京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18135号 / 医博第3855号 / 新制||医||1001(附属図書館) / 30993 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 亮, 教授 三森 経世, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19886号 / 医博第4135号 / 新制||医||1016(附属図書館) / 32963 / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 松田 文彦, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The focus of this diploma thesis is on mosaic numerical and structural chromosomal aberrations. In its theoretical part, general problems of mosaicism, its phenotypic effect, mechanisms of origin, related epigenetic modifications, and diagnostic options are described. The methodical part of the thesis then primarily refers to fluorescence in situ hybridization (FISH) and its application in the diagnostics of mosaicism. This method was used in the examination of 29 patients with numerical as well as structural abnormalities of autosomes or gonosomes with proven or suspected mosaicism. On the basis of this analysis, possible errors of measurement were determined and data for statistic evaluation were retrieved. For the examinations of three patients an alternative of the comparative genomic hybridization, the array CGH technique, was applied. The FISH method, although being based on random selection and human factor, proved sufficient sensitivity as well as specificity in the field of low-frequency mosaicism diagnostics. The main critical factors responsible for potential misinterpretation of the data arose from inherent characteristics of the biological material, incorrect targeting of the analysis, probe instability, bleed through effect and absence of mitosis during the structural aberrations analysis.

Tesis por compendio / [ES] La domesticación de los cítricos es un proceso en gran parte desconocido. Las investigaciones llevadas a cabo hasta la fecha indican que las variedades de cítricos que se comercializan en la actualidad son, en general, el producto de introgresiones ancestrales intraespecíficas e interespecíficas. Las introgresiones de tipo intraespecífico tuvieron lugar entre las poblaciones de dos subespecies antiguas de mandarinas y aportaron el comportamiento apomíctico que la mayoría de las mandarinas actuales y otros cítricos relacionados conservan todavía hoy. Una vez dichas mezclas genéticas o admixtures quedaron establecidas, otras introgresiones interespecíficas de pummelo establecieron nuevas características en el genoma de mandarina. Durante el proceso de domesticación, la variabilidad genética de estos cítricos se adquirió fundamentalmente a través de mutación espontánea, y mediante la práctica del injerto los genotipos carentes de semillas se extendieron rápidamente, acentuándose el papel de las mutaciones en la selección de cítricos como las naranjas y las mandarinas modernas. En este trabajo, mostramos que las mutaciones somáticas en cítricos se propagan siguiendo un patrón iterativo determinado por un modelo de ramificación simpodial y, en consecuencia, se agrupan en sectores a lo largo del árbol, donde algunas mutaciones quedan permanentemente fijadas. En este escenario, un árbol puede considerarse un mosaico genéticamente compuesto de diferentes genomas, en el que las ramas más jóvenes acumulan un número mayor de mutaciones. En promedio, nuestro árbol experimental de Clementina de 36 años muestra una tasa de mutación de 4.4 × 10-10 bp-1 yr-1, pudiendo contener en total entre 1500 y 5000 variantes y producir 1 mutación somática en cada meristemo axilar. Este número relativamente alto de mutaciones está en línea con el gran número de variedades derivadas de mutaciones espontáneas que se comercializan en cítricos. Para identificar caracteres esenciales adquiridos durante la domesticación, se han analizado los transcriptomas de la pulpa de frutos en desarrollo de la variedad silvestre e incomestible papeda de Ichang, la mandarina ácida Sun Chu Sha Kat y tres segregantes comestibles derivados de un cruce entre mandarinas comerciales. Basándonos en los resultados obtenidos, se propone que durante la transición de las papedas incomestibles a las mandarinas ácidas, la domesticación se caracterizó por una primera fase de cambios radicales en la expresión de los genes que regulan rutas fundamentales tanto del metabolismo primario como del secundario. Esta fase estuvo determinada principalmente por la estimulación de los procesos de crecimiento y la reducción de las defensas químicas, constituidas por compuestos de sabores desagradables. También se sugiere que en una segunda fase los atributos asociados a la palatabilidad de las mandarinas, especialmente la acidez, se mejoraron progresivamente a través de modificaciones específicas. Otros cambios de relevancia se relacionaron con la regulación de genes involucrados tanto en la síntesis de sustancias básicas de agradable aroma y sabor, como en la modulación de transportadores de azúcares. Por lo tanto, la transición entre las papedas incomestibles y las mandarinas de tipo ácido se caracterizó esencialmente por una