndard i amb translocacions robertsonianes. Mentre que l’estudi dels espècimens adults és realitzat mitjançant tècniques de morfometria geomètrica, l’estudi de les sèries ontogenètiques de ratolins juvenils implica un enfocament multi-mètode que inclou anàlisis histològics de seccions i superfícies òssies, i anàlisis de morfometria geomètrica. Els principals resultats de la present investigació són: i) l’al·lometria té un important efecte integrador sobre estructures morfològiques, la rellevància del qual augmenta a mesura que més translocacions robertsonianes s’acumulen; ii) l’estructura modular de la mandíbula en regió alveolar i branca ascendent és mantinguda independentment del nombre de translocacions robertsonianes; iii) la integració morfològica entre les regions cranials dorsal i ventral no és alterada per les translocacions robertsonianes; iv) l’organització de la caixa craniana en basicrani i cara en vista ventral, i en neurocrani i cara en vista dorsal, és normalment confirmada en tots els grups cromosòmics; v) la diferenciació cariotípica en espècimens adults, deguda a l’acumulació de metacèntrics, s’associa positivament amb la diversificació morfològica de les regions dorsal i ventral de la caixa craniana; vi) l’estructura de covariació morfològica de la mandíbula i la diferenciació fenotípica de la regió dorsal de la caixa craniana es correlacionen positivament amb el distanciament geogràfic entre grups cromosòmics; vii) els patrons de creixement mandibular difereixen entre espècimens salvatges amb el cariotip estàndard i aquells amb translocacions robertsonianes, encara que les diferències són més notables entre els ratolins salvatges estàndard i els ratolins de laboratori de la soca C57BL/6J; viii) les diferències entre grups respecte al creixement mandibular esdevenen més evidents després del deslletament; ix) la magnitud d’integració morfològica de la mandíbula disminueix al llarg del creixement postnatal en tots els grups. A la llum d’aquests resultats, les translocacions robertsonianes poden modificar la covariació morfològica de trets cranials i poden tenir certa influència sobre l’ontogènia de la mandíbula durant la vida postnatal inicial. En conseqüència, aquestes reordenacions cromosòmiques tindrien un paper important en l’evolució morfològica divergent.
El ratón doméstico de Europa occidental (Mus musculus domesticus Schwarz y Schwarz 1943) muestra una predisposición particularmente elevada hacia el acontecimiento y fijación de translocaciones robertsonianas; un tipo de reorganización cromosómica que comporta la fusión de cromosomas a nivel centrómerico y, por tanto, disminución del número diploide. Como resultado, esta subespecie presenta una elevada diversidad cariotípica. La acumulación de translocaciones robertsonianas tiene el potencial de dificultar el flujo genético. Consecuentemente, estas reordenaciones cromosómicas son consideradas potenciales factores desencadenantes de especiación cromosómica. Además, la restricción de intercambio genético entre poblaciones podría conducir a su divergencia morfológica. La presente tesis doctoral pretende profundizar en el papel que las translocaciones robertsonianas podrían tener en la covariación entre rasgos morfológicos, diversificación fenotípica de estructuras esqueléticas, y crecimiento de estas estructuras a lo largo de la ontogenia postnatal temprana, en poblaciones naturales del ratón doméstico de Europa occidental. El área de estudio incluye el sistema robertsoniano Barcelona de Mus musculus domesticus, caracterizado por siete cromosomas metacéntricos diferentes con una distribución clinal y constituido por poblaciones metacéntricas con números diploides de entre 27 y 39 cromosomas, y poblaciones circundantes de especímenes con el cariotipo estándar de 40 cromosomas. La presente tesis específicamente se centra en análisis comparativos de covariación morfológica y variación fenotípica de la mandíbula y caja craneana en especímenes adultos, y del patrón de crecimiento mandibular en series ontogenéticas de especímenes juveniles de entre la segunda y octava semana de vida posnatal. Además, el crecimiento mandibular es evaluado en una serie ontogenética de la cepa endogámica clásica de ratón doméstico (Mus musculus) C57BL/6J, con la intención de contextualizar las potenciales diferencias detectadas en crecimiento mandibular entre ratones con el cariotipo estándar y translocaciones robertsonianas. Mientras el estudio de los especímenes adultos es realizado mediante técnicas de morfometría geométrica, el estudio de las series ontogenéticas de ratones juveniles implica un enfoque multi-método que incluye análisis histológicos de secciones y superficies óseas, y análisis de morfometría geométrica. Los principales resultados de la presente investigación son: i) la alometría tiene un importante efecto integrador sobre estructuras morfológicas, la relevancia del cual aumenta a medida que más translocaciones robertsonianas se acumulan; ii) la estructura modular de la mandíbula en región alveolar y rama ascendente es mantenida independientemente del número de translocaciones robertsonianas; iii) la integración morfológica entre las regiones craneales dorsal y ventral no es alterada por las translocaciones robertsonianas; iv) la organización de la caja craneana en basicráneo y cara en vista ventral, y en neurocráneo y cara en vista dorsal, es normalmente confirmada en todos los grupos cromosómicos; v) la diferenciación cariotípica en especímenes adultos, debida a acumulación de metacéntricos, se asocia positivamente con la diversificación morfológica de las regiones dorsal y ventral de la caja craneana; vi) la estructura de covariación morfológica de la mandíbula y la diferenciación fenotípica de la región dorsal de la caja craneana se correlacionan positivamente con el distanciamiento geográfico entre grupos cromosómicos; vii) los patrones de crecimiento mandibular difieren entre especímenes salvajes con cariotipo estándar y con translocaciones robertsonianas, aunque las diferencias son más notables entre los ratones salvajes estándar y los ratones de la cepa C57BL/6J; viii) las diferencias entre grupos respecto al crecimiento mandibular devienen más evidentes después del destete; ix) la magnitud de integración morfológica de la mandíbula disminuye a lo largo del crecimiento posnatal en todos los grupos. A la luz de estos resultados, las translocaciones robertsonianas pueden modificar la covariación morfológica de rasgos craneales y pueden tener cierta influencia sobre la ontogenia de la mandíbula durante la vida posnatal inicial. En consecuencia, estas reordenaciones cromosómicas tendrían un papel importante en la evolución morfológica divergente.
