Dissertationen zum Thema „Chromosome inversions“

Les inversions paracentriques sont des anomalies chromosomiques généralement considérées comme inoffensives. Toutefois, des cas de porteurs de chromosomes remaniés issus d'inversions paracentriques ont été rapportés, soulignant la nécessité d'étudier le comportement méiotique de ces anomalies. Seules quelques études ont été pratiquées, utilisant la technique de fécondation croisée Homme-Hamster, le typage génétique des spermatozoïdes (sperm typing) ou l'hybridation in situ fluorescente (FISH) par marquages centromériques ou télomériques. Afin d'améliorer l'efficacité de l'étude méiotique des inversions paracentriques, nous avons développé l'utilisation des sondes BAC qui permettent une localisation chromosomique précise des points de cassures chromosomiques et l'identification de tous les produits méiotiques des inversions dans le sperme humain. Les points de cassures et la ségrégation méiotique de 3 inversions paracentriques, inv(5)(q13. 2q33. 1), inv(9)(q21. 2q34. 13) et inv(14)(q23. 2q32. 13), ont ainsi été déterminés. Les taux de recombinants observés dans ces 3 inversions varient de 3,72% à12,55%. Cette localisation des points de cassures et l'analyse des séquences d'ADN adjacentes sont essentielles pour mettre en évidence la formation de boucles d'inversion, pour déterminer le risque de recombinaison à terme, ainsi que pour étudier les mécanismes méiotiques de formation des recombinaisons. Ainsi, la présence de régions d'ADN riches en recombinaisons et en duplications inter-chromosomiques a été mise en évidence, en complément de la formation de recombinants chromosomiques. Par ailleurs, une nouvelle technique de FISH multi-couleurs 3D (3D MCB FISH) a été adaptée aux spermatozoïdes humains pour l'analyse in situ des ségrégations. Cette approche permet de visualiser in situ les inversions paracentriques et d'estimer la fréquence de tous les types de recombinants issus de boucles d'inversion, de recombinaisons U-loop ou de processus de cassure/fusion des chromatides-soeurs.

Human Artificial Chromosomes (HAC) are fascinating extrachromosomal molecules that stay independently from the host genome and are capable of segregating as efficiently as endogenous chromosomes. It has been proven that HAC are potential tools for both basic chromosome behavioural research and agents for gene therapy purposes. My DPhil project is divided into two main themes. The first theme was to develop a novel artificial chromosome in mouse embryonic stem cells. The second theme was to understand the factors affecting chromosome stability which may also affect the efficiency of artificial chromosome formation. so that our protocol for better HAC preparation can be refined. There are six results chapters in my thesis. The first three chapters described how I developed human artificial chromosomes in mouse embryonic stem cells. Initially, vectors containing a long stretch of human alphoid DNA were delivered to mouse cells using the Herpes Simplex Virus-I (HSV-l) amplicon system but the efficiency was low. Next, mouse pericentromeric and centromeric DNAs were employed for mouse artificial chromosome (MAC) via HSV-l system. However, the efficiency remained the same. Finally, I used the Microcell-Mediated Chromosome Transfer (MMCT) system to transfer HAC from HTl 080 cells into mouse ES cells and successfully established HAC in ES which were highly stable. The results obtained in this first part of my thesis suggested that to increase HAC formation efficiency it would be necessary to improve the techniques of HSV-I delivery and MMCT. Moreover, it would also be important to better characterize factors affecting chromosome behaviour. The last three results chapters focus on factors affecting chromosomes stability and improving the HSV -1 delivery system and MMCT. I undertook an in vivo study of whole cell fusion experiments with the aid of live cell irnaging system, and found that histone H2B proteins underwent a dynamic assembly/disassembly processes. Live cell imaging of MMCT suggested that the microcell delivery is a very slow process and the results may lead to a refinement of the MMCT protocol. I found it is possible to generate a single HAC using two HSV-l amplicons containing two different constructs, potentially doubling the HSV-l HAC capacity from 150 kb to 300 kb. The last chapter illustrated how the expression of non- coding centromeric satellites impaired chromosome stability in both human cultured and human embryonic stem cells. The findings revealed that non-coding centromeric RNA plays an important role on chromosome stability that might be important for artificial chromosome development.

Orientador: Louis Bernard Klaczko
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Drosophila mediopunctata é uma espécie pertencente ao grupo tripunctata, que tem como traço marcante um padrão de pigmentação abdominal, sob a forma de três pintas na região mediana dos últimos tergitos. Nesta espécie, este padrão é variável, havendo indivíduos com quatro fenótipos, que vão de zero a três pintas. Já se observou que esta variação tem determinação genética, com marcada influência do cromossomo II, e alta plasticidade fenotípica em resposta à temperatura de desenvolvimento. Neste trabalho, buscou-se caracterizar parte destas duas fontes de variação. Por um lado, foram estudadas as normas de reação da pigmentação a um gradiente térmico, investigando-se classes fenotípicas contrastantes. Devido ao desenho experimental, que buscou separar os efeitos desta variável de um possível papel das inversões do cromossomo II, foi possível evidenciar um forte efeito das classes fenotípicas utilizadas sobre a resposta das estirpes ao gradiente térmico, independente do cariótipo. Foram descritos, por polinômios, dois tipos de norma de reação relacionados ao fenótipo, ambos com forma de parábola, mas diferindo em relação ao coeficiente de curvatura. O grupo de estirpes de pigmentação clara apresentou uma curva côncava e o grupo escuro uma curva convexa. A norma de reação da taxa de desenvolvimento de ovo a adulto foi caracterizada a partir do mesmo procedimento. Entretanto, apesar dos efeitos significativos do cariótipo e da classe fenotípica, a homogeneidade das normas de reação descritas por regressões lineares não possibilitou uma interpretação clara destes efeitos. A plasticidade do caráter também foi investigada quanto ao período de desenvolvimento termo-sensível. Assim, foi possível determinar a porção final da fase de pupa como o período no qual ocorre a influência da temperatura sobre o fenótipo de pintas do adulto. Por outro lado, em relação à determinação genética do caráter, foram obtidas estimativas de herdabilidade para o número de pintas abdominais, em condições quase naturais. Visando estabelecer um parâmetro de comparação com outros trabalhos, foi estimada a herdabilidade do tamanho do tórax a partir do mesmo material. Os resultados deste experimento, apresentaram grande contraste entre os dois traços: as estimativas foram baixas ou não significativas para o tamanho do tórax e, em geral, altas e significativas para o número de pintas
Abstract: Drosophila mediopunctata belongs to the tripunctata species group, which has a typical abdomen pigmentation pattern, consisting of three dark spots in the last tergites. In this species, this pattern is variable, with the phenotypes ranging from zero to three spots. It has been noted that this variation has genetical determination, with strong influence from the second chromosome, and high phenotypic plasticity in response to the developmental temperature. In this work, we attempted to describe part of these two variation sources. On one side, the pigmentation reaction norm to a thermal gradient was studied, by investigating the influence of contrasting phenotypical classes. Given the experimental design, which was planned to separate the effects of this variable from a possible influence of the second chromosome inversions, it was possible to detect a strong effect of the phenotypical classes on the lineages response to the thermal gradient, independent of the kariotype. Two types of reaction norms, related to the phenotype, were detected and described by polynomial adjustment. Both had a parabolic shape, but with different curvature coefficients. The light pigmentation lineage group showed a concave curve, and the dark group had a convex curve. The reaction norm of development rate from egg to adult was described according to the same procedure. However, despite the significant effects of the karyotype and phenotypical classes, the homogeneity of reaction norms, described by linear regression, hindered a clear interpretation of these effects. The character plasticity was also investigated in respect to the developmental thermosensitive period. Thus, it was possible to determine that the period in which the temperature influence on the adult phenotype occurs is the last portion of the pupal phase. On another side, relative to the character genetic determination, heritability estimates for the number of abdominal spots were obtained, in nearly natural conditions. Aiming to establish a comparison parameter with other studies, the heritability of thorax length was estimated based on the same material. The results of this experiment reveal a great contrast between these trait estimates: for the thorax they were low or non-significant, and, in general, for the abdominal spot number, they were high and significant
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular

Bacteria are many, old and varied; different bacterial species have been evolving for millions of years and show many disparate life-styles and types of metabolism. Nevertheless, some of the characteristics regarding how bacteria organize their chromosomes are relatively conserved, suggesting that they might be both ancient and important, and that selective pressures inhibit their modification. This thesis aims to study some of these characteristics experimentally, assessing how changes affect bacterial growth, and how, after changing conserved features, bacteria might evolve. First, we experimentally tested what are the constraints on the horizontal transfer of a gene highly important for bacterial growth. Second, we investigated the significance of the location and orientation of a highly expressed and essential operon; and we experimentally evolved strains with suboptimal locations and orientations to assess how bacteria could adapt to these changes. Thirdly, we sought to understand the accessibility of different regions of the bacterial chromosome to engage in homologous recombination. And lastly, we constructed bacterial strains with chromosomal inversions to assess what effect the inversions had on growth rate, and how bacteria carrying costly inversions could evolve to reduce these costs. The results provide evidence for different selective forces acting to conserve these chromosome organizational traits. Accordingly, we found that evolutionary distance, functional conservation, suboptimal expression and impaired network connectivity of a gene can affect the successful transfer of genes between bacterial species. We determined that relative location of an essential and highly expressed operon is critical for supporting fast growth rate, and that its location seems to be more important than its orientation. We also found that both the location, and relative orientation of separated duplicate sequences can affect recombination rates between these sequences in different regions of the chromosome. Finally, the data suggest that the importance of having the two arms of a circular bacterial chromosome approximately equal in size is a strong selective force acting against certain type of chromosomal inversions.