drástica reprogramación de la expresión génica de rutas metabólicas fundamentales, mientras que las mandarinas modernas evolucionaron posteriormente mediante un refinamiento progresivo de las propiedades relacionadas con la palatabilidad. En su conjunto, estas observaciones sugieren que en cítricos, las tasas de mutación de los tejidos somáticos son consistentes con la idea de que las mutaciones desempeñaron un papel importante en las últimas fases de la domesticación. / [CAT] La domesticació dels cítrics és un procés en gran part desconegut. Les investigacions poratades a terme fins al moment indiquen que les varietats de cítrics comercialitzades hui en dia són, en general, el producte de introgressions ancestrals intraespecífiques i interespecífiques. Les introgressions de tipus intraespecífic van tindre lloc entre les poblacions de dues subespècies antigues de mandarina tot aportant el comportament apomíctic que la majoria de les mandarines actuals i altres cítrics relacionats encara conserven. Una vegada que aquestes barreges genètiques o admixtures es consolidaren, diferents introgressions interespecífiques de pummelo establiren noves característiques en el genoma de mandarina. Durant el procés de domesticació, la variabilitat genètica dels cítrics va ser assolida mitjançant mutació espontània i, per mitjà de la pràctica de l'empelt, genotips sense llavors es van estendre ràpidamente, accentuant el paper de les mutacions en la selecció de cítrics com les taronges i les mandarines modernes. En aquest treball, mostrem com les mutacions somàtiques en cítrics es propaguen seguint un patró iteratiu determinat per un model de ramificació simpodial i, en conseqüència, queden agrupades al llarg de l'arbre, on algunes d'aquestes mutacions romanen fixades. En aquest escenari, un arbre pot ser considerat un mosaic genèticament format per diferents genomes, en el qual les branques més joves acumulen un nombre major de mutacions. De mitjana, el nostre arbre experimental de Clementina mostra una ràtio de mutació de 4.4 × 10−10 bp−1 yr−1, arribant a incloure en total entre 1500 i 5000 variants i produint-hi 1 mutació somática en cada meristem axil·lar. Aquest nombre relativament elevat de mutacions està en línia amb el gran nombre de varietats provinents de mutacions espontànies que es comercialitzen en cítrics. Per tal d'abordar la identificació de trets essencials adquirits durant la domesticació, s'ha analitzat el transcriptoma de fruits en desenvolupament de la varietat silvestre i incomestible papeda d'Ichang, la mandarina àcida Sun Chu Sha Kat i tres segregants comestibles derivats d'un creuament entre mandarines comercials. Basant-nos en els resultats obtinguts, s'ha proposat que durant la transició de les papedes incomestibles a les mandariens àcides, la domesticació es va caracteritzar per una primera fase de canvis radicals en l'expressió dels gens que regulen rutes fonamentals del metabolisme primari i secundari. Aquesta fase va estar determinada principalment per l'estimulació dels processos de creixement i la reducció de les defenses químiques de gust desplaent. També es suggereix que en una segona fase atributs associats a la palatabilitat de les mandarines, especialment l'acidesa, van ser millorats progressivament mitjançant modificacions específiques. Altres canvis de rellevància van estar relacionats amb l'estimulació de gens involucrats tant en la síntesi de substàncies bàsiques d'agradable aroma i sabor, com en la modulació de transportadors de sucres. Per tant, la transició entre les papedes incomestibles i les mandarines àcides va estar caracteritzada essencialment per una dràstica reprogramació de l'expressió gènica de rutes metabòliques fonamentals mentre que les mandarines modernes evolucionaren posteriorment mitjançant un refinament progressiu de propietats relacionades amb la palatabilitat. En conjunt, aquestes observacions suggereixen que, en els cítrics, les ràtios de mutació dels teixits somàtics són consistents amb la idea que les mutacions tingueren un paper important en les últimes fases de la domesticació. / [EN] Citrus domestication is a largely unknown process. Research carried out to date indicates that current commercial citrus varieties are, in general, the product of ancestral intraspecific and interspecific introgressions. Intraspecific introgressions took place between the populations of two antique subspecies of mandarins and contributed to the apomictic behavior that most current mandarins and other related citrus still retain today. Once these genetic admixtures were established, interspecific pummelo introgression brought new traits to the admixtured mandarin genome. During domestication, genetic variability of these citrus occurred mainly through spontaneous mutations and with the practice of grafting, seedless genotypes expanded rapidly, emphasizing the role of mutations in the selection of citrus such as oranges and modern mandarins. In this work, we provide evidence that somatic mutations in citrus propagate following