The western European house mouse (Mus musculus domesticus Schwarz and Schwarz 1943) shows a particularly strong predisposition towards the occurrence and fixation of Robertsonian translocations; a type of chromosomal reorganization that entails centromeric fusion of chromosomes and, therefore, a decrease in diploid number. As a result, this house mouse subspecies displays great karyotypic diversity. The accumulation of Robertsonian translocations has the potential to hinder gene flow. Consequently, these chromosomal rearrangements are considered potential triggering factors of chromosomal speciation. Furthermore, restricted genetic exchange among populations could ultimately lead to their morphological divergence. The present PhD thesis intends to delve into the role that Robertsonian translocations may have on covariation among morphological traits, phenotypic diversification of skeletal structures, as well as on the growth of these structures over early postnatal ontogeny, in natural populations of the western European house mouse. The study area comprises the Barcelona Robertsonian system of Mus musculus domesticus, which is characterized by seven different metacentric chromosomes with a clinal distribution and includes metacentric populations with diploid numbers ranging between 27 and 39 chromosomes, as well as surrounding populations consisting of specimens with the standard karyotype of 40 chromosomes. The present research specifically focuses on comparative analyses of morphological covariation and phenotypic variation of the mandible and the cranium in adult specimens, as well as the pattern of mandible growth in ontogenetic series of juvenile specimens ranging from the second to the eighth week of postnatal life. Additionally, mandible growth is assessed in an ontogenetic series of the classical inbred strain of the house mouse (Mus musculus) C57BL/6J, with the aim of contextualizing the potential differences in mandible growth between wild mice with the standard karyotype and with Robertsonian translocations. While the study of the adult specimens is conducted by applying geometric morphometric techniques, the study of the ontogenetic series of juvenile mice involves a multi-method approach, including histological analyses of bone cross-sections and bone surface, as well as geometric morphometrics. The major results of the present research are the following: i) allometry has an important integrating effect over morphological structures, whose relevance increases as more Robertsonian translocations accumulate; ii) the modular structure of the mandible into the alveolar region and ascending ramus is maintained regardless of the number of Robertsonian translocations; iii) morphological integration between the dorsal and ventral cranial regions is not altered by Robertsonian translocations; iv) the modular organization of the cranium into the basicranium and face in ventral view, and into the neurocranium and face in dorsal view, is usually confirmed in all chromosomal groups; v) karyotypic differentiation in adult specimens, due to the accumulation of metacentrics, is positively associated with morphological diversification of the dorsal and ventral regions of the cranium; vi) the structure of morphological covariation of the mandible and the phenotypic differentiation of the dorsal region of the cranium positively correlate with geographic distancing among chromosomal groups; vii) mandible growth patterns differ between wild mouse specimens with the standard karyotype and those with Robertsonian translocations, although differences are more notable between the standard wild mice and laboratory mice of the C57BL/6J strain; viii) between-group dissimilarities in mandible growth become more evident after weaning; ix) strength of morphological integration of the mandible decreases over early postnatal ontogeny in all mouse groups. In light of these results, Robertsonian translocations can modify the morphological covariation of skull traits and can have certain influence over the ontogeny of the mandible during early postnatal life. Accordingly, these chromosomal rearrangements would play an important role in divergent morphological evolution.

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The Loricariidae family (Actinopterygii: Siluriformes) is morphologically diverse, has a number close to 900 valid species, distributed in seven subfamilies (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae and Hypostominae). However, cytogenetic studies in species of the family show evolutionary trends of karyotype diversification well defined for each of the subfamilies and the diploid number (2n) of 54 chromosomes is considered basal. Among the representatives of the subfamily Loricariinae, the variation of 2n is 36 to 74 chromosomes. Given these data, the Robertsonian rearrangements are the main mechanisms to explain the chromosome number variation in the subfamily. Rineloricaria is the most specious genus of Loricariinae, porting species with 2n = 36 to 2n = 70 chromosomes. However, little is known about what types of repetitive DNAs originate fission and fusion chromosome events. In this study, species of Rineloricaria from different rivers of the Paraná drainage were studied: Rineloricaria latirostris (Laranjinha river, Cinzas basin and Barra Grande river, Ivaí basin); Rineloricaria pentamaculata (Barra Grande and Juruba rivers, Tibagi basin); and, Rineloricaria stellata and Rineloricaria capitonia (Upper Uruguai river). The aim of this study was to characterize the karyotypes of populations/species of Rineloricaria and to check what types of repetitive DNAs may be related to Robertsonian events in the genus. In R. latirostris was detected 2n = 46 chromosomes for both populations, as well as for a triploid specimen from Laranjinha river. Rineloricaria pentamaculata had 2n = 56 chromosomes to populations from Barra Grande and Juruba rivers and a karyomorph in Barra Grande river with 2n = 54 chromosomes. Rineloricaria stellata had 2n = 54 chromosomes, while R. capitonia presented 2n = 64 chromosomes, both from the Uruguai river. The results using the chromosomal markers of 18S rDNA, 5S rDNA and TTAGGGn telomeric probe showed that these repetitive DNAs participated in end to end fusions of the st/a chromosomes in the karyotype diversification of R. latirostris. Vestiges of interstitial telomeric sites (ITS) were also detected in R. pentamaculata, karyomorph of 54 chromosomes from the Barra Grande river, suggesting chromosomal fusion to the diversification of this karyotype. The wide range of 2n between R. stellata and R. capitonia is compatible to the reproductive isolation of syntopic species and the diversification of R. capitonia can be explained by centric fusions. In addition to Robertsonian rearrangements, the pericentric inversions also assisted in the diversification of karyotypic formulas among the species/populations. In situ localization analysis using the transposable element Tc1-Mariner Like probe showed no evidence of the participation of transposon in chromosomal rearrangements and dispersion of multiple sites of 5S rDNA in Rineloricaria. Furthermore, analyzes of the Tc1-Mariner Like sequences showed intense molecular degeneration, especially in transposase domains. These results indicate the absence of activity of these sequences, which must be inert or serve to other genomic functions in the genus. Thus, this study discusses the telomeric instability, repetitive DNAs and the participation of rDNA gene families in karyotype diversification events in Rineloricaria.
A família Loricariidae (Actinopterygii: Siluriformes) é extremamente diversificada morfologicamente, conta com um número próximo a 900 espécies válidas, distribuídas em sete subfamílias (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae e Hypostominae). Não obstante, os estudos citogenéticos em representantes da família mostram tendências evolutivas da diversificação cariotípica bem definidas para cada uma das subfamílias, sendo considerado basal o número diploide (2n) de 54 cromossomos. Entre os representantes da subfamília Loricariinae a variação do 2n é de 36 a 74 cromossomos. Diante destes dados, os rearranjos Robertsonianos são os principais mecanismos para explicar a variação cromossômica numérica na subfamília. Rineloricaria é o gênero mais especioso de Loricariinae, com espécies apresentando 2n = 36 até 2n = 70 cromossomos. Contudo, pouco se sabe sobre quais os tipos de DNAs repetitivos originam os eventos de fissão e fusão cromossômica. Neste estudo, foram avaliadas espécies de Rineloricaria de diferentes rios do sistema hidrográfico do Paraná: Rineloricaria latirostris (rio Laranjinha, bacia do rio das Cinzas e rio Barra Grande, bacia do rio Ivaí); Rineloricaria pentamaculata (rio Barra Grande e rio Juruba, bacia do rio Tibagi); e, Rineloricaria stellata e Rineloricaria capitonia (Alto Rio Uruguai). O objetivo foi de caracterizar cariotipicamente as populações/espécies de Rineloricaria estudadas, além de verificar quais os tipos de DNAs repetitivos podem estar relacionados aos eventos Robertsonianos no gênero. Em R. latirostris foi detectado 2n = 46 cromossomos para ambas populações, além de um exemplar triploide para o rio Laranjinha. Rineloricaria pentamaculata apresentou 2n = 56 cromossomos para as populações dos rios Barra Grande e Juruba e um cariomorfo 2n = 54 cromossomos no rio Barra Grande. Rineloricaria stellata apresentou 2n = 54 cromossomos, enquanto R. capitonia detém 2n = 64 cromossomos, ambas do rio Uruguai. Os resultados com marcadores cromossômicos de rDNA 18S, rDNA 5S e sonda TTAGGGn evidenciaram que estes DNAs repetitivos participaram dos eventos de fusão terminal para terminal (end to end fusions) de cromossomos st/a na diversificação cariotípica de R. latirostris. Vestígios de sítios teloméricos intersticiais (ITS) foram evidenciados também em R. pentamaculata, cariomorfo de 54 cromossomos do rio Barra Grande, sugerindo fusão cromossômica para a diversificação deste cariótipo. A ampla variação de 2n entre R. stellata e R. capitonia é compatível para o isolamento reprodutivo das espécies sintópicas e pode ser explicado por fissões cêntricas na diversificação de R. capitonia. Além dos rearranjos Robertsonianos, as inversões pericêntricas também auxiliaram na diversificação de fórmulas cariotípicas entre as espécies/populações. A análise de localização in situ do elemento transponível Tc1-Mariner Like não mostrou evidências da participação deste transposon nos rearranjos cromossômicos e na dispersão dos sítios múltiplos de rDNA 5S em Rineloricaria. Ainda, as análises das sequências Tc1-Mariner Like evidenciaram intensa degeneração molecular, principalmente nos domínios transposase. Estes resultados indicam a ausência de atividade destas sequências, as quais devem ser inertes ou servir para outras funções genômicas no gênero. Desta forma, este estudo discute a instabilidade telomérica, DNAs repetitivos e a participação das famílias gênicas de rDNA nos eventos de diversificação cariotípica em Rineloricaria.