Les inversions cromosòmiques són variants estructurals on un fragment de genoma s'inverteix sense canviar-ne el contingut, i durant anys, els seus efectes subtils però importants han fascinat els biòlegs evolutius. De fet, les inversions van ser descobertes per primer cop fa cent anys en mosques de la fruita, i aviat es va fer evident la seva associació a processos evolutius com l'adaptació local i l'especiació. Tot i així, en el moment en què vivim de la genòmica i el big data, les inversions tendeixen a quedar fora de l'abast de les tecnologies més comunes i encara se sap poc d'elles. Durant els últims anys, el Projecte InvFEST ha tingut com a objectiu ampliar el nostre coneixement sobre les inversions humanes mitjançant la validació i genotipació d'una gran part de les inversions predites. En concret, aquest projecte ha generat un recurs molt útil format per 45 inversions comunes (d'entre 83 pb i 415 kpb) amb genotips d'alta qualitat per a un total de 550 individus de set poblacions diverses. En aquesta tesi s'utilitzen les dades poblacionals generades, junt amb les seqüències del Projecte 1000 Genomes, per a realitzar la primera anàlisi detallada de les propietats evolutives de les inversions polimòrfiques humanes. Per aconseguir-ho, s'han fet servir diferents mètodes que combinen models teòrics, simulacions i comparacions amb altres tipus de mutacions. A part d'obtenir una caracterització completa de les dades, els resultats confirmen que hi ha diferències importants entre inversions generades per diferents mecanismes. La distribució de freqüències de les 21 inversions creades per mecanismes no homòlegs (NH) és semblant a l'esperada per a variants neutres si es controlen els biaixos en la detecció, indicant que no es troben sota una forta selecció negativa. La recombinació s'inhibeix en tota la longitud de la inversió, sense que s'observi cap intercanvi de variants entre orientacions, i la inhibició podria estendre's alguna kilobase més enllà dels punts de trencament. Com a resultat, els nivells de variació genòmica es veuen força afectats per les inversions NH, tal i com prediuen les simulacions realitzades. Mentre que les inversions antigues i a freqüències intermèdies augmenten la variació nucleotídica, les inversions més recents poden crear l'efecte contrari. En canvi, la majoria de les inversions creades per recombinació homòloga no al·lèlica (NAHR) (19/24) han aparegut múltiples vegades de manera independent en diferents haplotips de la mostra. Els elevats nivells de recurrència es reflecteixen en diferents mesures: aquestes inversions estan enriquides en freqüències intermèdies, comparteixen polimorfismes nucleotídics entre orientacions, i es troben en baix desequilibri de lligament amb variants properes, dificultant-ne la seva detecció indirecta amb variants correlacionades. Finalment, per tal de trobar aquelles inversions candidates a tenir efectes funcionals, s'han explorat diverses senyals de selecció natural a partir de freqüències, diferenciació poblacional i patrons de variació nucleotídica. S'han identificat deu inversions candidates, tres d'elles de més de 1.5 milions d'anys i mantingudes a freqüències intermèdies, possiblement per selecció equilibradora, una d'elles també es troba a genomes d'hominins antics. Altres candidates sembla que han augmentat ràpidament de freqüència en algunes poblacions, que és consistent amb selecció positiva. Cal destacar que més de la meitat de candidates estan situades en regions gèniques, el qual suggereix que poden tenir un efecte funcional. Per tant, aquest treball ofereix una visió global de la dinàmica de les inversions i el seu paper com modificadors genòmics, obrint noves línies d'investigació.
Chromosomal inversions are structural variants that invert a fragment of the genome without usually modifying its content, and their subtle but powerful effects in natural populations have fascinated evolutionary biologists for a long time. Discovered a century ago in fruit flies, their association with different evolutionary processes, such as local adaptation and speciation, was soon evident in several species. However, in the current era of genomics and big data, inversions frequently escape the grasp of current technologies and remain largely overlooked in humans. During the last few years, the InvFEST Project has aimed to address the missing knowledge about human inversions by validating and genotyping a large fraction of predicted polymorphisms. In particular, it has generated one of the most useful data sets on human inversions, consisting of 45 common inversions (with sizes from 83 bp to 415 kbp) genotyped at high-quality in 550 individuals of seven populations of diverse ancestry. This thesis takes advantage of the available population-scale information, combined with whole-genome sequences available from the 1000 Genomes Project, to carry out the first detailed analysis of the evolutionary properties of human polymorphic inversions. The methods used combine theoretical models, simulations and empirical comparisons with other mutation types. Besides the complete characterization of the data set, the results confirm fundamental differences between inversions created by different mechanisms. The frequency distribution of the 21 inversions originated by non-homologous mechanisms (NH) is similar to that expected for neutral variants when controlling for detection biases, which indicates that they are not subjected to strong negative selection. Recombination is completely inhibited across the whole inversion length, with no clear genetic exchange found, and possibly over a few kbp beyond the breakpoints. As a result, NH inversions strongly affect local genome variation levels, as predicted by computer simulations, with older inversions increasing total nucleotide diversity, while younger ones at very high frequency could have the opposite effect. In contrast, most inversions created by non-allelic homologous recombination (NAHR) (19/24) have appeared independently in different haplotypes in the sample. These high recurrence levels are reflected in several measures: they are enriched in intermediate frequencies, share multiple nucleotide polymorphisms between orientations, and have little linkage disequilibrium with neighbouring variants, which limits their detection by tag SNP strategies. Finally, in order to find inversions that are functional candidates, different signatures of selection on inversions were explored based on their frequencies, population differentiation and sequence variation patterns. Ten candidates were revealed, with three of them found to be >1.5 million years old and maintained at intermediate frequencies, possibly by balancing selection. One of these was also found in archaic hominins. Other candidates seem to have reached high frequencies in a short period of time in some populations, consistent with positive selection. Notably, over half of the candidates are located within gene regions, which suggests that they may have functional effects. Thus, this work offers an overview of inversion dynamics and their role as genomic modifiers, opening interesting avenues of investigation.

Significant advances were performed in mapping and characterizing different types of structural variation in the human genome such as point mutations, insertions, deletions, etc. Nevertheless, there is still an important genetic component in multifactorial diseases that it is still unknown. Inversions are chromosome mutations in which a given loci change its orientation respect to a reference and present a balanced genetic material (neutral copy number). Our group developed tools in order to infer some variants of ancestral submicroscopic inversions (0.5 Mb – 5Mb) using computational methods with SNP arrays, and documented that inversions correlates with genetic expression. Recent studies shows that, some inversions, regarding to be neutral in copy-number, could affect to the expression of certain genes. Therefore, It is thought that submicroscopic inversions could have a contributive effect in certain diseases, there are not properly studied. Our target was try to determine the contribution of those inversions, with the phenotype of four complex disease models: Autism Spectrum Disorders (ASD), Schizophrenia Spectrum Disorders (SSD), Rheumatoid Arthritis (RA) and Ulcerative Colitis (UC), in available datasets (~27,000 samples). Our results have determined that: • The inversions 17q21.31 and 8p23 are related to ASD and SSD risk. Concretely, 17q21.31 are associated with multiplex ASD families (OR=1.20 p-value 9.52e-05). Meanwhile the inverted allel (17q), gives SSD risk (OR=0.86 p-value 0.007). • We better characterize 15q24 inversion found three putative inversion-induced haplotypes (NI, Ia and Ib). Homozygous for NI correlated with over-expression of MAN2C1 over many brain areas, where Ib homozygosity was highly under-expressed. • RA and UC, we found runs of homozygosity regions (RA OR=9.32 95%CI [2.66-63.45]) and 3p24.3 (UC OR=7.77 95% CI [2.08-54.56]). Additionally, we found haplotypes that could be inversions that are already associate to them, but we are pending to further validations.
S'han realitzat grans avenços en caracterització dels diferents tipus de variació en el genoma humà, com ara mutacions puntuals, insercions, delecions, etc., però encara existeix un component genètic de les malalties, determinada per l'heretabilitat, que és desconeguda. Les Inversions són mutacions cromosòmiques en què un fragment de cromosoma canvia la seva orientació respecte a la de referència i són difícils de genotipar per ser neutres en nombre de còpies. El nostre grup ha desenvolupat una eina per inferir algunes variants d’inversions submicroscòpiques ancestrals (0.5MB - 5MB) per mètodes computacionals utilitzant microarrays d’SNPs, i ha documentat que es correlacionen amb expressió gènica. El nostre objectiu ha estat intentar determinar la contribució de les inversions cromosòmiques al fenotip en quatre models de malalties complexa: autisme, esquizofrènia, artritis reumàtica i colitis ulcerosa. Realitzat estudis de cas control i de segregació en trios, els nostres resultats han determinat que: • Les inversions 17q21.31 i 8p23 s’associen a risc d’autisme i esquizofrènia. Concretament, la 17q21.31, s’associa especialment amb aquells individus provinents de famílies multiplex (OR=1.20 p-value 9.52e-05). Per altra banda, l’al•lel contrari (17q) confereix risc d’esquizofrènia (OR=0.86 p-value 0.007). • Hem caracteritzat tres haplotips relacionat amb la inversió de la regió 15q24 (NI, Ia i Ib) que segueixen un patró d'herència Mendeliana amb una alta frequència en Europeus (NI=20%, Ia=50%, Ib=30%). L'homozigositat per l'al•lel no invertit correlaciona amb la sobre-expressió de MAN2C1 en varies àrees del cervell (mentres que l'al•lel Ib està sota expresat). • Pel que fa a l’artritis reumàtica (RA) i la colitis ulcerosa (UC), hem trobat unes regions d’homozigositat 4q32 (RA OR=9.32 95%CI [2.66-63.45]) i 3p24.3 (UC OR=7.77 95% CI [2.08-54.56]) . Addicionalment, també hem trobat uns haplotips que podien ser inversions que també hi estarien associats però estan pendents de validacions addicionals.

Las inversiones cromosómicas son variantes estructurales donde un segmento de ADN cambia su orientación. Las inversiones cromosómicas reducen la recombinación homóloga y producen diferentes haplotipos en los cromosomas estándar e invertidos. Como resultado, influyen en la adaptación y la selección y desempeñan un papel en la susceptibilidad a las enfermedades humanas. Las inversiones se pueden estudiar con métodos experimentales y bioinformáticos. Los datos de SNP array se pueden usar para determinar genotipos de inversión mediante el uso de diferencias de haplotipos entre cromosomas invertidos y estándares. Sin embargo, estos métodos no están optimizados para grandes cohortes (con miles de individuos, como dbGaP o UK Biobank). Además, los métodos actuales solo pueden genotipar las inversiones con dos haplotipos y la clasificación es difícil de armonizar entre cohortes. Finalmente, se conoce que las inversiones cromosómicas afectan la expresión génica y la metilación del ADN. Sin embargo, no existen métodos precisos para evaluar globalmente el efecto de las inversiones en la expresión génica local o la metilación del ADN. El objetivo principal de esta tesis es desarrollar nuevos métodos robustos y escalables así como herramientas bionformáticas para estudiar los efectos fenotípicos y funcionales de las inversiones cromosómicas, superando las limitaciones existentes. Con este fin, he desarrollado un nuevo método para genotipar las inversiones cromosómicas que se puede usar en grandes cohortes, con inversiones con múltiples haplotipos y que utiliza haplotipos de referencia que permite el análisis conjunto de múltiples cohortes. En segundo lugar, he implementado un método multivariante basado en el análisis de la redundancia para estudiar los efectos de las inversiones cromosómicas en la metilación del ADN y la expresión génica locales. A continuación, he aplicado ambos métodos para estudiar el papel de las inversiones cromosómicas en dos grupos de enfermedades complejas: trastornos del neurodesarrollo y cáncer. Finalmente, he desarrollado un nuevo método para estudiar cómo las inversiones cromosómicas afectan los patrones de recombinación. Este método es aplicable a cualquier región genómica que contenga subpoblaciones con diferentes patrones de recombinación, lo que permite asociar estas subpoblaciones a rasgos fenotípicos.
Chromosomal inversions are structural variants where a segment changes its orientation. Chromosomal inversions reduce homologous recombination, producing different haplotypes in standard and inverted chromosomes. As a result, they influence adaptation and selection and play a role in susceptibility to human diseases. Inversions can be studied using experimental and bioinformatic methods. SNP array data can be used to call inversion genotypes by using haplotype differences between inverted and standard chromosomes. However, these methods are not optimized for large cohorts (thousands of individuals from existing databases such as dbGaP or UK Biobank). Also, current methods can only genotype inversions with two haplotypes and the inversion calling is difficult to be harmonized among cohorts. Finally, it is recognized that chromosomal inversions affect gene expression and DNA methylation. However, there are no accurate methods to globally assess the effect of inversions on local gene expression or DNA methylation. The main aim of this thesis is to develop new robust and scalable methods and bioinformatic tools to study the phenotypic and functional effects of chromosomal inversions by overcoming the existing limitations. To this end, I have developed a new method to genotype chromosomal inversions that can be used in large cohorts, inversions with multiple haplotypes and that uses reference haplotypes allowing the integrative analysis of multiple cohorts. Second, I have implemented a multivariate method based on redundancy analysis to study the effects of chromosomal inversions on local DNA methylation and gene expression. Then, I applied both methods to study the role of chromosomal inversions in two groups of complex diseases: neurodevelopmental disorders and cancer. Finally, I developed a new method to study how chromosomal inversions affect recombination patterns. This method is extendable to any genomic regions containing subpopulations with different recombination patterns, allowing associating these subpopulations to phenotypic traits.