an iterative pattern determined by the sympodial branching model and consequently are grouped in sectors along the tree, where some of them remain fixed. In this scenario, a tree can be considered a mosaic genetically composed of different genomes, in which younger branches accumulate greater number of mutations. On average, our 36-yr-old experimental Clementine tree shows a mutation rate of 4.4 × 10-10 bp-1 yr-1, may carry a total of 1,500 to 5,000 variants and produces 1 somatic mutation per axillary meristem. This relatively high number of mutations is in line with the large number of varieties derived from spontaneous mutations that are commercialized in citrus. To identify key traits elicited by citrus domestication, we analyzed transcriptomes from developing fruit pulp of wild inedible Ichang papeda, Sun Chu Sha Kat sour mandarin and three palatable segregants derived from a cross between commercial mandarins. Based on these results, we propose that during the transition from inedible papedas to sour mandarins, domestication involved a first phase of major changes in the expression of genes regulating central pathways of primary and secondary metabolism. This stage was mainly characterized by both the stimulation of growth processes and the reduction of distasteful chemicals defenses. It is also suggested that in a second phase, edible attributes of mandarins, especially acidity, were progressively improved through specific modifications. Other relevant changes included upregulation of genes involved in the synthesis of key substances of pleasant aroma and flavor, and the modulation of sugar transporters. Thus, the transition from inedible papeda to sour mandarin was essentially defined by a drastic reprogramming of gene expression of fundamental metabolic pathways, while modern mandarins evolved later through progressive refinement of palatability properties. Taken together, these observations suggest that the rates of mutations occurring in citrus somatic tissues are consistent with the idea that they have played an important role during the later steps of citrus domestication. / This research is co-funded by the Ministerio de Ciencia, Innovación y Universidades (Spain) through grant RTI2018-097790-R-100 and by the European Union through the European Regional Development Fund (ERDF) of the Generalitat Valenciana 2014- 2020, through grants IVIA 51915 and 52002 / Pérez Román, E. (2022). Mutation Rate and Gene Expression in Genetic Admixtures of Domesticated Citrus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191452 / Compendio

The primary aim of this study was to determine the effectiveness of array-based technology for detecting and quantifying the presence of mosaicism. This aim was achieved by studying individuals having mosaicism for Down syndrome. SNP arrays were performed on 13 samples from individuals with mosaicism for trisomy 21, 13 samples from individuals with normal chromosome 21complements (negative controls) and 5 samples from individuals with full or partial trisomy 21 (positive controls). In addition, BAC arrays were processed on 6 samples from individuals with mosaicism for trisomy 21, 3 negative controls and 1 positive control. These studies have shown that array-based technology is effective for detecting mosaicism that is present in 20% or more cells with the results being consistent for both platforms. We also demonstrated the strength of array-based technology to identify previously unrecognized chromosomal mosaicism. A second aim of this study was to gain insight regarding the effect that trisomy 21 has on telomere attrition and the frequency of chromosomal instability. This study provides the first reported measure of both chromosome-specific telomere lengths and the frequency of acquired chromosome abnormalities in trisomic cells and isogenic euploid cells obtained from the same individuals. A chromosome-specific telomere length assay was performed on lymphocytes obtained from 24 young individuals with mosaicism for Down syndrome. While differences in overall telomere signal intensities were observed between the euploid and trisomic cells within a person, strikingly similar profiles for chromosome-specific telomere intensities were observed between the cell types within a person. Analyses were also completed on lymphoblast samples obtained from 8 older individuals with mosaicism for Down syndrome, including 5 individuals without dementia and 3 individuals with dementia. In the older study subjects, a significant inverse correlation was observed between telomere length and the frequency of micronuclei, suggesting that telomeric shortening is leading to an increased frequency of chromosomal instability, possibly through dicentric chromosome formation. However, further studies of more individuals, especially additional analyses of older individuals, are needed. These future studies may help to identify genomic regions of interest and serve to inform investigators of potential candidate genes in the etiology of dementia.