A displasia frontonasal (DFN) compreende quadros de aparência facial variável, sendo clinicamente caracterizada por dois ou mais dos seguintes sinais: hipertelorismo ocular com consequente alargamento da base nasal; fissura facial mediana afetando o nariz ou o nariz e lábio superior e, por vezes, o palato; fissura alar (uni ou bilateral); ponta nasal ausente; crânio anterior bífido oculto, e implantação em 'V' dos cabelos na fronte. A DFN pode ser vista como um defeito de desenvolvimento que pode ocorrer por si só ou como parte do quadro clínico de várias síndromes. A maioria dos casos de DFN é esporádica, e em raras circunstâncias foram observadas alterações cromossômicas em alguns indivíduos. Até o momento, quatro genes foram relacionados à patogênese molecular de algumas das síndromes com DFN, EFNB1, associado a uma forma de DFN ligada ao X e os genes ALX1, ALX3 e ALX4, todos associados a formas de DFN com herança autossômica recessiva. Embora esteja claro haver heterogeneidade etiológica, na maioria dos casos de DFN a causa não é conhecida, dificultando o adequado aconselhamento genético aos pacientes e seus familiares. Sendo assim, realizamos estudos com diferentes estratégias metodológicas buscando melhor compreender as possíveis causas genéticas da DFN. Ao todo foram analisados 10 pacientes: um caso familial de DFN leve com herança aparentemente autossômica dominante, um caso clinicamente sugestivo de mutação em ALX1, e oito casos de DFN associada a atraso de desenvolvimento com ou sem outras anomalias, dos quais um apresentava um rearranjo de novo aparentemente balanceado entre os cromossomos 4 e 12. Optamos por realizar sequenciamento dos genes previamente relacionados a fenótipos com DFN em todos os casos; para aqueles em que não foram detectadas mutações patogênicas, realizamos análise de variações de número de cópias (CNV) por microarray de polimorfismos de base única e, para o paciente com rearranjo cromossômico, realizamos o mapeamento do ponto de quebra por hibridação in situ fluorescente. Constatamos uma mutação em heterozigose no gene ALX4 co-segregando com o fenótipo do caso familial, sendo esta a primeira descrição de alteração em tal gene causando uma forma de DFN com herança dominante, e sugerimos pela primeira vez um mecanismo de dominância negativa. No caso sugestivo de mutação em ALX1, o diagnóstico foi confirmado através da identificação de uma mutação em homozigose neste gene do paciente; este caso consiste no 3o da literatura mundial e evidencia pela primeira vez que mutações em ALX1 não necessariamente levam a atraso de desenvolvimento ou deficiência intelectual. Os estudos citogenéticos e moleculares dos pontos de quebra do paciente com rearranjo cromossômico sugeriram os genes ARAP2 e CAND1 como possíveis responsáveis por seu quadro clínico, enquanto o estudo de CNVs nos indivíduos com DFN associada a atraso de desenvolvimento apontou os genes DNAJB12 e ENOX2 como possíveis candidatos para explicar o fenótipo de dois dos pacientes. É preciso que novos estudos sejam realizados a fim de melhor compreender o significado de tais achados e a real contribuição de cada gene para o desenvolvimento craniofacial humano e para a etiologia da DFN. Para os casos em que não foram identificadas alterações conclusivas no presente estudo, embora causas ambientais não possam ser descartadas, é preciso que seja investigada também a existência de fatores genéticos e epigenéticos não detectáveis pelas metodologias utilizadas, bem como a hipótese de mosaicismo somático. Nossos resultados, além de corroborarem o envolvimento dos genes ALX1 e ALX4 em fenótipos com DFN, sugerem também novos genes candidatos: ARAP2, CAND1, DNAJB12 e ENOX2
Frontonasal dysplasia (FND) is a rare group of disorders that comprises cases with a variety of facial appearances, and is clinically characterized by two or more of the following signs: ocular hypertelorism with consequent broadening of the nasal root; median facial cleft affecting the nose and/or upper lip and palate; clefting of the alae nasi (uni or bilateral); lack of formation of the nasal tip; anterior cranium bifidum occultum; and a V-shaped frontal hairline. FND is a developmental defect that can occur alone or as part of several syndromes. Most cases of FND are sporadic, and in rare circumstances chromosomal alterations were observed in affected individuals. To date, four genes have been related to the molecular pathogenesis of some syndromes with DFN, one (EFNB1) is associated with an X-linked form while the 3 others (ALX1, ALX3 and ALX4) are associated with autosomal recessive forms. Although it is clear that FND is etiologic heterogeneous, the causative mechanism is unknown in most cases which makes it hard to give proper genetic counseling to patients and their families. In order to get new insights into the genetic mechanisms leading to FND, we performed studies with different methodologies. Altogether, 10 patients were analyzed: a familial case of a mild form of FND with an apparently autosomal dominant inheritance pattern, a case clinically suggestive of mutation in ALX1, and eight cases of FND associated with developmental delay with or without other anomalies, one of which with an apparently balanced de novo rearrangement between chromosomes 4 and 12. We chose to sequence the genes previously associated with FND phenotypes in all cases; for those in which pathogenic mutations were not detected, we conducted an analysis of copy number variations (CNV) by single nucleotide polymorphisms microarrays; for the patient with chromosomal rearrangement, we also mapped the breakpoints by using fluorescence in situ hybridization. We found a heterozygous mutation in ALX4 co-segregating with the phenotype of the familial case; this is the first description of mutation in this gene causing a form of FND with dominant inheritance pattern, and we suggested for the first time a dominant negative mechanism. In the case suggestive of mutation in ALX1, the diagnosis was confirmed by the identification of a homozygous mutation in this gene; this is the third case of the literature and shows for the first time that mutations in ALX1 are not necessarily related to developmental delay or intellectual disability. Breakpoints cytogenetic and molecular studies done with the patient with chromosomal rearrangement suggested ARAP2 and CAND1 genes as causative candidates for his condition, while the study of CNVs in individuals with FND associated with developmental delay pointed DNAJB12 and ENOX2 genes as possible candidates to explain the phenotypes of two of the patients. Further studies are necessary to better understand the significance of such findings and the actual contribution of each of these genes to human craniofacial development and the etiology of FND. Although environmental causes cannot be ruled out, it should also be investigated the existence of genetic and epigenetic factors as well as the possibility of somatic mosaicism, among the cases negative for the molecular approaches used in our study. Our results corroborate the involvement of ALX1 and ALX4 in FND phenotypes, and suggest new candidate genes: ARAP2, CAND1, DNAJB12 and ENOX2.