La variación estructural contribuye de forma substancial a la diversidad genética, pero su asociación con rasgos complejos y enfermedades no se entiende del todo y merece una caracterización detallada. Esto es especialmente cierto para las inversiones, cuyas consecuencias funcionales han permanecido ocultas, con algunas pocas excepciones. A pesar del creciente interés en caracterizar todo tipo de variantes genómicas, las inversiones se han dejado a menudo de lado debido a la presencia de regiones repetitivas en sus puntos de rotura sumado a su naturaleza balanceada. El proyecto InvFEST ha tratado de superar este desafío técnico mediante el desarrollo de nuevos métodos para genotipar inversiones. Gracias a este esfuerzo, un total de 111 inversiones comunes en humanos han sido genotipadas con precisión en un gran número de individuos de diversas poblaciones, convirtiéndose en el recurso más completo y fiable de este tipo de variación disponible hasta la fecha. En la era actual de la medicina de precisión, el análisis de loci de carácteres cuantitativos (QTL) ha surgido como un enfoque clave para determinar cómo los polimorfismos genéticos influyen en la expresión génica y, a su vez, en los rasgos fenotípicos. Por lo tanto, esta tesis aprovecha al máximo los datos generados para realizar por primera vez una cuantificación sistemática del impacto funcional de las inversiones polimórficas humanas. Los resultados muestran que las inversiones pueden afectar la expresión génica ya sea manteniendo haplotipos diferenciados, alterando o reoganizando la estructura de los genes, creando nuevos transcritos de fusión, o actuando a través de cambios en los patrones epigenéticos. Sorprendentemente, la mitad de las inversiones analizadas actúan como QTL principales o están en un desequilibrio de ligamiento (LD) con QTL centinelas para la expresión génica y los cambios epigenéticos en diferentes tejidos y líneas celulares, lo que sugiere que las inversiones están enriquecidas en efectos funcionales. En particular, esta influencia sobre fenotipos moleculares es aún más acusada para las inversiones largas (>100 kb), ya que están involucradas en el 80% de los QTL identificados. Aunque se sabe que las variantes estructurales tienen una mayor probabilidad de estar asociadas con niveles de expresión y rasgos complejos, los efectos detectados para las inversiones podrían reflejar un compromiso entre cambios de expresión beneficiosos y el posible impacto negativo sobre la fertilidad debido a la producción de gametos desequilibrados por recombinación. Además, las regiones de las inversiones presentan un enriquecimiento de señales de estudios de asociación a nivel de todo el genoma, y 14 de ellas están en alto LD con variantes asociadas a diferentes caracteres, lo que apoya su posible implicación en fenotipos humanos. Finalmente, se han investigado en detalle dos inversiones interesantes. HsInv0102 invierte un exón alternativo no codificante del gen RHOH. Curiosamente, esta inversión también está asociada con los niveles de la proteína RhoH y se ha estimado que el alelo invertido actúa como un locus protector con efectos moderados sobre la susceptibilidad al cáncer sanguíneo. Por otra parte, HsInv0124 regula la expresión de varios genes en la región IFITM, como IFITM2 e IFITM3, a través de cambios en los patrones de modificación de histonas. Además, bajo condiciones de infección, esta inversión tiene un efecto más generalizado sobre la expresión de genes relacionados con la respuesta inmune, lo que indica que puede desempeñar un papel importante en la defensa contra las infecciones víricas. En su conjunto, estos hallazgos ilustran el posible impacto funcional de las inversiones en el genoma humano y ayudan a desentrañar la relación que existe entre estas variantes y la variabilidad fenotípica.
Structural variation contributes substantially to the genetic diversity, but its association with complex traits and diseases is not well understood and deserves detailed characterisation. This is particularly true for chromosomal inversions, whose functional consequences have remained elusive in humans, with very few notable exceptions. Despite the rising interest in identifying all types of genomic variants, inversions have been often set aside due to the presence of repetitive regions in their breakpoints along with their balanced nature. The InvFEST Project has tried to overcome this technical challenge by developing unique methods for inversion genotyping. Thanks to this effort, a total of 111 polymorphic human inversions have been accurately genotyped in a large number of individuals from diverse populations, becoming the most complete and reliable resource of this type of variation available to date. In the current era of precision medicine, quantitative trait loci (QTL) analysis has emerged as a key approach to determine how genetic polymorphisms influence gene expression and, in turn, phenotypic traits. Thus, this thesis makes the most of the great amount of data generated to perform for the first time a systematic quantification of the functional impact of human polymorphic inversions. The results show that inversions can affect gene expression by maintaining differentiated haplotypes, disrupting or reorganizing gene structures, creating novel fusion transcripts or acting through changes in epigenetic patterns. Strikingly, half of the inversions analysed act as lead QTLs or are in high linkage disequilibrium (LD) with top QTLs for gene expression and epigenetic changes across different tissues and cell lines, which suggests that inversions are enriched for functional effects. In particular, this influence on molecular phenotypes is even stronger for long inversions (>100kb), which are involved in 80% of the QTL associations. Although structural variants are known to have a higher chance to be associated with expression levels and complex traits, the detected inversion effects may reflect a trade-off between beneficial expression changes and potential negative costs on fertility due to production of unbalanced gametes by recombination. Furthermore, inversions present an enrichment of genome-wide association studies (GWAS) signals in their surrounding area, and 14 of them are in high LD with trait-associated variants, supporting their potential implication in human phenotypes. Finally, the phenotypic consequences of two interesting inversions have been investigated in detail. HsInv0102 inverts an alternative non-coding exon from the RHOH gene. Interestingly, this inversion is also associated with RhoH protein levels and the inverted allele could act as a moderate protective locus on blood cancer susceptibility. On the other hand, HsInv0124 regulates the expression of several genes in the IFITM region, including IFITM2 and IFITM3, through changes in histone modification patterns. Moreover, under infection conditions, this inversion has a pervasive effect on the expression of genes related with immune response, indicating that it may play an important role in defense against viral infections. All together, these findings illustrate the potential functional impact of inversions on the human genome and help to uncover previously missing variants related to phenotype variability.

In summary, this thesis presents the localisation and identification of genetic variants of which some are disease associated and some considered to be neutral. Knowledge of the basic mechanisms behind human disorders is important both from a biological and medical point of view. The thesis is based on four papers of which the first two clarify the genetic basis of autosomal dominant aplasia of lacrimal and salivary glands (ALSG). ALSG is a rare disorder with high penetrance and variable expressivity characterized by dry mouth and eyes. In paper I, we located the ALSG gene to a 22 centiMorgan region on chromosome 5 through a genome-wide linkage scan with microsatellite markers in two families. Mutations were found in the gene encoding fibroblast growth factor 10 (FGF10) situated in the linked chromosome 5 region. Mice having only one copy of the FGF10 gene (Fgf10+/- mice) have a phenotype similar to ALSG, providing an animal model for the disorder. In paper II, we describe two additional patients with ALSG and missense mutations in FGF10, providing further genotype-phenotype correlations. The aim of paper III was to identify a gene involved in autosomal recessive severe congenital neutropenia (SCN), also referred to as Kostmann syndrome. The disease is characterized by a very low absolute neutrophil count and recurrent bacterial infections. Affected individuals from the family with SCN originally described by Dr Kostmann were genotyped with whole-genome SNP arrays. Autozygosity mapping identified a shared haplotype spanning 1.2 Mb on chromosome 1q22. This region contained 37 known genes, of which several were associated with myelopoiesis. Our finding contributed to the identification of the gene mutated in Kostmann syndrome. In paper IV a cytogenetic inversion on chromosome 10 was mapped and characterized. Sequence- and haplotype analysis of carriers from four non-related Swedish families revealed identical inversion breakpoints and established that the rearrangement was identical by descent. A retrospective study of karyotypes together with screening of large sample sets established that the inversion is a rare and inherited chromosome variant with a broad geographical distribution in Sweden. No consistent phenotype was found associated with the inversion. Genetic research increases the understanding of our genomes and makes it possible to discover variants contributing to disease. Identification of such genetic variants further enables studies of gene function and pathogenesis. The finding of the disease associated variants in this thesis will eventually contribute to improved diagnosis, prognosis, risk assessment and a future treatment of patients.

There are numerous different white spotting patterns in the horse, including two of particular interest tobiano and sabino. In the mouse, genetic variation in the gene KIT causes many white spotting patterns. Due to the phenotypic similarity among white spotting patterns in horses and mice, KIT was investigated as the cause of the tobiano and sabino spotting patterns in horses. Initially, the KIT cDNA sequences from horses with several spotting patterns were compared. Three single nucleotide polymorphisms (SNPs) were identified, though none were associated with a spotting pattern. Three novel splicing variants were also observed: exon 17 skipping, exon 18 skipping and alternative splicing of exon 3. Families segregating for a sabino spotting pattern (designated Sabino 1) and exon 17 skipping were discovered. Sequencing revealed a SNP (KI16+1037) within intron 16 that was completely associated with skipping of exon 17. Using a PCR-RFLP for KI16+1037, linkage was discovered for sabino spotting (LOD=9.02 for =0) and presence of the Sabino 1 allele detected in seven breeds. While all horses with this SNP exhibited the Sabino 1 phenotype, some horses with a sabino phenotype did not possess the SNP. This is most likely due to genetic heterogeneity of the phenotype. Fluorescent in situ hybridization (FISH) was used to investigate the possibility of chromosome inversion in the region of KIT. A chromosomal inversion was discovered spanning ECA3q13 to 3q21 using BAC clones containing KIT and other genes in the same region. The ECA3q inversion was completely associated with Tobiano in the eight horses tested by FISH. This inversion may disrupt regulatory sequences of the KIT gene and thereby cause tobiano spotting. Spotting patterns are important to horse breeders for aesthetic as well as economic reasons. Spotting patterns in the horse may also be an interesting scientific model. The two genetic variants discovered in this work are good examples of genetic diversity due to mechanisms other than SNPs. Study of these variants may be valuable for examining the effects of the KIT gene on health traits. In particular, the KIT gene directs many functions of the mast cell, a cell that is involved in the etiology of inflammation.

Species belonging to the Saccharomyces ‘sensu stricto’ group present a good model for studying evolution due to their genetic versatility and availability of genomic data. Chromosomal rearrangements play a very important role in evolution of eukaryotes as well as prokaryotes as they can affect phenotypic characteristics and modes of speciation. In nature these rearrangements are most likely caused by highly mobile genetic elements, such as retrotransposons. Karyotypic changes can cause alterations in gene transcription causing genomic instabilities by inactivating or over-expressing particular genes. Only few studies looked at the importance of chromosomal rearrangements and their role on global and local gene expression. The aim of this PhD project is to investigate the impact of chromosomal inversions and translocations on fitness adaptation, gene expression and speciation. I first demonstrated that a single gene inversion (of DAL2) within a co-expressed gene cluster can cause an alteration of the expression of inverted gene as well as the neighbouring genes, ultimately leading to a phenotypic change. I also showed that small and large size pericentric and paracentric inversions do not always alter the gene expression and in general have no effect on growth rate. It was also shown that effect of large inversion on gene expression is not always localized within the inversion but occurs globally. Finally, it is demonstrated that chromosomal translocations can be responsible for the reproductive isolation of Saccharomyces paradoxus and Saccharomyces cariocanus.