Le rétinoblastome est une tumeur rare qui touche la rétine du jeune enfant. L’inactivation bi-allélique du gène RB1 est à l’origine du développement tumoral. RB1 est le premier gène suppresseur de tumeur qui ait été identifié et la prédisposition au rétinoblastome constitue un véritable paradigme de la prédisposition aux cancers. Dans les formes non prédisposées génétiquement, les deux mutations apparaissent dans une cellule rétinienne unique ; le rétinoblastome est alors unilatéral. Dans les formes à prédisposition génétique, la première mutation est constitutionnelle et la deuxième est somatique. La mutation constitutionnelle est une néo-mutation pré- ou post- zygotique dans les formes sporadiques, alors qu’elle est héritée dans les formes familiales. Dans les formes avec prédisposition génétique, le diagnostic est plus précoce que dans les formes sans prédisposition et la bilatérisation du rétinoblastome est généralement la règle. Néanmoins, de rares familles présentent une pénétrance réduite et une variabilité phénotypique se traduisant par la coexistence de patients atteints de rétinoblastome bilatéral ou unilatéral, d’apparentés porteurs sains et d’apparentés présentant des rétinomes. Les mécanismes responsables de la variabilité phénotypique intrafamiliale sont inconnus et l’existence de facteurs génétiques modulant le phénotype tumoral est probable.L’origine de la variabilité de l’expression phénotypique du rétinoblastome peut être la résultante (i) de l’existence de mutations en mosaïque, (ii) de mutations de RB1 et (iii) de facteurs modificateurs génétiques indépendants du locus de RB1. Trois axes distincts et originaux basés sur ces origines possibles de variabilité phénotypique ont été développés pour caractériser les relations génotype/phénotype dans le rétinoblastome. Premièrement, les conséquences d’une mosaïque somatique ont été illustrées grâce à l’étude d’une famille ayant bénéficié de cinq diagnostics prénatals. Dans ces familles, certains fœtus porteurs de l’allèle à risque identifié par une approche indirecte basée sur l’étude de microsatellites au locus de RB1, n’étaient pas porteurs de la mutation du parent atteint, lui-même atteint d’un rétinoblastome bilatéral. Ainsi, nous avons démontré la présence d’une mosaïque somatique et gonadique chez ce parent lourdement atteint. La conséquence de l’existence de patients présentant une mosaïque dans le cadre du conseil génétique a été discutée. La suite de nos travaux a pris en compte ces résultats afin de limiter les biais que pourrait induire la présence de mutations en mosaïque dans des études de corrélation génotype/phénotype dans le rétinoblastome. Deuxièmement, l’association de grandes délétions emportant RB1 avec des retards psychomoteurs chez des patients atteints de rétinoblastome a été étudiée. Une approche de CGH hautement résolutive, ciblée sur le locus de RB1, a été mise en place afin de caractériser le rôle des gènes contigus de RB1 dans ce syndrome. Ainsi, cette approche a permis de définir une zone à risque de retard psychomoteur que nous proposons comme seuil d’alerte pour le généticien. Cette zone définit un gène, PCDH8 d’expression cérébrale exclusive, comme un excellent candidat au retard psychomoteur. Enfin, troisièmement, une approche « gène candidat » reposant sur l’étude du SNP309 du promoteur de MDM2, a été mise en œuvre. / Retinoblastoma is the most common intraocular childhood cancer and occurs when both alleles of the RB1 gene are inactivated in the retina. In patients without genetic predisposition, the two mutations occurred in a single unique retinal cell. In subjects with a genetic predisposition to retinoblastoma, the first RB1 mutation is found in the germline and the second appears as a somatic mutation. Germline carriers usually develop bilateral or multifocal tumors and the diagnosis is earlier. However, some rare families exhibit low penetrance and variable expressivity of the disease because bilaterally affected, unilaterally affected, and unaffected mutation carriers are known to coexist. The existence of genetic modifiers in retinoblastoma therefore appears highly probable and must be considered. The lack of penetrance and the variable expressivity could be the sum of three independent causes. The presence of a mosaic can affect the phenotype, the nature of the mutation can drive low penetrance and particular phenotype like psychomotor delay in case of large