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os cromossomos sexuais se originam independentemente de um par de homólogos autossômicos e em várias linhagens apresentam características comuns, tais como acúmulo de vários tipos de DNA repetitivo, restrição da recombinação e perda ou ganho de genes devido á diferenciação morfológica e genética entre os cromossomos sexuais X e Y ou Z e W. Estas características representam um exemplo fascinante de convergência evolutiva. Em Orthoptera, o sistema cromossômico sexual comumente encontrado na maioria das espécies estudadas é do tipo X0♂/XX♀. Entretanto, sistemas cromossômicos sexuais derivados dos tipos neo-XY♂/XX♀ e neo- X1X2Y♂/X1X1X2X2♀ são também observados, surgindo repetidamente por fusões cêntricas e em tandem, inversões e dissociações envolvendo cromossomos sexuais ancestrais e autossomos. O presente trabalho teve três objetivos. Primeiro, entender o possível papel dos DNAs repetitivos na estrutura/diversificação dos cromossomos sexuais simples e derivados, a partir do isolamento e mapeamento físico de sequências, tais como, famílias multigênicas, DNA satélite (DNAsat) e microssatélites, nas espécies Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis. Segundo, testar e comparar transcrição diferencial de DNAsat entre diferentes tecidos, sexos e espécies a partir de transcriptomas de Gryllus assimilis, G. bimaculatus, G. firmus e G. rubens, com o objetivo de entender os possíveis papéis funcionais destas sequências na regulação gênica, modulação da cromatina e como componentes funcionais de importantes estruturas como telômeros, centrômeros e cromossomos sexuais. Terceiro, a partir de transcriptomas de espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus) prospectar genes codificadores de proteínas relacionados com a determinação sexual, envolvidos com o fitness reprodutivo e genes enviesados do sexo, responsáveis pelas diferenças fenotípicas entre machos e fêmeas, e tentar elucidar de uma maneira comparativa os fatores evolutivos atuando nestes loci. Origem de novo de cromossomos sexuais mediante rearranjos cromossômicos, assim como acúmulo de DNA repetitivo que levaram a diferenciação entre cromossomos sexuais são relatados em C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). Estas características observadas em grilos representam outro caso notável de convergência evolutiva devido os cromossomos sexuais não relacionados compartilharem muitas propriedades entre táxons distantes. Acúmulo surpreendente de loci de DNAsat foi encontrado no neo-Y altamente diferenciado de E. surinamensis, incluindo 39 DNAsat representados em excesso neste cromossomo, que é a maior diversidade de DNAsat até agora relatada para cromossomos sexuais. Foi documentado que, particularmente os DNAsat, contribuíram grandemente para o aumento de tamanho genômico entre G. assimilis e E. surinamensis. Um achado interessante foi a identificação de DNAsat conservados entre espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus), mas transcritos diferencialmente. Os dados relativos à presença de DNAsat no genoma de G. assimilis foram discutidos em um contexto evolutivo, com dados transcricionais permitindo comparações entre os sexos e entre os tecidos quando possível. Foram discutidas hipóteses para a conservação e transcrição de DNAsat em Gryllus, que podem resultar do seu papel na diferenciação sexual no nível da cromatina, na formação da heterocromatina e na função centromérica. Outra descoberta foi a identificação de genes determinantes do sexo e outros genes relacionados ao fitness reprodutivo, como a biossíntese de hormônios de insetos e ritmo circadiano entre espécies de Gryllus. Os efetores e os alvos downstream das vias de determinação do sexo foram previamente identificados em outros insetos, mas nunca em Orthoptera. Usando G. assimilis como modelo para estudar genes enviesados do sexo foi possível identificar um conjunto de genes altamente expressos que podem explicar diferenças fenotípicas entre os sexos. Estimou-se que os genes codificadores de proteínas relacionadas com a diferenciação sexual e com o fitness reprodutivo evoluem mais rapidamente do que os genes não reprodutivos (genes housekeeping) como resultado de uma forte seleção positiva nos primeiros. Além disso, foi encontrado que as espécies estudadas apresentam níveis excepcionalmente elevados de duplicações gênicas. As descobertas sugerem que as duplicações gênicas podem desempenhar um papel na expressão de genes enviesados do sexo no grilo de campo G. assimilis, uma espécie que no futuro provavelmente irá fornecer informações sobre genômica funcional e epigenética da determinação do sexo.
Sex chromosomes have arisen independently from an ordinary autosomal pair and in several lineages they present common characteristics, such as accumulation of distinct classes of repetitive DNAs, restriction of the recombination and loss or gain of genes due to the morphological and genetic differentiation between the sexual chromosomes X and Y or Z and W. These characteristics represent a fascinating example of evolutionary convergence. In Orthoptera, the X0♂/XX♀ sex-determining system is considered modal but eventually, diverse sex chromosome systems evolved several times, such as neo-XY♂/XX♀, X1X20♂/X1X1X2X2♀ and even neo- X1X2Y♂/X1X1X2X2♀. It was found that particularly centric fusions (i.e., Robertsonian translocations) and tandem fusions with autosomes, dissociations and inversions contributed to the formation of neo-sex chromosomes in Orthoptera. The present work had three objectives. First, get insights of the role of repetitive DNAs in the structure/diversification of simple and derivative sex-chromosomes by isolation and physical mapping of repetitive DNA sequences, such as multigene families, satellite DNA (satDNA) and microsatellites using Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis, as models. Second, looking at differential satDNA transcription between different tissues, sexes, and species from transcriptomes of Gryllus assimilis, G. bimaculatus, G. firmus and G. rubens, I tried to understand the possible functional roles of these sequences in gene regulation, chromatin modulation and as functional components of important structures such as telomeres, centromeres and sex chromosomes. Third, using transcriptomes from cricket species (Gryllus assimilis, G. bimaculatus and G. firmus), I searched for genes encoding proteins related to sexual determination, reproductive fitness and sex-biased genes which are responsible for the phenotypic differences between males and females. I also tried to elucidate in a comparative way the evolutionary factors acting at these loci. De novo origin of sex chromosomes by chromosomal rearrangements, as well as repetitive DNA accumulation that led to the differentiation between sex chromosomes are reported for C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). These features observed in crickets represent another remarkable case of evolutionary convergence because unrelated sex chromosomes share many common properties among distant taxa. Especially astonishing accumulation of satDNAs loci was found in the highly differentiated neo-Y, including 39 satDNAs over-represented in this chromosome, which is the greatest satDNAs diversity yet reported for sex chromosomes. It has been documented that, particularly the satDNA, contributed greatly to the increase in genomic size between G. assimilis and E. surinamensis. An interesting finding was the identification of satDNA conserved among species of crickets (Gryllus assimilis, G. bimaculatus and G. firmus), but differentially transcribed. The data regarding satDNA presence in G. assimilis genome was discussed in an evolutionary context, with transcriptional data enabling comparisons between sexes and across tissues when possible. I discussed hypotheses for the conservation and transcription of satDNAs in Gryllus, which might result from their role in sexual differentiation at the chromatin level, heterochromatin formation, and centromeric function. Another finding was the identification of sex-determining genes and other genes related to reproductive fitness, such as biosynthesis of insect hormones and circadian rhythm among Gryllus species. The effectors as well as downstream targets of sex-determination pathways have been previously identified in other insects but never in Orthoptera. Using G. assimilis to study sex-biased genes I identified a set of highly expressed genes that might account for phenotypic differences between sexes. Furthermore, I estimated that proteinencoding reproductive genes evolve faster than non-reproductive genes as result of strong positive selection at those loci. It was documented that the species studied harbor exceptionally high levels of gene duplications. The findings suggest that gene duplications may play a role in sex-biased genes expression in the field cricket G. assimilis, a species likely to yield insights into the functional genomics and epigenetics of sex determination.