Les translocations chromosomiques et les autres types de réarrangements peuvent, bien qu'associées à un phénotype normal, mener à la transmission d'un contenu génétique déséquilibré à la descendance. Il n'est pas possible de distinguer les chromosomes équilibrés des déséquilibrés, ce qui empêche toute sélection dans le cadre d'une fécondation in vitro (FIV). Nous avons ainsi mené une série de projets de recherche dont le but a été de mettre en évidence des caractéristiques spécifiques de ces spermatozoïdes déséquilibrés, afin de pouvoir les distinguer au cours d'une FIV. Nous avons montré que les spermatozoïdes déséquilibrés présentaient des taux de fragmentation de l'ADN plus élevés, étaient moins denses, et avaient un volume nucléaire supérieur. Ces constatations ont mené au développement d'une procédure de sélection des spermatozoïdes équilibrés chez les porteurs de réarrangements chromosomiques. En utilisant le hypo-osmotic swelling test (HOST), nous avons montré qu'une morphologie flagellaire spécifique était associée à un contenu chromosomique équilibré. Nous proposons d'utiliser cette procédure de sélection dans le cadre d'une ICSI, afin d'améliorer le pronostic reproductif chez les couples concernés. Nous proposons également d'évaluer la proportion de spermatozoïdes déséquilibrés chez chaque patient porteur de réarrangement chromosomique. En effet, bien que ce taux varie d'un patient à l'autre, il est stable dans le temps pour un patient donné. Il est de plus un élément déterminant dans le choix d'une des options de prise en charge : reproduction naturelle, insémination artificielle avec test de migration survie (TMS), ICSI avec sélection par HOST, et diagnostic préimplantatoire (DPI)
Chromosomal translocations and other balanced rearrangements, although usually associated with a normal phenotype, can lead to the transmission of an abnormal unbalanced genetic content to the offspring. Balanced and unbalanced spermatozoa are indistinguishable, making it impossible to select them prior to in vitro fertilization. We conducted a series of research projects aimed at evidencing specific characterics for unbalanced spermatozoa, in a way to ultimately distinguish them from balanced ones during in vitro fertilization. We showed that unbalanced spermatozoa had higher DNA fragmentation rates, were less dense, and that their nuclear volume was higher. This led to developing a selection procedure for balanced spermatozoa in rearrangement carriers. Using the hypo-osmotic swelling test (HOST), we showed that a specific flagellar morphology was associated with balanced chromosomal status. We suggest using this procedure prior to ICSI in order to improve the reproductive prognosis in those patients. We also suggest evaluating the proportion of unbalanced spermatozoa in every patient with a chromosomal rearrangement. Although this proportion varies among patients, it is stable over time for a given patient. We believe this is a decisive element in choosing between the different available options : natural conception, artificial insemination with discontinuous gradient centrifugation, ICSI with HOST-based selection, and pre-implantation genetic diagnosis (PGD)

Nombrosos estudis han constatat el valor adaptatiu del ric polimorfisme d’inversions cromosòmiques al drosofíl· lid D. subobscura. No obstant això, fins ara es coneixien molt poc les bases moleculars que hi ha darrere del seu manteniment a les poblacions naturals. En cercar loci candidats, un experiment previ de xoc tèrmic va quantificar els nivells de la proteïna Hsp70 en soques homocariotípiques dels ordenaments OST, O3+4+8 i O3+4. Inesperadament, els individus de l’ordenament càlid O3+4 mostraven nivells incrementats d’aquesta proteïna, en absència d’estrès tèrmic, que no augmentaven després del xoc tèrmic. Malauradament, en el moment en què es va dur a terme l’experiment hi havia moltes incògnites sobre l’organització molecular del locus Hsp70IR a D. subobscura. Els resultats prèviament esmentats, van donar peu al present treball de tesi, els objectius del qual són localitzar el locus Hsp70IR al cariotip i conèixer-ne l’organització genòmica, característiques moleculars i expressió gènica en diversos ordenaments cromosòmics d’interès que inclouen la regió genòmica on es troba la família gènica hsp70: O3+4+16+2, O3+4+8, O3+4 i OST. Gràcies a la seqüència d’un clon de la genoteca d’una línia OST i a còntigs del genoma de D. subobscura, hem pogut dissenyar una sonda a partir de la regió codificant de hsp70 que ens ha permès determinar la localització del locus on es troba aquesta família gènica (Hsp70IR) mitjançant hibridació in situ (ISH). Paral· lelament, hem pogut completar la seqüenciació d’una regió de 9-10 kb al locus Hsp70IR en 12 línies isogèniques per als ordenaments esmentats i a les espècies properes D. madeirensis i D. guanche per aclarir l’evolució d’aquest locus en els darrers 1,8 - 2,8 milions d’anys (Ma). Els resultats de la ISH van mostrar un únic punt d’hibridació a la banda 94A del segment distal (SI) del cromosoma O que coincidia els 4 cariotips estudiats: O3+4+16+2, O3+4+8, O3+4 i OST. Les seqüències corresponents a les 12 línies isogèniques i a D. madeirensis i D. guanche indiquen que en aquestes tres espècies del clúster subobscura, el locus Hsp70IR consta de dues còpies paràloges de 2,5 – 3,0 kb en orientació divergent i separades per una regió espaiadora central no duplicada de 0,5 – 1,4 kb. Les dues còpies mostren un elevat grau de conservació entre els diferents ordenaments i espècies analitzats, mentre que la regió espaiadora central és altament polimòrfica. Entre els aspectes més rellevants de l’anàlisi del polimorfisme, destaquem l’elevada conservació de les regions codificadores (CDSs) i els diferents elements reguladors en cis (CREs) al promotor proximal de tots els gens hsp70 analitzats, que indicarien que aquests són funcionals a totes les línies estudiades, i que la seva regulació podria ser similar. Curiosament, a nivell de seqüència, les regions paràlogues del promotor proximal i el CDS tendeixen a ser significativament més similars dins del mateix ordenament i, en alguns casos, dins la mateixa línia, probablement com a resultat de conversió gènica ectòpica. Per últim, hem dut a terme la quantificació dels nivells basals de mRNA i proteïna en mascles i femelles adults de sis línies isogèniques per a l’ordenament fred OST i sis per a l’ordenament càlid O3+4. La quantificació dels nivells de mRNA indica que els nivells són similars entre els dos ordenaments però en canvi aquests difereixen entre mascles i femelles de l’ordenament càlid O3+4. Així mateix, la quantificació dels nivells de la proteïna Hsp70 suggereix que no hi ha diferències entre sexes ni entre els dos ordenaments, però en canvi observem una interacció significativa entre sexe i ordenament. Aquests resultats, tant per a mRNA com per a proteïna, indiquen que l’expressió de hsp70 podria estar influïda pel sexe.
Numerous studies have confirmed the adaptive value of the rich chromosomal inversion polymorphism in the drosophilid D. subobscura. However, until recently very little was known about the molecular basis behind its maintenance in natural populations. In search of candidate loci, a previous heat shock experiment quantified Hsp70 protein levels in homokaryotypic strains for the OST, O3+4+8 and O3+4 arrangements. Unexpectedly, individuals of the warm climate-associated O3+4 arrangement showed increased levels in absence of thermal stress that did not boost after the heat shock. Unfortunately, by the time this experiment was performed there was very little data available on the molecular organization of the Hsp70IR locus in D. subobscura. The previously mentioned results led to the present thesis work, whose objectives are to locate the Hsp70IR locus in the karyotype and to know the genomic organization, molecular characteristics and gene expression patterns in several representative chromosomal arrangements that comprise the genomic region where the hsp70 gene family is located: O3+4+16+2, O3+4+8, O3+4 and OST. Using the sequence of a clone from an OST line genomic library and contigs from the unassembled genome of D. subobscura, we designed a probe from the hsp70 coding region that enabled us to determine the location of the locus by in situ (ISH) hybridization. Concomitantly, we completed the sequencing of a 9-10 kb region in the Hsp70IR locus in 12 lines isogenic for the aforementioned arrangements and in D. madeirensis and D. guanche to shed light on the evolution of this locus in the last 1.8 - 2.8 million years (myr). ISH results showed a single hybridization site in the 94A band in the distal segment (SI) of the O chromosome coincident in the 4 studied karyotypes: O3+4+16+2, O3+4+8, O3+4 and OST. The sequences corresponding to the 12 isogenic lines and to D. madeirensis and D. guanche indicated that in these three species of the subobscura cluster, the Hsp70IR locus consists of two 2.5 - 3.0 kb long paralogous copies in divergent orientation separated by a 0.5 - 1.4 kb nonduplicated central spacer region. The two copies show a high degree of conservation between the different gene arrangements and species analyzed, while the central spacer region is highly polymorphic. Among the most relevant aspects of polymorphism analyses, we highlight the high degree of conservation in the coding regions (CDSs) and the cis-regulatory elements (CREs) in the proximal promoter of all the analyzed hsp70 genes, which might indicate that these are functional in all studied lines, and that their regulation might be similar. Curiously, at the sequence level, the paralogous 5'-UTR and CDS regions tend to be significantly more similar within the same arrangement and, in some cases, within the same line, probably as a result of ectopic gene conversion. Lastly, we carried out the quantification of basal hsp70 mRNA and protein levels in adult males and females of six lines isogenic for the cold climate-associated OST and six for the warm climate-associated O3+4 arrangements. Basal mRNA quantification results indicate that the two arrangements exhibit similar levels, yet significant differences are observed between males and females of the warm O3+4 arrangement. Regarding the quantification of basal Hsp70 protein levels, these suggest that there are no differences between sexes nor between the two arrangements, but instead we observe a significant interaction between sex and arrangement. Overall, the results for both, mRNA and protein data, indicate that hsp70 expression might be influenced by sex.

Mise en evidence d'organisations affectant des fractions importantes du chromosome bacterien, de structure polarisee le long de l'axe origine-terminus de replication. Les fourches de replication progressent a l'inverse de la normale et ont une velocite tres diminuee sur environ 20% du chromosome. Les zones contraintes pour la replication sont non divisibles par des inversions, ce sont des remaniements genomiques conservatifs. Chaque zone contrainte abrite au moins deux de ces sites de pause, de meme polarite et de sequences voisines. En utilisant un mutant "terminaison-moins", les auteurs ont etabli que le caractere defavorable des inversions affectant ces memes regions n'est pas une consequence de l'augmentation du temps de replication due aux sites de pause inverses