genomic deletions and genetic modifier factors could modulate the phenotype. These three major keys have been studied in order to highlight the relations between the phenotype and the genotype. Firstly, the consequences of mosaicism have been illustrated by a prenatal diagnosis concerning a couple with a bilateral retinoblastoma-affected male patient who requested five successive prenatal diagnoses and in whom RB1 mutation mosaicism had important implications. Implications of mosaicism in genetic counseling have been discussed and taken into consideration in order to limit bias in the two following genotype/phenotype studies. Secondly, the association between whole germline monoallelic deletions of the RB1 gene and psychomotor delay was studied by a high-resolution CGH array focusing on RB1 and its flanking region. Comparative analysis detected a four megabase critical interval including a candidate gene, protocadherin 8 (PCDH8). PCDH8 is thought to function in signaling pathways and cell adhesion in a central nervous system-specific manner, making loss of PCDH8 one of the probable causes of psychomotor delay in RB1-deleted patients. Thirdly, a candidate gene approach based on partners that are necessary for the development of the tumor attempted to find possible genetic modifiers. MDM2, which increases p53 and pRB catabolism, was therefore a prominent candidate. The minor allele of MDM2 that includes a 309T>G transversion (SNP rs2279744) in the MDM2 promoter is known to enhance MDM2 expression. In family-based association analyses performed in 70 retinoblastoma families, the MDM2 309G allele was found to be statistically significantly associated with incidence of bilateral or unilateral retinoblastoma among members of retinoblastoma families under a recessive model (Z = 3.305, two-sided exact P = .001). The strong association of this genotype with retinoblastoma development designates MDM2 as the first modifier gene to be identified among retinoblastoma patients

O imprinting genômico, processo regulado epigeneticamente segundo o qual os genes se expressam de acordo com sua origem parental (paterna ou materna), está envolvido no desenvolvimento placentário. Na região cromossômica 11p15.5 encontram-se vários genes importantes para o desenvolvimento fetal e da placenta, os quais são regulados por duas principais regiões controladoras de imprinting (ICR1 e 2) onde se encontram as regiões diferencialmente metiladas H19DMR e KvDMR1, respectivamente. O imprinting genômico e a inativação aleatória do cromossomo X são processos epigenéticos presentes em mamíferos placentários. O presente trabalho teve como objetivo principal verificar a presença de mosaicismo do perfil epigenético entre tecidos extraembrionários de estágios precoces da gravidez (primeiro trimestre), e em vilosidade coriônica de placentas a termo (terceiro trimestre). Foram coletadas amostras de 10 gestações de primeiro trimestre (vilosidade coriônica, âmion, membrana de cordão umbilical e tecido embrionário) e 14 de terceiro trimestre (vilosidade coriônica), das quais 10 foram consideradas como controles e quatro utilizadas para estudo de mosaicismo restrito à vilosidade coriônica (coleta de amostras de todos os cotilédones). Após extração do DNA, foi utilizado o Método de Digestão Enzimática Sensível à Metilação Associada à PCR em Tempo Real para o estudo do padrão de metilação da KvDMR1 e da H19DMR em diferentes tecidos do primeiro trimestre gestacional e em tecido placentário do terceiro trimestre. O padrão de inativação do cromossomo X foi avaliado em todos os cotilédones de duas placentas a termo, de fetos do sexo feminino, por meio do ensaio do receptor de andrógeno humano (HUMARA assay), utilizando eletroforese capilar, e com acréscimo de um novo marcador de inativação do cromossomo X (ICX1). Na análise estatística foram utilizados o teste t não pareado, teste de Turkey e teste t pareado. A média de metilação da KvDMR1 das amostras de vilosidade coriônica do primeiro trimestre gestacional foi estatisticamente diferente da média de metilação do terceiro trimestre. Enquanto que a metilação da H19DMR não apresentou diferença estatística entre amostras de vilosidade coriônica do primeiro e do terceiro trimestre gestacionais. Com relação ao mosaicismo, a KvDMR1 não apresentou variação com relação ao tamanho ou a posição dos cotilédones, enquanto que a H19DMR apresentou diferença estatisticamente significativa