FAPESP: 2014/02038-8

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Karyological analysis of Geophagus brasiliensis (Quoy and Gaimard, 1824) was performed on 81 specimens from six localities, three geologically recent lakes and three stream collection sites. Techniques included conventional staining with Giemsa, NOR banding, C-banding and in situ hybridization (FISH) with 5S rDNA and 18s rDNA probes. The diploid number was 2n = 48 chromosomes, and fundamental number varied between 50-52. We observed four different karyotypes, based on heteromorphisms presented by the first chromosome pair and were not related to sex, NOR location or collection site. This heteromorphism is related to differences in the ratio arms, which led to variations in the karyotypic formulae (3sm +18 st +26 t; 2sm +20 st +26 t; 4sm +18 st +26 t). This heteromorphism may be related to chromosome rearrangements, such as pericentromeric inversions, deletions, and unequal crossing-over, which together with other processes, such as Muller s ratchet, background selection and dosage compensation caused size alterations in some chromosomes. The number of NORs varied within and between specimens, however most individuals had NOR bands in more than one chromosome pair, a distinctive feature of the Doce River populations. The 18S rDNA probe confirmed the presence of NORs in more than two chromosomes. The location of the 5S rDNA probe remained conserved in all samples, marking a pair of chromosomes. The heterochromatin blocks occurred predominantly in the centromeric / pericentromeric chromosomes, and this a characteristic of the Cichlidae family. Heterochromatin blocks in interstitial regions were observed in two pairs of chromosomes. The presence of two subtelocentric chromosomes, with fully heterochromatic small arms is a diagnostic feature of the populations of the Doce River Basin. We conclude that the populations of G. brasiliensis of the Rio Doce Basin present unique characteristics, as evidenced by four configurations of the first pair of chromosomes and different results obtained by banding techniques. Results suggest differential viability of the chromosomal variations described in this study.
A análise cariotípica de Geophagus brasiliensis (Quoy & Gaimard, 1824) foi realizada em 81 espécimes de seis localidades da bacia do rio Doce. Foram usadas as técnicas de coloração convencional com Giemsa, bandeamento NORs, bandeamento C e hibridização in situ (FISH) com sondas rDNA 18s e rDNA 5S. O número diplóide foi de 2n=48 cromossomos, com variação do número fundamental entre 50-52. Foram observados quatro diferentes cariótipos, com base em heteromorfismos apresentados pelo primeiro par cromossômico e não foram associados ao sexo, à NOR nem ao local de coleta. Esse heteromorfismo está relacionado com diferenças de razão de braços, o que acarretou variações nas fórmulas cariotípicas encontradas (3sm+18st+26t; 2sm+20st+26t; 4sm+18st+26t). Este heteromorfismo pode estar relacionado com rearranjos cromossômicos, como inversões pericentroméricas, deleções, e crossing-over desiguais, as quais, associadas a outros processos, como catraca de Muller, seleção de fundo e compensação de dosagem, determinaram a alteração do tamanho de alguns cromossomos. O número de NORs observadas teve variações intra e inter-individuais, contudo a maioria dos indivíduos apresentou marcações em mais de um par cromossômico, uma característica única das populações de G. brasiliensis da bacia do rio Doce. A sonda de rDNA 18S confirmou a presença de NORs em mais de dois cromossomos. A localização da sonda de rDNA 5S manteve-se conservada em todas as amostras, marcando par de cromossomos telocêntricos. Os blocos de heterocromatina ocorreram predominantemente nas regiões centroméricas/pericentromérica, sendo essa uma característica da família Cichlidae. Blocos de heterocromatina em regiões intersticiais foram observados em dois pares de cromossomos. A presença de dois subtelocêntricos apresentando seus braços menores totalmente heterocromáticos é uma característica diagnóstica das populações da bacia do rio Doce. Conclui-se que as populações de G. brasiliensis da bacia do rio Doce apresentam A análise cariotípica de Geophagus brasiliensis (Quoy & Gaimard, 1824) foi realizada em 81 espécimes de seis localidades da bacia do rio Doce. Foram usadas as técnicas de coloração convencional com Giemsa, bandeamento NORs, bandeamento C e hibridização in situ (FISH) com sondas rDNA 18s e rDNA 5S. O número diplóide foi de 2n=48 cromossomos, com variação do número fundamental entre 50-52. Foram observados quatro diferentes cariótipos, com base em heteromorfismos apresentados pelo primeiro par cromossômico e não foram associados ao sexo, à NOR nem ao local de coleta. Esse heteromorfismo está relacionado com diferenças de razão de braços, o que acarretou variações nas fórmulas cariotípicas encontradas (3sm+18st+26t; 2sm+20st+26t; 4sm+18st+26t). Este heteromorfismo pode estar relacionado com rearranjos cromossômicos, como inversões pericentroméricas, deleções, e crossing-over desiguais, as quais, associadas a outros processos, como catraca de Muller, seleção de fundo e compensação de dosagem, determinaram a alteração do tamanho de alguns cromossomos. O número de NORs observadas teve variações intra e inter-individuais, contudo a maioria dos indivíduos apresentou marcações em mais de um par cromossômico, uma característica única das populações de G. brasiliensis da bacia do rio Doce. A sonda de rDNA 18S confirmou a presença de NORs em mais de dois cromossomos. A localização da sonda de rDNA 5S manteve-se conservada em todas as amostras, marcando par de cromossomos telocêntricos. Os blocos de heterocromatina ocorreram predominantemente nas regiões centroméricas / pericentromérica, sendo essa uma característica da família Cichlidae. Blocos de heterocromatina em regiões intersticiais foram observados em dois pares de cromossomos. A presença de dois subtelocêntricos apresentando seus braços menores totalmente heterocromáticos é uma característica diagnóstica das populações da bacia do rio Doce. Conclui-se que as populações de G. brasiliensis da bacia do rio Doce apresentam características únicas, associadas à existência de quatro configurações do primeiro par cromossômico e aos diferentes resultados obtidas nas técnicas de bandeamento realizadas. Os resultados também sugerem uma viabilidade diferenciada das variáveis cromossômicas descritas nesse trabalho.