Introducción El calentamiento global afecta de modo distinto a diferentes especies. Una de las especies en las que se ha documentado una respuesta genética a la adaptación térmica es Drosophila subobscura. Las clinas latitudinales en la frecuencia de muchas inversiones cromosómicas descritas en esta especie en las poblaciones originales del Paleártico y el descubrimiento de patrones clinales paralelos pocos años después de la colonización de América del Sur y del Norte han proporcionado una de las pruebas más convincentes de que las clinas de inversiones son el producto de la selección natural. Sin embargo, se desconoce el tipo de selección responsable del mantenimiento del polimorfismo cromosómico de inversiones asociado a las clinas. Tradicionalmente se han propuesto tres hipótesis selectivas para dar cuenta del polimorfismo cromosómico, las cuales abarcan distintas unidades de selección: el cromosoma, los genes coadaptados ("supergenes") y los genes individuales. Objetivos Para llegar a entender los mecanismos de selección en la especie D. subobscura, en este trabajo se ha estudiado: 1. La distribución de las ordenaciones cromosómicas a lo largo de un gradiente térmico; 2. La variación nucleotídica en seis genes incluidos en las tres ordenaciones cromosómica más frecuentes; 3. La base genética de la preferencia térmica y de la tolerancia al calor en líneas isocromosómicas. Resultados y Conclusiones Las frecuencias de las ordenaciones cromosómicas en general están correlacionadas con el gradiente de temperatura, formando clinas latitudinales. La ordenación OST se correlaciona positivamente con la latitud y su frecuencia aumenta conforme se avanza desde el sur hacia el norte. Inversamente, la frecuencia de O3+4+7 muestra una correlación negativa con la latitud: alcanza su máxima frecuencia en el sur de Europa y desaparece en el norte. La ordenación O3+4 también exhibe una correlación negativa con la latitud. Estas correlaciones indican que la ordenación OST está adaptada al frío, mientras que las otras ordenaciones pueden considerarse adaptadas a temperaturas más elevadas. La variación nucleotídica de las ordenaciones más frecuentes se analizó en dos poblaciones españolas distantes latitudinalmente. Aunque las frecuencias de las inversiones difieren entre ambas poblaciones, no se han detectado sin embargo diferencias nucleotídicas dentro de cada inversión entre las poblaciones. Se ha detectado flujo genético entre las diferentes inversiones, pero éste no es suficiente para evitar la existencia de diferenciación genética significativa entre las inversiones para todos los genes analizados. La diferenciación genética entre las ordenaciones también se detectó mediante el análisis de desequilibrio de ligamiento. Aparte de la baja tasa de recombinación entre las inversiones, la epistasis en eficacia entre algunos genes podría también contribuir a la diferenciación observada. Mediante la aplicación de diversas pruebas de neutralidad, se ha podido detectar la huella de la selección prácticamente en todos los genes analizados, ya sea en regiones codificadoras o no codificadoras. La hipótesis de la adaptación local es la que se ajusta mejor a nuestros datos, o sea, las inversiones mantienen complejos de genes coadaptados en inversiones de D. subobscura. Nuestros resultados corroboran que las ordenaciones del cromosoma O afectan la preferencia térmica en adultos en un gradiente termal producido en el laboratorio, y que moscas que llevan la ordenación OST adaptada al frío muestran una preferencia térmica hacia temperaturas más bajas que aquellas que tienen las ordenaciones O3+4 y O3+4+8 adaptadas al calor. Sin embargo, estas ordenaciones cromosómicas no tienen ningún efecto sobre la tolerancia al calor en adultos y, por lo tanto, podemos suponer que no hay covarianza genética entre ambos rasgos. La preferencia térmica y la tolerancia al calor en las líneas isocromosómicas de D. subobscura parecen pues ser genéticamente independientes, lo que podría impedir una respuesta coherente del comportamiento y la fisiología (es decir, la coadaptación) a la selección térmica.
Background Global warming is affecting many wild species in different ways. One of the species demonstrating thermal adaptation on the population genetic level is Drosophila subobscura. Latitudinal clines in the frequency of many chromosomal inversions of this species were well documented in the original Palearctic populations, and the discovery of parallel clinal patterns a few years after the colonization of South and North Americas provided compelling evidence that the clines evolved by natural selection. However, the selective process maintaining inversions in populations is not yet clear. Traditionally three selective hypotheses have been advanced to explain the maintenance of the chromosomal polymorphism, according to the level of operation of natural selection: chromosome, coadapted genes (“supergenes”) and individual genes. Objectives To distinguish between different hypotheses the following aspects were studied in D. subobscura: 1. The distribution of chromosomal arrangements along the thermal gradient; 2. The nucleotide variation in six genes inside the three most frequent chromosomal inversions; 3. The genetic basis of thermal preference and heat shock tolerance in isochromosomal lines. Results and conclusions The frequencies of the most abundant chromosomal arrangements in general correlated with temperature gradient, forming latitudinal clines. The arrangement OST positively correlated with latitude and its frequency increased from the south to the north. At the same time the frequency of O3+4+7 shows a negative correlation with latitude and reaches its maximum frequency in the south of Europe disappearing in the north. The O3+4 arrangement has a negative correlation with the latitude. Therefore, the arrangement OST is supposed to be cold-adapted while the other arrangements are considered to be warm-adapted. The nucleotide variation of the most frequent chromosome arrangements was analyzed in two distant Spanish populations situated along a latitudinal gradient. No within-inversion genetic differences were detected among populations, which suggest that the gene content along the gradient is rather constant for the various chromosomal arrangements and genetic flow is high. Although gene flux between different inversions was detected, significant genetic differentiation among inversions for all genes was found. Genetic differentiation between arrangements was also detected by linkage disequilibrium analysis, showing significant associations between informative sites when comparing arrangement pairs, which could be explained by low recombination rate between inversions and probable epistasis between some genes. The footprints of selection nearly in all genes, either in coding or noncoding parts, were detected using several neutrality tests. The Local Adaptation hypothesis is the one that fits better to our data and would explain the maintenance of the coadapted gene complexes within inversions in D. subobscura. Our results corroborate that arrangements on chromosome O affect adult thermal preference in a laboratory temperature gradient, with cold-climate OST carriers displaying a lower thermal preference than their warm-climate O3+4 and O3+4+8 counterparts. However, these chromosome arrangements did not have any effect on adult heat tolerance and, hence, we putatively discard a genetic covariance between both traits arising from linkage disequilibrium between genes affecting thermal preference and genes of heat shock resistance. Therefore, thermal preference and heat tolerance in the isochromosomal lines of D. subobscura appear to be genetically independent, which might potentially prevent a coherent response of behavior and physiology (i.e., coadaptation) to thermal selection. If this pattern were general to all chromosomes, then any correlation between thermal preference and heat resistance across latitudinal gradients would likely reflect a pattern of correlated selection rather than genetic correlation.

Drosophila subobscura és una espècie amb un polimorfisme cromosòmic molt ric, adaptatiu i que actualment està responent al canvi climàtic. Malgrat això, encara es desconeixen els mecanismes selectius que mantenen les inversions a les poblacions i fan que aquestes siguin adaptatives. Existeixen tres hipòtesis descrites per tal d’explicar el manteniment de les inversions a les poblacions: la hipòtesi de la Coadaptació de Dobszhansky, la hipòtesi de la selecció supergènica de Wassermann i la hipòtesi de l’Adaptació local de Kirkpatrick i Barton. L’objectiu del present treball ha estat estudiar a D. subobscura el paper de les inversions cromosòmiques en l’adaptació a l’ambient i com es mantenen a les poblacions naturals. D’aquesta manera, es va analitzar el contingut genètic de les 4 inversions més freqüents del cromosoma O en set poblacions europees, localitzades al llarg d’un gradient latitudinal. S’han utilitzat marcadors moleculars com ara loci microsatèl•lits i gens candidats a l’adaptació tèrmica. Els resultats han mostrat una alta uniformitat genètica de les inversions cromosòmiques al llarg de la clina latitudinal, suggerint que el contingut genètic d’un determinat ordenament cromosòmic és constant a totes les poblacions. A més, en comparar diferents inversions, s’ha trobat una gran diferenciació genètica, essent l’OST el mes diferent. El gran desequilibri de lligament trobat entre dos dels gens, localitzats a més de 14Mb de distància i dins i fóra, respectivament de la inversió O7, podria estar suggerint l’existència d’interaccions epistàtiques entre ells. El conjunt dels nostres resultats indiquen que la hipòtesi de l’adaptació local és la que millor explicaria el manteniment de les inversions del cromosoma O de D. subobscura. Per altra banda també, s’ha analitzat la relació de diferents ordenaments cromosòmics amb la termotolerància i la resposta al xoc tèrmic. Els nivells basals d’HSP70 en els individus homocariotips O3+4 són molt elevats i equivalents als nivells de proteïna mesurats després d’un xoc tèrmic en tots els homocariotips independentment del seu ordenament. Això, sumat a que presenten una major termotolerància i termopreferència en relació als individus portadors de l’ordenament OST, podria estar relacionat amb la clina latitudinal de freqüència que presenta d’aquest ordenament.
Drosophila subobscura is a species with a highly rich inversion polymorphism, which has been shown to be adaptive and responding to global warming. However, the selective pressures acting on inversion how are they maintained in the populations are still unclear. Three main hypotheses have been proposed to explain the adaptive value of inversions: the Coadaptation hypothesis by Dobszhansky, the supergene selection postulated by Wassermann and the Local adaptation hypothesis of Kirkpatrick and Barton. In the present work we aimed to study in D. subobscura the role of chromosomal inversions in the adaptation to the environment and how are they maintained in the populations. To discriminate between the different hypotheses, the genetic content of the four more frequent arrangements of the O chromosome was analyzed in seven different populations located along a latitudinal cline. Different molecular markers such as microsatellite loci and candidate genes for thermal adaptation were used. The results showed a high genetic uniformity of inversions along the latitudinal cline, suggesting that the genetic content of each arrangement it is constant in all populations. Moreover, when comparing different arrangements, a high differentiation was found, being the OST the most different. The strong linkage disequilibrium found between two of the genes, despite being located 14Mb apart and inside and outside O7 inversion respectively could suggest the existence of epistatic interactions between them. Thus, the Local Adaptation hypothesis is the one that fits better our data and would explain the maintenance of the arrangements of the O chromosome of D. subobscura. Moreover, we have studied the relationship of the different chromosomal arrangements with the thermotolerance and the heat shock response. The results showed that O3+4 outbred individuals presented very high basal values of Hsp70 levels, equivalent to those measured after a heat shock in all the homokaryotypes independently of the arrangement. This, together with higher thermotolerance and thermopreference when comparing with the individuals carrying the OST arrangement, could be related whit the latitudinal cline of frequency that the O3+4 arrangement presents.