na média de metilação com relação ao tamanho dos cotilédones e ao posicionamento nos quadrantes; em consequência da hipometilação em cotilédones pertencentes a uma das placentas estudadas. Não foram observadas diferenças estatisticamente significativas na média de metilação da KvDMR1 e da H19DMR entre diferentes tecidos das amostras do primeiro trimestre gestacional. No entanto, a comparação entre tecidos pareados de um mesmo indivíduo mostrou que a metilação não é correspondente entre os tecidos. Os dados obtidos mostram que o imprinting genômico provavelmente é um processo dinâmico, que evolui ao longo da gestação, estando relacionado a formação e ao amadurecimento da placenta. No presente estudo foi possível verificar que cotilédones de uma mesma placenta apresentam diferentes padrões de inativação do cromossomo X. Diferenças que podem ser explicadas pela expansão clonal das células trofoblásticas progenitoras com o cromossomo X paterno ou o cromossomo X materno inativo. Devido à variabilidade epigenética, exames em tecidos placentários devem considerar as diferenças intra-placentárias e as diferenças entre tecidos embrionários e extraembrionários. / Genomic imprinting, a mechanism of allele-specific expression depending on parental origin, is an epigenetic process that regulates the expression of many genes involved in placental development. Several important genes for fetal and placental growth are located on the human chromosome region 11p15.5, which are regulated by two imprinting control regions (ICR1 e 2), which have the differentially methylated regions H19DMR and KvDMR1, respectively. Genomic imprinting and random inactivation of X chromosome are two epigenetic processes present in placental mammals. The present study aimed to verify the presence of epigenetic mosaicism between extra-embryonic and embryonic tissues from early stages of pregnancy (first trimester), and in chorionic villi of term placentas (third trimester). Samples were collected from 10 pregnancies in the first trimester (chorionic villous, amnion, umbilical cord membrane, and embryonic tissue) and 14 from third trimester (chorionic villus sampling), of which 10 were considered as controls and four used to study mosaicism restricted to chorionic villi (sampling of all cotyledons). After DNA extraction, we used real time PCR associated to enzymatic restriction with a methylation sensitive enzyme to study the methylation pattern of KvDMR1 and H19DMR in different tissues from first trimester and placental third trimester tissue. The pattern of X chromosome inactivation was evaluated in all cotyledons from two term placentas of female fetuses, using the human androgen receptor (HUMARA) assay, capillary electrophoresis, and adding a new X chromosome inactivation (ICX1) marker. Unpaired and paired t and Turkey tests were used in statistical analysis. The average methylation of KvDMR1 of chorionic villi samples in first trimester was statistically different from average methylation of the third trimester. While the methylation of H19DMR showed no statistically significant difference between chorionic villi samples in the first and third trimester of pregnancy. In relation to the mosaicism, the KvDMR1 methylation did not vary in respect to the size or position of the cotyledons, while H19DMR showed statistically significant difference in average methylation relative to the size of the cotyledons, to the position in quadrants, due to the hypomethylation in cotyledons from one studied placenta. There were no statistically significant differences in the mean methylation KvDMR1 and H19DMR among different tissues from the first trimester of pregnancy, however, the comparison between paired tissues from the same individual showed that the methylation is different between tissues. The data from this study showed that genomic imprinting is probably a dynamic process and evolved across human pregnancy. This process is probable connected to placenta formation and maturation. We observed different patterns of X chromosome inactivation in cotyledons from the same placenta. This difference could be explained by clonal expansion of a limited number of trophoblastic progenitor cells with either an inactive maternal or paternal X chromosome. Due to the epigenetic variability, placental tissue examinations must consider the differences intra-placental and differences between embryonic and extra-embryonic tissues.