La déficience intellectuelle (DI) est définie par un QI < 70. La DI, répartie en formes non syndromiques et en formes syndromiques, est observée dans 3 % de la population. Des anomalies chromosomiques sont identifiées dans 15 % des DI syndromiques. Les translocations chromosomiques réciproques (TR) apparemment équilibrées sont observées chez 1 individu sur 1000 et seul 6 % des patients avec une TR de novo apparemment équilibrée ont une DI. Plusieurs mécanismes chromosomiques peuvent expliquer la DI syndromique associée à une TR : (i) un microremaniement déséquilibré identifié par l'utilisation de techniques plus résolutives, (ii) la formation d'un gène de fusion, (iii) un effetde position, (iv) la modification d’une région soumise à une empreinte parentale, (v) une interruption d'un gène au niveau d'un ou des deux points de cassure, (vi) une mutation génique sans rapport avec la TR, (vii) ou encore une cause acquise ou multifactorielle. Nous rapportons l'étude de 12 patients avec DI et porteurs d'une TR de novo apparemment équilibrée. L'analyse systématique par puces à ADN de ces individus a été réalisée avec une résolution de 25 kb. Un déséquilibre infracytogénétique au niveau des points de cassure ou ailleurs dans le génome a été observé chez 3/12 patients. Chez les 9 patients sans anomalies sur puces à ADN, nous avons étudié les points de cassure des remaniements de novo apparemment équilibrés. En dehors de la technique de marche sur le chromosome par FISH, deux autres approches ont été mises en oeuvre : (i) l'Array-Painting qui correspond à l'hybridation sur puces à ADN de chacun des dérivés chromosomiques préalablement séparés par Cytométrie en Flux, (ii) et le séquençage haut débit (WGS - Whole Genome Sequencing). Grâce à l'Array-Painting, nous avons identifié (i) chez 2 patients, des interruptions de gènes pouvant expliquer leur phénotype, à savoir les gènes : KIF1A, AUTS2 et EPHA6 ; (ii) et chez 1 patiente, un point de cassure entraînant une dérégulation de la transcription du gène MEF2C. L'étude par WGS a permis (i) chez 1 patiente, de diagnostiquer un déséquilibre plus complexe que celui observé par puce à ADN ; (ii) chez 2 patients, de mettre en évidence unchromothripsis, qui pourrait avoir un impact dans les pathologies constitutionnelles par interruption de gènes et/ou par effet de position ; (iii) et chez 2 autres patients, de caractériser précisément les points de cassure. Ainsi, grâce aux résultats obtenus par ces différentes techniques, plusieurs mécanismes physiopathologiques responsables de DI sont mis en évidence permettant un conseil génétique adéquat. Cependant, aucun mécanisme chromosomique commun ne peut être identifié hormis le chromothripsis observé chez patients. Finalement, ce travail nous permet principalement de comparer les techniques mises en oeuvre qui se sont avérées complémentaires. En conclusion, nous proposons une démarche diagnostique pour explorer un remaniement chromosomique apparemment équilibré chez des patients à phénotype anormal
Intellectual disability (ID) is defined by an IQ <70. ID, observed in 3% of the population, and displays heterogeneous origins, including acquired etiology (toxicologic, pathologic, traumatic) or genetic disorders with non-syndromic and syndromic forms. Numerical or structural chromosomal abnormalities are observed in 15% of patients with ID. Reciprocal balanced chromosomal translocations (RT) are observed in one individual in 1000. However, only 6% of patients carrying a de novo apparently balanced RT present ID. The relation between these balanced rearrangements and ID could be explained by different mechanisms namely (i) subtle rearrangement, (ii) gene fusion, (iii) position effect, (iv) disturbance of parental imprinting, (v) gene disruption at the breakpoints, (vi) mutation in gene unrelated to the translocation, (vii) or acquired or multifactorial cause. We report a chromosomal study of 12 patients with DI and carrying a de novo apparently balanced reciprocal translocation. A systematic analysis by microarrays was performed in all individuals (using a resolution of 25 kb). For three patients, a microdeletion was observed at the breakpoints or elsewhere in the genome. For the 9 remaining cases, we hypothesize that the phenotype is due to a disruption of gene(s) located at the breakpoint(s). In this context, we studied the breakpoints of the apparently balanced de novo rearrangements in these patients. Outside FISH walking, two approaches have been implemented namely Array-Painting, which combines flow chromosome sorting in an attempt to isolate derivative chromosomes from each other and DNA microarrays as well as Whole Genome Sequencing (WGS). Using Array-Painting, we identified (i) in 2 patients, a gene disruptions: in the KIF1A, AUTS2 and EphA6 genes; (ii) and in 1 patient, a breakpoint resulting in deregulation of transcription of the gene MEF2C. The WGS technology has permitted (i) in 1 patient, to diagnose more complex imbalance than that observed by micro-array; (ii) in 2 patients, to show a chromothripsis, (iii) and 2 other patients, to characterize precisely breakpoints. In conclusion, taking together, these results highlight different physiopathological mechanisms responsible for DI allowing adequate genetic counseling. However, no common chromosomal mechanism can be identified except for chromothripsis observed in 2 patients. In addition, this work allows us especially to compare the used techniques which seem to be complementary. Finally, we propose a pipeline to elucidate the etiology of the abnormal phenotype in patients carrying an apparently balanced rearrangement

Rearrangements involving the mixed lineage leukemia gene (MLL) are found in majority of human infant leukemias (>60% of ALLs, 35% of AMLs) and are associated with dismal prognosis of these patients. They are also found in a small percentage of childhood and adult leukemias (10% cases), making MLL-rearranged leukemias ideal for this study. We performed induction of the leukemic MLL-ENL chromosomal translocation in hematopoietic cells at different developmental stages i.e., fetal liver (FL, E12.5) and bone marrow (BM, P60) to develop disease models that can recapitulate human infant and adult leukemias respectively. After evaluating several models to induce MLL-ENL recombination, we have reproducibly obtained leukemia in adult animals with the interferon inducible Mx1-Cre line. The embryonic model of MLL-ENL leukemia was also developed with partial success and it is more aggressive compared to the adult leukemia. In conclusion, we have developed a novel embryonic leukemia model to study infant leukemia ontogeny.