Thesis (MSc)--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: This study examines the phylogeography of the southern African vlei rat, Otomys irroratus using the mtDNA cyt b gene and chromosomal data derived using G-, and C-banding, Ag-NOR staining and fluorescence in situ hybridization (FISH using flow sorts of Myotomys unisulcatus). A total of N = 102 specimens were used from the Western Cape, Eastern Cape, Northern Cape, Free State, Mpumalanga and KwaZulu-Natal provinces of South Africa. Of the N = 102, N = 55 comprised fresh material while N = 47 comprises museum material obtained from the Durban Natural Science Museum of South Africa. Cytogentic analysis of N = 55 specimens collected from seven localities in South Africa revealed intra-specific variation resulting from two rearrangements, namely pericentric inversions and heterochromatin variation. Of the 55 specimens that were analyzed 47% contained inversions or centromeric shifts on four autosomes (OIR1, OIR4, OIR6 and OIR10) which were present singly in specimens (i.e. none of the specimens contained all four inversions concurrently). These inversions were present in both homozygous and heterozygous state over a wide geographic range suggesting that they are floating polymorphisms. Given the potential role of inversions in post-mating isolation (through production of aneuploid gametes), the prevalence of inversions as floating polymorphisms in the vlei rats suggest that they are probably retained in the population through suppression of recombination in the inverted regions of the chromosomes. In addition, differences between populations is due to the presence or absence of heterochromatic arms (and not inversions), which cause variation in the NFa (40 – 49) and supernumerary B chromosomes, resulting in the variation in diploid number (2n = 28 – 32). Analysis of N = 55 specimens revealed Ag-NORs on 7 autosomal pairs 1, 2, 5, 7, 8 and 9 proximal to the centromere on the short arm of the chromosome. Pair 8 also displayed Ag-NOR at the distal end of the long arm of the chromosome in individuals from the Free State province. Pair 3 showed two Ag- NORs occurring proximal to the centromere on the short arm and on the terminal end of the long arm, respectively. I obtained 953bp of mtDNA cyt b from fresh material and 400bp from museum material. Using maximum parsimony and Bayesian inference two main clades were retrieved. Clade A specimens occur mainly in the Western and Eastern Cape provinces of South Africa. Clade B specimens occur in the Eastern Cape, Free State, KwaZulu-Natal, Northern Cape and Mpumalanga provinces of South Africa. The mean sequence divergence between the main clades (A and B) is 7.0% and between sub-clades comprising clade B is 4.8%, while within clade A the sequence divergence was 1.91%. Nested clade analysis revealed allopatric fragmentation within O. irroratus. Chromosomal characters also support the two evolutionary lineages as clade A has pericentric inversions which occur as floating polymorphisms which are absent in clade B. Clade B in turn is fixed for a complex tandem fusion rearrangement which is absent from clade A. Divergence date estimates indicate that the two clades separated around 1.1 MYA, which coincides with climate changes since the late Pliocene/Pleistocene epochs. Cladogenesis within this species complex could therefore have been influenced by habitat fragmentation. A full taxonomic review of O. irroratus is therefore warranted by this study.
AFRIKAANSE OPSOMMING: Die suider Afrikaanse vlei rot, Otomys irroratus word gekenmerk deur fenotipiese konservatisme regoor die spesie se verspreiding en het groot chromosomale variasie met diploied chromosoom getalle wat reeks vanaf 2n = 23 tot 2n = 32. Hierdie variasie binne O. irroratus het gelei tot die beskrywing van drie chromosomale groupe naamlik die A sitotipe wat gekenmerk word deur 'n akrosentriese komplement. Die tweede groep wat die B sitotipe genoem word besit ten minste agt chromosoom pare met heterokromatiese kort arms, onderwyl die derde group (die C sitotipe) vier chromosoom pare het met heterokromatiese kort arms. Hierdie studie bestudeer die bevolkings genetika struktuur van O. irroratus deur 102 monsters te analiseer wat gekollekteer was regoor die spesie se verspreiding binne Suid-Afrika en die mitochondriale merker sitokroom b sowel as chromosoom fluoressent hibridisasie te gebruik. Ek het 55 monsters van sewe lokaliteite binne Suid-Afrika sitogeneties geanaliseer deur gebruik te maak van G- en C-bandbepaling asook die hibridisasie patrone geproduseer deur die vloeisorteerde chromosoome van Myotomys unisulcatus. Die analise het gewys dat 47% van die monsters perisentromeriese inversies besit het, wat slegs aangetref was of die outosome OIR1, OIR4, OIR6 en OIR10. Hierdie inversies was nooit almal teenwoordig binne dieselfde monster nie en was gevind in beide heterosigotiese en homosigotiese vorm. Die inversies kom ook voor oor 'n wye verspreiding wat daarop aandui dat dit swerwende polymorfisme is. Omdat inversies lei tot die produksie van aneuploiede gamete speel hulle 'n belangrike rol in post-parings reproduktiewe isolasie, die verskyning van swerwende inversies binne vlei rotte dui dus daarop dat hulle onderhou word binne populasie verband deur die onderdrukking van rekombinasie in die gedeeltes van die chromosoom. Verdere verskille tussen populasies behels die voorkoms of afwesigheid van heterochromatiese kort arms wat (nie inversies) wat lei tot die variasies in die Nfa (40 – 49). Die variasie in diploied getal (2n = 28 – 32) is eksklusief as gevolg van B chromosoome. Ag-NOR banding het ook gewys dat daar twee evolusionêre lyne binne O. irroratus voorkom. Verder het filogenetiese analise van al die monsters verkryg deur volgorde-bepaling met behulp van maksimale parsimonie en Bayesian afleiding twee klades geidentifiseer. Klade A diere kom voor in die Wes en Oos-Kaap provinsies van Suid-Afrika terwyl klade B diere voorkom in die Oos-Kaap, Vrystaat, KwaZulu-Natal, Noord-Kaap en Mpumalanga provinsies onderskeidelik van Suid-Afrika. Die gemiddelde volgorde-bepalings verskille beloop 7% tussen die twee hoof klades (A en B) en tussen sub-klades 4.8%, terwyl binne klade A die verskille slegs 1.91% beloop het. Analise van die verwantskap tussen die klades het gewys dat allopatriese fragmentasie heel waarskynlik gelei het tot die populasie genetiese struktuur binne O. irroratus. Chromosoom karakters onderskraag die twee evolusionêre lyne waar klade A slegs perisentriese inversies besit wat swerwend wat ontbreek in klade B. Klade B op sy beurt besit 'n komplekse tandemme fusie wat glad nie voorkom in klade A nie. Molekulêre datering het verder gewys dat die twee klades omtrent 1.1 miljoen jaar gelede versprei het, wat ooreenstem met die klimaats veranderinge wat sedert die Peioceen en Pleistoceen plaasgevind het. Klade vorming binne die spesies komples kan daarom as gevolg van habitat fragmentasie plaasgevind het. Hierdie studie dus noodsaak 'n volle taksonomiese ondersoek van O. irroratus ten einde vas te stel hoeveel spesies binne die komplex voorkom.

In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

Orientador: Louis Bernard Klaczko
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O presente trabalho tem como objetivo caracterizar a variação do tamanho e forma das asas de populações de Drosophila melanogaster em três pontos ao longo de uma grande amplitude latitudinal na costa brasileira. O trabalho foi feito a partir de coletas realizadas no Recife, Rio de Janeiro e Porto Alegre, e os seguintes aspectos foram abordados: 1) variação geográfica; 2) variação temporal; 3) herdabilidade; e 4) a influência de inversões cromossômicas. Para este fim, o método da elipse foi aplicado a imagens digitalizadas das asas, e foram analisados: o tamanho das asas, sua forma e as posições dos pontos de junção e das extremidades das veias (caracterizadas por suas coordenadas angulares é radiais, essas padronizadas pelo tamanho da asa). Os resultados obtidos mostraram que a variação de tamanho em D. melanogaster no Brasil segue a tendência mundial de formação de clines latitudinais, com indiv.íduos maiores sendo encontrados a latitudes também maiores. No entanto, a herdabilidade e a variação temporal entre múltiplas coletas realizadas no Recife e no Rio de Janeiro não apresentou um padrão regular nítido. O único efeito consistente e significativo de inversões cromossômicas que pudemos observar foi o de In(3R)Payne sobre o tamanho corporal, sendo também consistente com achados prévios descritos na literatura. Entretanto, não detectamos efeito significativo de interação genótipo-ambiente, quer entre coletas, quer entre localidades
Abstract: The present work aims to characterize the variation of wing size and shape in Drosophila melanogaster populations from three localities distributed along a wide latitudinal range of the Brazilian coast. The work was performed based on collections made in the cities of Recife, Rio de Janeiro and Porto Alegre. The aspects studied were: 1) geographic variation; 2) temporal variation; 3) heritability; and 4) the influence of chromosomal inversions. To this end, the ellipse method was applied to digitized images of the wings. We analyzed wing size, wing shape and the position of vein junctions and extremities (characterized by their angular and radial coordinates, the latter being standardized by wing size). The results obtained showed that size variatiorn in Brazilian D. melanogaster follows the worldwide tendency toward the formation of latitudinal clines, with larger individuaJs being found at higher latitudes. However, the heritability and temporal variation among multiple collections performed in Recife and Rio de Janeiro did not show a clear regular pattern. The only consistent and significant effect of chromosomal inversions that we could observe was that of In(3R)Payne on body size, which is also consistent with previous findings reported in the literature. However, we did not detect a significant effect of genotype-environment interactions, neither among collections, nor among localities
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular

Orientadores: Louis Bernard Klaczko, Ana Maria L. Azeredo-Espin
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O grupo tripunctata e o segundo maior grupo neotropical de Drosophila em numero de espécies e foi incluído na radiação tripunctata ¿ que inclui outros grupos próximos. O segundo cromossomo de Drosophila medipunctata (espécie que pertence ao grupo tripunctata) e altamente polimorfico para inversões. Trabalhos anteriores sugeriram a presença de interação genotipo-ambiente para tamanho da asa, mas não para forma. Experimentos de laboratório foram realizados para testar os efeitos combinados de temperatura e inversão cromossômica no tamanho e forma da asa de D. mediopuncta. A morfologia da asa foi analisada por métodos de morfometria geométrica. Os resultados mostraram que tamanho e forma da asa são influenciados por temperatura, sexo e cariótipo. Também foram encontradas evidencias que sugeriram a existência entre os efeitos de cariótipo e temperatura para forma da asa, mas nao para tamanho, indicando a presença de interação genotipo-ambiente para forma da asa em D. mediopunctata. Sugerimos que outros fatores ecológicos ¿ tais como densidade de larvas ¿ ou variação sazonal de conteúdo genético das inversões poderia explicar os resultados anteriores. Alem disso, uma nova hipótese filogenética para a radiação tripunctata e sugerida com base em dados de seqüências dos genes mitocôndrias das subunidades I e II da citocromo oxidase, e a variação de tamanho e forma da asa e descrita com bases na nova hipótese filogenética. As arvores filogenéticas foram reconstruídas por métodos de parcimônia, máxima verossimilhança e inferência Bayesiana. Os resultados rejeitaram a hipótese de monofila para o grupo tripunctata, enquanto esta hipótese não foi rejeitada para a radiação tripunctata e outros grupos específicos dentro da radiação. Ambos tamanho e forma da asa apresentaram sinal filogenético e diferentes padrões de tamanho e forma puderam ser identificados para cada um dos agrupamentos mais importantes detectados pela analise filogenética
Abstract: The tripunctata group is the second largest Neotropical group of Drosophila in number of species and was included in the tripunctata radiation ¿ which comprises other closely related groups. The second chromosome of Drosophila mediopunctata (a species that belongs to the tripunctata group) is highly polymorphic for inversions. Previous work suggested the presence of genotype-environment interaction for wing size but not for shape. We performed experiments in the laboratory to test for the joint effects of temperature and chromosome inversions on size and shape of the wing in D. mediopunctata. Wing morphology was analyzed by methods of geometric morphometrics. Our findings show that wing size and shape are influenced by temperature, sex, and karyotype. We also found evidence suggestive of an interaction between the effects of karyotype and temperature on wing shape (but not for size), indicating the existence of genotype-environment interaction for wing shape in D. mediopunctata. We suggest that other ecological factors ¿ such as larval crowding ¿ or seasonal variation of genetic content within inversions may explain the previous results. Moreover, we suggest a new phylogenetic hypothesis for the tripunctata radiation based on sequences of mitochondrial genes of cytochrome oxidase subunits I and II, and describe wing size and shape variation in different species based on this new phylogenetic hypothesis. Phylogenetic trees were reconstructed by parsimony, maximum likelihood and bayesian inference. Results reject the monophyly hypothesis for the tripunctata group whereas monophyly is not rejected for the tripunctata radiation and other specific groups within the radiation. Both wing size and shape displayed phylogenetic signal and different patterns of size and shape could be identified for each of the most important clusters detected by phylogenetic analysis
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular

La détermination sexuelle chez les vertèbres repose sur différents mécanismes génétiques et epigenetiques. Chez les vertebres hétérothermes, la détermination du sexe de l'embryon ou de la larve par la température existe dans de nombreuses espèces. Chez l'amphibien pleurodèles waltl, une température d'élevage de 32c inverse le sexe des larves femelles génétiques. Cette inversion est fonctionnelle et permet ainsi l'obtention de thermoneomales phénotypiques. Nous avons étudié les bases génétiques de la détermination du sexe chez le pleurodèle en recherchant des gènes homologues de sry, déterminant testiculaire des mammifères. Une séquence sox (sry-box containing gène) a été clonée chez le pleurodèle mais elle ne joue sans doute aucun rôle dans la détermination sexuelle. Dans un deuxième temps, nous avons étudié l'activité de l'aromatase chez le pleurodèle. La régulation de l'expression de cette enzyme, qui catalyse la conversion des androgènes en strogenes, constitue un événement-clef de la différenciation sexuelle chez les vertebres hétérothermes. L'activité aromatase a été étudiée dans les conditions de différenciation sexuelle normale et sous l'effet de traitements thermiques et hormonaux. Des traitements de larves en cours de différenciation sexuelle par divers inhibiteurs, comme des inhibiteurs de l'aromatase, ou d'hormones, comme l'stradiol et la dihydrotestosterone, ont également été entrepris. Les résultats obtenus montrent le rôle primordial de l'stradiol et de l'aromatase dans la différenciation sexuelle chez le pleurodèle. La dihydrotestosterone pourrait également intervenir de façon importante dans la différenciation male.