O imprinting genômico, processo regulado epigeneticamente segundo o qual os genes se expressam de acordo com sua origem parental (paterna ou materna), está envolvido no desenvolvimento placentário. Na região cromossômica 11p15.5 encontram-se vários genes importantes para o desenvolvimento fetal e da placenta, os quais são regulados por duas principais regiões controladoras de imprinting (ICR1 e 2) onde se encontram as regiões diferencialmente metiladas H19DMR e KvDMR1, respectivamente. O imprinting genômico e a inativação aleatória do cromossomo X são processos epigenéticos presentes em mamíferos placentários. O presente trabalho teve como objetivo principal verificar a presença de mosaicismo do perfil epigenético entre tecidos extraembrionários de estágios precoces da gravidez (primeiro trimestre), e em vilosidade coriônica de placentas a termo (terceiro trimestre). Foram coletadas amostras de 10 gestações de primeiro trimestre (vilosidade coriônica, âmion, membrana de cordão umbilical e tecido embrionário) e 14 de terceiro trimestre (vilosidade coriônica), das quais 10 foram consideradas como controles e quatro utilizadas para estudo de mosaicismo restrito à vilosidade coriônica (coleta de amostras de todos os cotilédones). Após extração do DNA, foi utilizado o Método de Digestão Enzimática Sensível à Metilação Associada à PCR em Tempo Real para o estudo do padrão de metilação da KvDMR1 e da H19DMR em diferentes tecidos do primeiro trimestre gestacional e em tecido placentário do terceiro trimestre. O padrão de inativação do cromossomo X foi avaliado em todos os cotilédones de duas placentas a termo, de fetos do sexo feminino, por meio do ensaio do receptor de andrógeno humano (HUMARA assay), utilizando eletroforese capilar, e com acréscimo de um novo marcador de inativação do cromossomo X (ICX1). Na análise estatística foram utilizados o teste t não pareado, teste de Turkey e teste t pareado. A média de metilação da KvDMR1 das amostras de vilosidade coriônica do primeiro trimestre gestacional foi estatisticamente diferente da média de metilação do terceiro trimestre. Enquanto que a metilação da H19DMR não apresentou diferença estatística entre amostras de vilosidade coriônica do primeiro e do terceiro trimestre gestacionais. Com relação ao mosaicismo, a KvDMR1 não apresentou variação com relação ao tamanho ou a posição dos cotilédones, enquanto que a H19DMR apresentou diferença estatisticamente significativa na média de metilação com relação ao tamanho dos cotilédones e ao posicionamento nos quadrantes; em consequência da hipometilação em cotilédones pertencentes a uma das placentas estudadas. Não foram observadas diferenças estatisticamente significativas na média de metilação da KvDMR1 e da H19DMR entre diferentes tecidos das amostras do primeiro trimestre gestacional. No entanto, a comparação entre tecidos pareados de um mesmo indivíduo mostrou que a metilação não é correspondente entre os tecidos. Os dados obtidos mostram que o imprinting genômico provavelmente é um processo dinâmico, que evolui ao longo da gestação, estando relacionado a formação e ao amadurecimento da placenta. No presente estudo foi possível verificar que cotilédones de uma mesma placenta apresentam diferentes padrões de inativação do cromossomo X. Diferenças que podem ser explicadas pela expansão clonal das células trofoblásticas progenitoras com o cromossomo X paterno ou o cromossomo X materno inativo. Devido à variabilidade epigenética, exames em tecidos placentários devem considerar as diferenças intra-placentárias e as diferenças entre tecidos embrionários e extraembrionários. / Genomic imprinting, a mechanism of allele-specific expression depending on parental origin, is an epigenetic process that regulates the expression of many genes involved in placental development. Several important genes for fetal and placental growth are located on the human chromosome region 11p15.5, which are regulated by two imprinting control regions (ICR1 e 2), which have the differentially methylated regions H19DMR and KvDMR1, respectively. Genomic imprinting and random inactivation of X chromosome are two epigenetic processes present in placental mammals. The present study aimed to verify the presence of epigenetic mosaicism between extra-embryonic and embryonic tissues from early stages of pregnancy (first trimester), and in chorionic villi of term placentas (third trimester). Samples were collected from 10 pregnancies in the first trimester (chorionic villous, amnion, umbilical cord membrane, and embryonic tissue) and 14 