En la mayoría de las leucemias humanas del lactante (> 60% de los casos de ALL, 35% de los casos de AML), se encuentran reordenamientos relacionados con el gen de la leucemia de linaje mixto (mixed lineage leukemia, MLL), que se asocian con un mal pronóstico de estos pacientes. También se encuentran en un pequeño porcentaje de casos de leucemias infantiles y de adultos (10% de casos), por lo que las leucemias con reordenamientos en MLL resultan ideales para este estudio. Hemos realizado la inducción de la translocación cromosómica leucémica MLL-ENL en las células hematopoyéticas en diferentes etapas de desarrollo, en el hígado fetal (fetal liver, FL E12.5) y en la médula ósea (bone marrow, BM P60), para desarrollar modelos de enfermedades que puedan recapitular las leucemias humanas del lactante y adultas, respectivamente. Después de evaluar varios modelos para inducir la recombinación de MLL-ENL, hemos generado de forma reproducible, leucemia en animales adultos con la línea de Mx1-Cre inducible por interferón. El modelo embrionario de leucemia inducida por MLL-ENL también se desarrolló con éxito parcial y es más agresivo en comparación con la leucemia en adultos. En conclusión, hemos desarrollado un nuevo modelo de leucemia embrionaria para estudiar la ontogenia de la leucemia del lactante.

This Thesis describes experiments carried out to identify the chromosome abnormalities, and complete karyotypes of a panel of breast cell lines by 24-colour fluorescence in situ hybridisation (FISH). 24-colour FISH allows all 24 human chromosomes to be identified simultaneously. Using 24 different combinations of up to five fluorochromes each chromosome is hybridised with a unique probe mixture. Computer software identifies wavelength differences between the different probe mixtures and colours each chromosome a different colour. In this way a translocation composed of three chromosomes will hybridise in three different colours, and the component chromosomes can be easily identified. The karyotypes of the breast cell lines were compared to each other, to previous G-banding karyotypes, and to fresh breast tumours. The cell lines fall into two main categories, those with near-diploid modal chromosome number and few rearrangements; and those with near-triploid modal chromosome number and many rearrangements. The 24-colour karyotypes can elucidate complex chromosome rearrangements intractable to G-banding analysis. This was clearly illustrated in several cell lines with complex co-amplification of two chromosomes, for example co-amplification of 8 and 11 in MDA-MB-134; and 8 and 17 in ZR-75-30. Also 24-colour FISH allows the elucidation of highly complex marker chromosomes in, for example, SK-BR-3, which involve six or more different chromosomes. The rearrangements identified in the breast tumour cell lines compared well with those identified in fresh breast tumours and in breast tumour cell lines by conventional cytogenetics and 24-colour FISH. Translocations of chromosomes 8;11 and 1;20 are equally as frequent in breast cell lines as in the primary tumours, and 8, 16 and 11 are the chromosomes most frequently involved in translocations in both the cell lines and the primary tumours.

In this study the question of nonrandomness in the distribution of human constitutional rearrangements was evaluated. The distribution of breakpoints were analysed in three groups of reciprocal translocations and three groups of inversions, subdivided according to method of ascertainment of cases for study. In addition, one data set of structural aberrations obtained from sperm chromosomes was also analysed. The method of statistical analysis, based on the binomial distribution, was developed specifically to allow testing distributions in chromosome segments as small as chromosome bands. The distribution of breakpoints was analysed in all data sets using this method, in addition to testing for overall nonrandomness using goodness of fit statistics. Nonrandomness in breakpoint distributions was found in reciprocal translocations (rcp) and inversions ascertained through abnormalities and through incidental events. However, random distribution was observed in incidentally ascertained de novo rearrangements as well as in sperm chromosome aberrations. The nonrandomness in the distribution of rcp breakpoints can be largely attributed to a bias in ascertainment of cases based on the phenotypic manifestations of chromosomal imbalance resulting from a rearrangement. A dependence of the probability of producing specific types of balanced or unbalanced progeny on the position of breakpoints is a likely explanation for the nonrandomness produced in breakpoint distributions. However, some bands including, 5q35, 7p22, 9p22, 13ql4, and 17q25, were observed in different ascertainment groups, excluding selection bias as a likely explanation for this observation. These bands may represent true sites of nonrandom rearrangement due to some factor associated with an underlying DNA sequence or structural characteristic of chromatin that predisposes to rearrangement at specific sites. The nonrandomness observed in the distribution of inversion breakpoints is most likely the product of a founder effect. Many identical inversions in apparently unrelated individuals have been found suggesting that a few ancestral mutations have become widespread in the population. A large data set of incidentally ascertained de novo inversions is required to distinguish between sites of frequent breakage and nonrandomness produced by the ascertainment of related cases. All evidence considered together, indisputable predisposition to rearrangement at specific sites was not found in this study. Furthermore, an overall random association of constitutional rearrangement breakpoints in bands with known oncogenes and fragile sites was observed. However, the possibility of oncogenes and fragile sites as factors involved in constitutional rearrangements in a few isolated cases cannot be excluded. Nonrandomness was found when distribution of breakpoints in light and dark G bands was compared. An excess of breakpoints in some light G bands was observed even after a conservative correction for a possible pattern recognition bias which may lead to the overascertainment of breakpoints in light G bands.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

In this thesis, chromosome I rearrangements were used to study the organization of essential genes and regions important for chromosome behaviour in the nematode Caenorhabditis elegans. To facilitate the genetic mapping of mutations in essential genes, rearrangements were isolated using a procedure designed to recover derivative chromosome I duplications shortened by gamma radiation from existing duplications. Sixty-two duplications were isolated in this way. These duplications, along with three deletions isolated in this study and 9 existing deletions of the region, divided the left half of chromosome I into at least 24 regions. Protocols were developed and used to rapidly map mutations into the regions defined by the breakpoints. The techniques and results described demonstrate the feasibility of carrying out a similar analysis on the whole genome. The majority of duplications behaved as if they were free; that is they segregated independently of the euploid chromosome set. While size was an important determinant of mitotic stability, clear exceptions to a size - stability correlation were observed. For example, despite its larger size, hDp72 was lost during cell division more frequently than hDpl8, suggesting features of chromosome structure were important. Shortening of duplications in the unc-11 dpy-5 region caused greater reductions in mitotic stability than similar sized shortenings in the dpy-5 unc-13 region. Therefore, specific sequences appear to influence duplication stability. Some free duplications were also observed to break spontaneously. Breakage occurred at different frequencies for different duplications and correlated with mitotic instability. The meiotic properties of four translocations involving chromosome I were examined. No recombination was observed in any of the translocation heterozygotes along the left (let-362 - unc-13) portion of chromosome I. By isolating a half-translocation chromosome as a free duplication, I mapped the breakpoints of three of the translocations. The boundaries of cross-over suppression coincided with the physical breakpoints. These results agree with the proposal that DNA sequences at the right end of chromosome I are essential for homologue recognition followed by meiotic synapsis and recombination. The published data of other translocations and duplications indicates that each of the other five C. elegans chromosomes has DNA sequences localized to one end that are required for homologue recognition and recombination.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Apparently balanced chromosome rearrangements (ABCRs), mainly reciprocal translocations and inversions, are common in our species and are present both in patients with clinical abnormalities and in phenotypically normal individuals. The four main features that are thought to explain clinical abnormalities in patients with ABCRs are: (i) breakpoint-mediated gene disruption, (ii) breakpoint-associated genomic imbalances, (iii) additional chromosomal complexity and (iv) genomic imbalances unrelated to the breakpoints. The work presented in'this thesis represents one of the first systematic studies to ascertain the occurrence ofthe above four features in both phenotypically normal and abnormal carriers ofABCRs. . Molecular cYtogenetic analyses by FISH and/or array painting and by array CGH were applied in the characterisation ofABCRs in 31 phenotypically normal individuals (control cohort) and in 16 phenotypically abnormal patients (patient cohort). The occurrence ofthe above four features was assessed in both cohorts and the results were compared in an attempt to determine if the ABCRs in these two groups are molecularly distinct. Genomic imbalances both at the breakpoints and unrelated to the breakpoints I and additional chromosomal complexity were present in 25% ofthe cases in the . patient cohort, but in none ofthose in the control cohort. Surprisingly, breakpoint-mediated gene disruption was equally frequent in both cohorts. However, there was a difference in the type ofgenes involved, with those ofthe patient cohort being more commonly involved in development and function of the nervous system. These observations suggest that there are molecular differences in the two groups ofcarriers. Furthermore, among the four features analysed, genomic imbalances both at the breakpoints and elsewhere in the . genome appear to be the main cause ofphenotypic abnormalities in carriers of ABCRs; nevertheless disease candidate genes have also been identified and future studies will assess their contribution to the abnormal phenotypes. In summary, the application ofmolecular technique~ is an invaluable approach to characterise genomic regions involved in chromosome rearrangements and other genomicregions unrelated to the breakpoints, thus aiding in the understanding of~e contribution ofthese genomic regions to human disease and to human variation'.