Abstract In sympatric Drosophila species, songs produced by male wing vibration during courtship are an effective mechanism preventing interspecific matings and maintaining sexual isolation between different species. These songs can vary greatly even between closely related species. The aim of this study was to localise X chromosomal and autosomal genes affecting species differences in male courtship song and to study their interaction in the D. virilis group species. Various genes were probed by in situ hybridisation on the X chromosomes of six species of the group, which enabled us to use localised RFLP markers in QTL studies, as well as to compare gene arrangements of different species. Genetic analyses of differences between the songs of D. virilis and D. littoralis showed that species-specific song traits are affected both by X chromosomal and autosomal genes. The X chromosomal gene(s) having a major impact on pause and pulse length in male song were found to be located at the proximal region of the chromosome. Precise localisation of the song genes was, however, not possible due to multiple chromosome rearrangements restricting recombination between RFLP markers located on this area. The same problem was faced when studying hybrids between D. flavomontana and D. montana with less diverged X chromosomal gene arrangements. Interaction between the X chromosomal and autosomal song genes in determining male song traits was studied in four species belonging to the virilis and montana phylads of D. virilis group. The long pauses in courtship song were found to be mainly caused by X chromosomal song genes (or maternal / cytological factors), while pulse length was determined by X chromosomal genes interacting with autosomal genes. This confirms the important role of X chromosomal gene(s) in song evolution in the montana phylad species. The direction of dominance in hybrid songs suggests that the songs of the montana phylad species have been affected by directional selection favouring shorter pulses and longer pauses between sound pulses during their evolution. The levels and patterns of DNA polymorphism in an X-linked fused (fu) gene was studied in different D. montana populations. These studies revealed that D. montana populations are significantly but not completely isolated, and that a selective sweep at fu (or at a gene linked to fu) may be the reason for the reduced levels and patterns of variability of this gene in Finnish D. montana populations. The methods used in this study will be utilized to study variation in 'song genes' in the future.

La première partie de cette thèse rappelle les résultats expérimentaux concernant l'influence de la température sur le sex ratio des populations de pleurodèles issues de croisements intra et interspécifiques. L’analyse de ces résultats a permis la construction d'un modèle théorique de l'inversion sexuelle chez pleurodèles waltl et pleurodèles poireti. Ce modèle postule notamment l'inactivation des chromosomes sexuels. L’action de la température sur la différenciation sexuelle du pleurodèle a tout d'abord suggéré une médiation des protéines de choc thermique (HSPS). Les premiers essais de mise en évidence de ces HSPS chez les larves soumises à un traitement thermique ont été infructueux. En revanche une surexpression protéique a été détectée chez des adultes, non pas après un choc thermique mais consécutivement à une biopsie. Cette réponse est reproductible puisque plus de la moitié des adultes ont réagi en synthétisant cinq protéines de 110Kd, 48Kd, 46Kd, 44Kd et 15Kd dans les tissus immédiatement adjacents à la biopsie. Trois de ces protéines ont été utilisées comme antigènes. Les antisérums récoltés ont été testés sur des extraits protéiques d'organes adultes provenant de males et de femelles. L’un des antisérums (is48) reconnait un antigène de 55Kd (p55) uniquement dans les ovocytes. La seconde partie de la thèse décrit l'isolement et la caractérisation de cet antigène spécifique du sexe femelle, par conséquent d'un intérêt potentiel pour les études entreprises au laboratoire et pour tester le modèle d'inversion sexuelle exposé dans la première partie, ce qui nécessite l'accès à des marqueurs génétiques des chromosomes sexuels. La purification de p55 a permis de démontrer qu'il s'agit d'un mucopolysaccharide qui pourrait avoir un rôle dans la mise en place de la matrice extracellulaire grâce a une interaction avec des lectines ovocytaires

Les duplicacions segmentàries (DSs), o també anomenades duplicons o Low copy Repeats (LCRs), són regions de coma mínim 1 kb amb un alt nivell d'identitat (>90%), que estan presents almenys dues vegades en el genoma. La regió 8p23.1 consta de 6.5 Mb a la part distal del braç curt del cromosoma 8 i està flanquejada per duplicacions segmentàries. Degut a la seva arquitectura genòmica aquesta regió és susceptible a patir reordenaments mediats per recombinació homòloga no al·lèlica entre les DSs, com per exemple la inversió polimòrfica de 8p23.1 [inv(8)(p23)], present en un de cada quatre individus de la població general europea i japonesa, així com d'altres reorganitzacions menys corrents.
El treball realitzat en aquesta tesi doctoral pretén aprofundir en la caracterització de la complexa arquitectura genòmica d'aquesta regió. En la nostra primera aproximació a l'estudi de les DSs que flanquegen la regió cromosòmica 8p23.1, es va identificar una nova família gènica específica de primats, la família gènica FAM90A.Així, bona part d'aquesta tesi doctoral està centrada en l'anàlisi de l'origen, formació, evolució i expansió de FAM90A en els homínids.

Per altra banda també s'ha analitzant en detall la variabilitat de FAM90A com a variant en número de còpia (CNV) en diferents poblacions humanes.
Finalment, s'ha establert la freqüència de la inversió que afecta a 8p23.1 en població espanyola. També s'ha procedit a genotipar diversos individus homozigots per la inversió i s'ha predit l' estatus de la inversió en 150 individus del projecte HapMap i s'ha analitzat l'efecte que té aquesta reorganització sobre els nivells d'expressió dels gens de la regió.
Segmental duplications (SDs), also known as duplicons or Low Copy Repeats (LCRs), are regions of a minimum of 1 kb with a high sequence identity level (>90%), which are present at least two times in the genome. The 8p23.1 region extends 6.5 Mb at the distal part of the short arm of chromosome 8 and it is flanked by segmental duplications. Due to its genomic architecture the region is prone to suffer rearrangements mediated by non-allelic homologous recombination between these SDs, such as the polymorphic inversion of 8p23.1 [inv(8)(p23)], which is present in one out of every four of European and Japanese general population individuals, as well as other less frequent rearrangements.
The aim of the work presented in this doctoral thesis is to get insights in the characterization of the genomic architecture of this complex region. Our first approach to study the SDs flanking 8p23.1 region resulted in the identification of a novel gene family which is primate specific, the FAM90A gene family. Thus, this doctoral thesis is mainly focused on the analysis of the origins, formation, evolution and expansion of FAM90A in hominoids.

It has also been analyzed in detail the variability of FAM90A as a copy number variant (CNV) in different human populations.
Finally, it has been established the frequency of the inversion affecting 8p23.1 region in the Spanish population. Several homozygous inverted individuals have been genotyped and the status for the inversion has been predicted for 150 HapMap individuals, as well as the effect of this rearrangement on the gene expression levels of the genes contained in the region.