Chromosomų struktūros persitvarkymai gali lemti įvairius žmogaus sveikatos sutrikimus. Netgi chromosominės ligos, pvz., Dauno ar Ternerio sindromai, gali būti nulemtos ne chromosomų skaičiaus, bet chromosomų struktūros persitvarkymų. Chromosomų trūkiai gali vykti bet kurioje chromosomos dalyje, tai lemia įvairius chromosomų struktūros pokyčius, tačiau tik dalis jų yra suderinami su gyvybinėmis funkcijomis ir yra nustatomi postnataliai. Chromosomų struktūros persitvarkymai, priklausomai nuo to, subalansuoti ar nesubalansuoti, lemia įvairias dismorfines anomalijas arba vaisingumo problemas. Jų nustatymas yra svarbus genetiniam konsultavimui. Šio drabo tikslas- įvertinti chromosomų struktūros persitvarkymų, nustatytų VUL SK MGC 2002 – 2008 m., įvairovę ir genetinę jų reikšmę. Tam, kad būtų įvertinta chromosomų struktūros persitvarkymų įvairovę, buvo analizuojami 2002-2008 metais atlikti kariotipo tyrimai. Remiantis rezultatais galima teigti, kad dažniausias chromosomų struktūros pakitimas yra reciprokinė translokacija, kuri dažniausiai lemia nevaisingumą bei savaiminius persileidimus. Didžiausia chromosomų struktūros pakitimų įvairovė nustatyta X chromosomoje.Rutininė citogenetinė kariotipo analizė yra svarbi ir tais atvejais, kai atliekamas FISH tyrimas naudojant tam tikrai genetinei sričiai specifinį žymenį, kadangi skirtingi chromosomų struktūros persitvarkymai gali lemti panašų fenotipą.
Chromosome structural rearrangements could cause various human health problems. Even Down’s or Turner’s syndromes, which are usually determined by chromosome number change, in some cases could be caused by chromosome structure abnormalities. Structure rerrangements of autosomes, depending on whether it is balanced origin or not, are responsible for various dysmorphic abnormalities or fertility problems. Chromosome breakpoints can occur in any part of chromosome and form any type of rearrangement, but only part of them could be compatible with vital functions and detected postnatally. Chromosome structural rearrangements in many cases are unique and only particular ones are more common. The objective of this work was to assess the diversity of chromosome structural rearrangements and their implication to the human genetic. Cytogenetic analysis of karyotype was performed using G-banding and FISH techniques. Cytogenetic analyses of 76 patients using routine cytogenetic analysis and 20 patients using FISH method have been performed. In order to assess the variety of chromosome structural rearrangements, the results of karyotype analyses performed in Department of Human and Medical Genetics, Faculty of Medicine, Vilnius University during the period of 2002–2008 were reviewed. On the basis of obtained results a conclusion can be drawn that translocation is the most frequent chromosome structure rearrangement type, comprising 44,3% of all our cases. X chromosome is the most... [to full text]

The cause of the disease in two familial and two sporadic cases of aniridia associated with chromosome 11 rearrangements has been analysed. Since the rearrangements apparently leave the PAX6 gene intact, a cosmid contig encompassing PAX6 was assembled and facilitated mapping of the four breakpoints. All were found to lie distal of PAX6 at distances of between 20 and 150kb beyond the 3' end of the gene. Phenotypically these patients are indistinguishable from other aniridia cases, suggesting the involvement of PAX6 haploinsufficiency. The site of the most distal breakpoint has been defined by isolating chromosome 11 fragments which cross it. These fragments contain regions of conservation between species and sequencing reveals potential open reading frames, raising questions about the function of the likely gene at this position. A possible position effect mechanism involving disconnection of PAX6 from adjacent control elements has been explored. These elements may be locus-specific or more general chromatin organising elements and may be detected by DNaseI hypersensitive site mapping in appropriate cell types. Cell lines which appeared suitable for this purpose have been characterised. An alternative possibility involves disturbance of PAX6 expression by the incoming chromosomal region in each rearrangement, through an inappropriate chromatin conformation or the presence of negative control elements. The detailed physical map generated for the aniridia-associated breakpoint region will allow further exploration of the position effect mechanism. These aniridia-associated rearrangements contribute to a growing list of possible position effect cases in humans and other mammals. Extensive study of position effects in fruit flies and yeast shows that gene inactivation can result from a switch in chromatin environment between euchromatin and heterochromatin. In humans, the phenomenon of position effect may also involve disturbing the usual chromatin environment of a gene, reflecting the heterogeneity of the human genome in terms of chromatin structure and transcriptional permissiveness.

Allopolyploids form through the hybridization of two or more diploid genomes. A challenge to reproduction in allopolyploids is that pairing can occur between homologous chromosomes or homeologous chromosomes (i.e.different subgenomes.). Crossover between homeologous chromosomes can result in chromosome rearrangements that lower fertility and overall fitness. Rearrangements can alter the dosage of either entire chromosomes or just parts of chromosomes. Understanding the frequency and extent of rearrangements will help to explain the evolution and genome stabilization of agriculturally important allopolyploid species. Pyrosequencing is a useful tool in the study dosage changes in allopolyploids because it allows quantification of the relative contribution from each progenitor species at any given locus. Here we use pyrosequencing to analyze resynthesized Brassica napus allopolyploids and their progeny. Targets for pyrosequencing were identified using a bioinformatic approach taking advantage of recently-released Brassica genome sequence. SNPs identified through bioinformatics were confirmed through molecular biology. Markers along the A3/C3 homeolog pair were used to identify the occurrence of novel homeologous exchanges during meiosis in the parent plant, and segregation patterns arising from dosage changes in the parent. We identify a higher frequency of homeologous rearrangements at the distal end of the chromosomes. We also observe that the presence of a dosage change in a parent increases the likelihood that the chromosome bearing the dosage change will undergo subsequent rearrangements in neighboring loci.