At the core of evolutionary biology stands the study of divergence between populations and the formation of new species. This dissertation applies a diverse array of theoretical and statistical approaches to study how chromosomes evolve. In the first chapter, I build models that predict the amount of neutral genetic variation in chromosomal inversions involved in local adaptation, providing a foundation for future studies on the role of these rearrangements in population divergence. In the second chapter, I use a large dataset of the geographic variation in frequency of a chromosomal inversion to infer natural selection and non-random mating, revealing that this inversion could be implicated in strong reproductive isolation between subpopulations of a single species. In the third chapter, I use coalescent models for recombining sex chromosomes coupled with approximate Bayesian computation to estimate the recombination rate between X and Y chromosomes in European tree frogs. This novel approach allows me to infer a rate so low that would have been hard to detect with empirical methods. In the fourth chapter, I study the theoretical conditions that favor the evolution of a chromosome fusion that reduces recombination between locally adapted alleles.
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Tese de mestrado em Biologia Humana e do Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
As síndromes de malformação congénitas são um dos principais grupos de patologias que afetam neonatos e crianças em países desenvolvidos. Muitos destes casos têm como base genética os arranjos genómicos ou cromossómicos. No entanto, por norma, devido à complexidade inerente às síndromes de malformação, é difícil e laborioso identificar com exatidão a alteração molecular que lhes deu origem. Aliado à inexistência atual de um genoma humano completamente anotado, torna-se complicado a compreensão e a previsão das consequências fenotípicas dos rearranjos cromossómicos. As inversões cromossómicas são rearranjos que ocorrem quando dois pontos de quebra ocorrem num mesmo cromossoma e são reinseridos invertidos, sem alteração de número de cópia. Normalmente as inversões são subclínicas, sem um fenótipo clínico associado. Se estes forem transmitidos a mais de 1% de uma dada população, tratam-se de polimorfismos. Se um rearranjo afectar transcritos ou a arquitetura genética junto dos pontos de quebra, perturbando assim o normal funcionamento dos genes, sobretudo os de expressão indispensável, este estará envolvido na etiologia de uma patologia potencialmente grave. Comparado com outros rearranjos cromossómicos, são poucas as inversões atualmente detalhadamente caracterizadas, frequentemente devido a dificuldades técnicas relacionadas com regiões repetitivas, frequentes nos pontos de quebra das inversões. Metodologias clássicas de citogenética são de baixa resolução e por vezes incapazes de identificar determinadas anomalias estruturais. As tecnologias atualmente mais avançadas para o estudo de rearranjos incluem microarrays genómicos, ideal na análise de variações no número de cópias, e a sequenciação de próxima geração (NGS), mais concretamente sequenciação pangenómica, para a generalidade dos rearranjos cromossómicos. Esta última tem a particularidade de ser eficiente na identificação de alterações crípticas, de oferecer uma potencial resolução bastante elevada (em certos casos nucleotídica) e de gerar grande quantidade de dados rapidamente. Das plataformas NGS existentes, as mais aptas para a análise de inversões envolvem a construção de bibliotecas mate-pair de grandes insertos, cuja distância entre pares de leitura é de 2 a 6 kb, permitindo superar dificuldades técnicas com zonas repetitivas e pequenas alterações junto aos pontos de quebra. Esta tese pretende identificar as alterações moleculares responsáveis pela síndrome de malformação congénita num indivíduo portador de uma inversão cromossómica pericêntrica aparentemente equilibrada de origem materna. O caso índex, portador da síndrome de malformação, apresenta acentuado atraso de desenvolvimento mental e psicomotor, dismorfia facial e perturbações do espectro do autismo. Ele tem muito baixo peso e altura para a idade. Foram também diagnosticadas cardiopatias, criptorquidia, escoliose e hipotonia generalizada. Estudos citogenéticos detetaram a existência de uma inversão pericêntrica no cromossoma 2, também encontrada na mãe. Os pais têm fenótipo aparentemente normal. Primeiramente, procedeu-se à identificação de alterações estruturais desequilibradas no indivíduo índex. Foram detetaram várias alterações de número de cópia, na maioria pequenas (< 100kb) e sem envolver genes OMIM, com a exceção da duplicação de 590 kb em 2q21.1. Os genes na duplicação não aparentam estar relacionados com o fenótipo observado. Ademais, foi detetado uma duplicação de 610 kb no pai nesta mesma região genómica, sugerindo que se trata de uma alteração de origem paterna, muito provavelmente não-patogénica e possivelmente de natureza polimórfica. Sequenciação pangenómica de grandes insertos (large-insert whole-genome sequencing) usando ácido desoxirribonucleico (ADN) do caso índex foi realizada para a identificação dos pontos de quebra da inversão no cromossoma 2. Uma vez delimitado a região dos pontos de quebra por NGS, foram desenhados oligonucleotídeos específicos para a amplificação dos fragmentos de junção e, seguidamente, procedeu-se à análise de segregação familiar e determinação nucleotídica dos pontos de quebra através de sequenciação Sanger. O estudo do perfil de expressão genética foi feito com Human Transcriptome Assay (HTA 2.0) da Affymetrix, utilizando ácido desoxirribonucleico (ARN) da linha celular linfoblastóide do indivíduo índex. Os dados obtidos por NGS permitiram a redefinição da localização genómica da inversão. O cariótipo do caso índex foi assim redefinido como 46, XY, inv(2)(p16.1q14.3)mat. Os pontos de quebra da inversão no cromossoma 2, no caso índex e na sua mãe, foram determinados. Estes localizam-se na posição chr2:55,935,064 e chr2:123,767,685 (GRCh37), respetivamente, nas bandas p16.1 e q14.3. Na sequência invertida ocorreu a deleção de 5 bases. Os pontos de quebra da inversão são iguais em ambos os indivíduos, sem quaisquer alterações detetadas nos fragmentos de junção. Segundo a nomenclatura baseada em citogenética de próxima geração, esta inversão é descrita como seq[GRCh37] inv(2)(pter→2p16.1(55,935,06{1-3})::2q14.3(123,767,68{3-1})→2p16.1 (55,935,06{5-4})::2q14.3(123,767,68{4-5})→qter). Os pontos de quebra não interrompem diretamente genes conhecidos. Em inv2p16.1, este é flanqueado a 5’ pelo gene polirribonucleotídeo nucleotidiltransferase 1 (PNPT1; chr2:55,861,198-55,921,045, GRCh37; OMIM *610316), e a 3’ pelo gene proteína 1 da matriz extracelular tipo-fibulina contendo EGF (EFEMP1; chr2:56,093,097-56,151,298, GRCh37; OMIM *601548) a 158 kb. O PNPT1 está envolvido na cadeia respiratória mitocondrial. Mutações em homozigotia foram associadas com deficiência na fosforilação oxidativa (OMIM #614932), originando nomeadamente encefalopatias, e com a surdez hereditária autossómica recessiva 70 (OMIM #614934). Em murganhos, tem expressão acentuada na cóclea. EFEMP1 é essencial para a correta formação de fibras elásticas em tecido conjuntivo, tendo elevada expressão nos pulmões e esófago em murganhos, e baixa no cérebro e coração. Mutações neste gene estão descritas como causa genética da distrofia da retina de Doyne (OMIM #126600), patologia autossómica dominante com início na segunda década de vida, causando perda progressiva de visão. O ponto de quebra em inv2q14.3 situa-se numa região pobre em genes. O gene translina (TSN; chr2:122,513,120-122,525,428, GRCh37; OMIM *600575) franqueia o ponto de quebra proximamente a 1240 kb, enquanto o gene tipo-proteína associada à contatina 5 (CNTNAP5; chr2:124,782,863-125,672,953, GRCh37; OMIM *610519) localiza-se 1020 kb distal do ponto de quebra. TSN codifica uma proteína que reconhece sequências-alvo em junções de pontos de quebra de translocações em doentes com leucemia, e está envolvido no mecanismo de reparação de ADN e transporte de ARN em neurónios. Em murganhos, expressa-se preferencialmente no tecido adiposo. O CNTNAP5 produz uma proteína que atua no sistema nervoso como moléculas de adesão celular e de recetor na comunicação intercelular. Em murganhos, expressa-se predominantemente no sistema nervoso. Existe suspeita de que mutações pontuais possam conferir suscetibilidade a comportamentos do espectro do autismo. Quanto à expressão genética, os resultados mostraram que os genes que flanqueiam a inversão não aparentam ter nível de expressão significativamente alterada comparativamente com os controlos. O estudo aprofundado de expressão a nível genómico está a decorrer. Os restantes genes próximos dos pontos de quebra da inversão relevaram baixa probabilidade de serem as alterações causadoras do fenótipo, nomeadamente a nível das doenças associadas. Tendo em conta os resultados obtidos, especialmente a confirmação da origem materna da inversão, esta alteração não aparenta ser a principal e única causa molecular do fenótipo. Ademais, esta conclusão é suportada pela pouca sobreposição clínica dos genes flanqueadores com a síndrome de malformação congénita, e da expressão génica aparentemente não alterada. Assim, atualmente, a relação causal entre o fenótipo observado e a inversão no cromossoma 2 foi excluída. Esta inversão é muito provavelmente não-patogénica por si só. Até ao momento e com os dados disponíveis, não foi possível identificar genes candidatos nem as alterações moleculares por detrás da síndrome de malformação congénita no caso índex. Informação médica disponível exclui influência de fatores ambientais na embriogénese. Futuramente, sugere-se recorrer à sequenciação do exoma, visto que tem uma sensibilidade muito superior para a deteção de pequenas em exões, potencialmente não detestáveis pelas abordagens até ao momento utilizadas. Adicionalmente, o estudo nos restantes membros da família permitirão obter uma melhor visão sobre a segregação familiar.
Congenital malformation syndromes can be caused by genomic and/or chromosome rearrangements. It is difficult to establish the underlying causes of malformations because of their high level of complexity. Although balanced chromosome inversions are in most cases subclinical, those disrupting transcripts or affecting the genomic architecture at breakpoint regions may well be pathogenic. Currently, the lack of a fully annotated human genome hinders the predictability of the phonotypic consequences of such rearrangements. The aim of this study is the identification of potential candidate genes for a malformation syndrome in an individual with an apparently balanced maternally inherited pericentric chromosome inversion inv(2)(p16.1;q14.3)mat. The proband has severe congenital malformation with multiple psychomotor and developmental anomalies, dismorphism and autistic features. The parents are phenotypically normal. Classical cytogenetic methods are of low resolution, often in the magnitude of a 5 to 10 Mb. Whole-genome Next-Generation Sequencing (NGS) of large-insert sequencing library (liWGS) has the capability to detect structural rearrangements with incomparably higher resolution, including cryptic alterations. As consequence, it was applied for the identification of inv(2)(p16.1;q14.3) breakpoints in the proband. Familial segregation analysis and definition of the inversion breakpoints at a nucleotide resolution were performed by amplification of junction fragments and Sanger sequencing. Genome and transcriptome array analysis were also carried out, for detection of additional genomic alterations and for gene expression profiling, respectively. Additionally, a possibly polymorphic duplication at 2q21.1, inherited from his father, was found. No apparent pathogenic genomic imbalances were identified in the proband. The inversion breakpoints are located at chr2:55,935,064 and chr2:123,767,685 (GRCh37), respectively, in 2p16.1 and 2q14.3. The inv2p16.1 breakpoint is flanked 14 kb proximal by the gene polyribonucleotide nucleotidyltransferase 1 (PNPT1; chr2:55,861,198-55,921,045, GRCh37; OMIM *610316) and 172 kb distal by EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1; chr2:56,093,097-56,151,298, GRCh37; OMIM *601548). PNPT1, highly expressed in mice cochlea, has been associated with deafness (OMIM #614934) and with combined oxidative phosphorylation deficiency (OMIM #614932), both autosomal recessive. Meanwhile, the autosomal dominant Doyne honeycomb retinal dystrophy (OMIM #126600) is reported to be associated with mutations in EFEMP1. This gene is essential for the formation of elastic fibers in connective tissue. The 2q14.3 breakpoint is in a gene-poor region. Located 1.2 Mb proximal to the breakpoint is translin (TSN; chr2:122,513,120-122,525,428, GRCh37; OMIM *600575). Involved in DNA damage repair and RNA trafficking in neurons, TSN codes for a protein that specifically binds to breakpoint junctions of translocations in acute leukemia. The gene contactin-associated protein-like 5 (CNTNAP5; chr2:124,782,863-125,672,953, GRCh37; OMIM *610519) is localized 1 Mb, distal. CNTNAP5 is involved in cell adhesion and intercellular communication. Susceptibility to autistic syndromes has been suspected. The above described breakpoints at nucleotide resolution are the same in the proband’s mother, and did not directly disrupt any gene. Publicly available clinical information on alterations affecting the inversion flanking genes revealed no major similarity with the proband’s phenotype. Furthermore, no significant alteration in their expression level was observed. In-depth analysis of genome-wide expression data is in progress. Based on these findings, the causal relationship between clinical phenotype and the inv(2)(p16.1;q14.3) is most likely excluded, since the inversion is most likely non-pathogenic. Therefore it is not yet possible to identity the underlying genetic cause of the malformation syndrome reported in this subject. Whole-exome sequencing is proposed as a future task to detect the disease causing alteration. This study highlights the application of NGS-based methodology, with its capability in mapping chromosome inversion breakpoints at a very high resolution. Large scale application of this approach will represent a hallmark in the characterization of congenital malformations associated with structural chromosomal abnormalities.

A. de novo chromosomal aberration in a female with severe mental retardation and dysmorphic features has been characterized cytogenetically (Dill et al. 1987). The patient’s karyotype was described as 46,XX,inv dup(8)(p12→23.1). Previous Southern blot dosage analysis of the patient’ s DNA with a probe from the D8S7 locus, which maps to 8p23→8pter (Wood et al. 1986), demonstrated that the patient was monosomic for this locus. This dosage abnormality was interpreted as a consequence of the chromosomal rearrangement, suggesting that the aberrant chromosome was a duplication deficiency chromosome. We have reinvestigated this patient using fluorescent in situ hybridization using cosmids from a flow sorted chromosome 8 library as well as an 8p painting probe mixture generated by Mu element mediated PCR. Both the normal and the inversion duplication chromosome p arms are uniformly labelled by the 8p painting probe mixture. Hybridization of a cosmid from the D8S7 locus results in a hybridization signal on the normal chromosome 8 and a complete lack of signal on the inversion duplication chromosome 8. Hybridization of a cosmid from the D8S133 locus, localized to 8p21→8cen using a hybrid cell panel (Wagner et al. 1991), provides a single hybridization signal on the normal chromosome 8 and a double hybridization signal on the aberrant chromosome. The pattern produced by this double signal is suggestive of an inversion duplication chromosome. These studies directly confirm both the origin of the extra chromosomal material and that the duplication chromosome has undergone deletion.

碩士
國立臺灣大學
生態學與演化生物學研究所
100
Chromosomal inversion polymorphisms have been demonstrated to play adaptive roles in natural populations by capturing local co-adapted alleles within recombination-suppressed regions of inversion heterozygotes. On the other hand, a less studied but important role of inversion polymorphisms is that recombination suppression by heterozygous chromosomal rearrangement may accumulate recessive deleterious mutations and thus cause a great amount of mutation load. Deleterious mutations will be eliminated when homozygotes and in turn increase heterozygosity of various inversions. The Afrotropical population of Drosophila melanogaster with high chromosomal inversion heterozygosity and high ratio of recessive lethals provides an ideal material to test any association between them by examining the accumulation pattern of recessive lethals. Recessive lethals are predicted to locate near the inversion breakpoints where the recombination is greatly suppressed by inversion heterokaryotypes. By using recombination and deficiency mappings, 14 recessive lethal alleles from eight lethal-bearing third chromosomes (each from eight distinct isofemale lines, respectively) were identified. All of recessive lethals were mapped into the regions close to (less than 3 centi-Morgan) the inversion breakpoints which were polymorphic in the African population. This result clearly shows that recessive lethal alleles are accumulated by recombination suppression and distributed non-randomly along inverted chromosomes. The data also provide the strong association between chromosomal inversions and the accumulation of recessive deleterious mutations.

Speciation is a constantly ongoing process whereby reproductive isolating baririer build up over time until groups of organisms can no longer exchange genes with each other. Adaptation is thought to play a major role in the formation of these barriers, although the genetic mechanisms and geographic mode underlying the spread of barriers due to adaptive evolution is poorly understood. Critically, speciation may occur in stages through the formation of intermediate partially reproductively isolated groups. The idea of such widespread ecotypes has been the subject of great controversy over the last century. Even so, we have relatively little understanding about whether widespread ecotypes exist, wheather they are reproductively isolated, and how adaptive alleles are distributed among partially isolated groups. In this dissertation, I examined these issues in widespread coastal perennial and inland annual ecotypes of the yellow monkeyflower, Mimulus guttatus. First, I determined that coastal and inland populations comprise distinct ecotypic groups. I then determined that these ecotypes are adapted to their respective habitats through genetically based flowering time and salt tolerance differences. I assessed the genetic architecture of these adaptations through quantitative trait loci (QTL) analysis and determined the geographic distribution of the underlying alleles through latitudinally replicated mapping populations. I quantified the contribution of these loci to adaptation in the field through the incorporation of advance generation hybrids in reciprocal transplant experiments. In the process, I discovered a widespread chromosomal inversion to be involved in the adaptive flowering time and annual/perennial life-history shift among the ecotypes. Overall, the results of this study suggest that widespread reproductively isolated ecotypes can form through the spread adaptive standing genetic variation between habitats and that chromosomal rearrangements can integral to this process.

Dissertation