from third trimester (chorionic villus sampling), of which 10 were considered as controls and four used to study mosaicism restricted to chorionic villi (sampling of all cotyledons). After DNA extraction, we used real time PCR associated to enzymatic restriction with a methylation sensitive enzyme to study the methylation pattern of KvDMR1 and H19DMR in different tissues from first trimester and placental third trimester tissue. The pattern of X chromosome inactivation was evaluated in all cotyledons from two term placentas of female fetuses, using the human androgen receptor (HUMARA) assay, capillary electrophoresis, and adding a new X chromosome inactivation (ICX1) marker. Unpaired and paired t and Turkey tests were used in statistical analysis. The average methylation of KvDMR1 of chorionic villi samples in first trimester was statistically different from average methylation of the third trimester. While the methylation of H19DMR showed no statistically significant difference between chorionic villi samples in the first and third trimester of pregnancy. In relation to the mosaicism, the KvDMR1 methylation did not vary in respect to the size or position of the cotyledons, while H19DMR showed statistically significant difference in average methylation relative to the size of the cotyledons, to the position in quadrants, due to the hypomethylation in cotyledons from one studied placenta. There were no statistically significant differences in the mean methylation KvDMR1 and H19DMR among different tissues from the first trimester of pregnancy, however, the comparison between paired tissues from the same individual showed that the methylation is different between tissues. The data from this study showed that genomic imprinting is probably a dynamic process and evolved across human pregnancy. This process is probable connected to placenta formation and maturation. We observed different patterns of X chromosome inactivation in cotyledons from the same placenta. This difference could be explained by clonal expansion of a limited number of trophoblastic progenitor cells with either an inactive maternal or paternal X chromosome. Due to the epigenetic variability, placental tissue examinations must consider the differences intra-placental and differences between embryonic and extra-embryonic tissues.

Orientadores: Andréa Trevas Maciel-Guerra, Vera Lúcia Gil da Silva Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T23:41:30Z (GMT). No. of bitstreams: 1 Latuf_JulianadePaulo_M.pdf: 2398402 bytes, checksum: cbd1c5c98ab12541607b0be2b5ca8120 (MD5) Previous issue date: 2013 / Resumo: Quadros de ambiguidade genital e esterilidade com cariótipo 46,XY podem ser devidos a mosaico com linhagem 45,X não detectável no cariótipo em linfócitos de sangue periférico. Quando essa linhagem não é detectada, esses indivíduos deixam de ser investigados em relação a uma série de problemas clínicos. Este trabalho teve como objetivo verificar se a hibridação in situ com fluorescência (FISH) em células de mucosa oral poderia ser empregada para detectar criptomosaicismo com linhagem 45,X em indivíduos com cariótipo 46,XY. A casuística foi composta por 19 homens saudáveis com idades entre 20 e 30 anos e cinco pacientes com distúrbios da diferenciação do sexo (DDS) com idades entre 5 e 23 anos, quatro com mosaico 45,X/46,XY e um com disgenesia testicular 46,XY associada a déficit de crescimento. Após confirmar que os jovens saudáveis tinham cariótipo 46,XY em 50 metáfases de linfócitos de sangue periférico, foi realizada análise por FISH com sondas específicas para os cromossomos X e Y em 1.000 núcleos interfásicos de linfócitos de sangue periférico e 1.000 de mucosa oral, seguida de comparação da proporção de núcleos contendo apenas o sinal do cromossomo X nos dois tecidos. A mesma análise foi feita nos cinco pacientes com DDS. A distribuição da proporção dos núcleos interfásicos de linfócitos e mucosa oral contendo apenas o sinal do X nos jovens saudáveis foi compatível com a distribuição normal, e número superior a 12:1.000 em linfócitos e 13:1.000 em mucosa oral devem ser considerados indicativos de mosaicismo em nosso laboratório. A frequência desses núcleos nos dois tecidos não diferiu significativamente (p=0,6855). Nos cinco pacientes com DDS a frequência de núcleos contendo apenas o sinal do X diferiu significativamente da observada em indivíduos normais em linfócitos (p=0,0008) e mucosa oral (p=0,0008). No paciente com cariótipo prévio 46,XY a linhagem 45,X foi confirmada por FISH em metáfases, e em um dos casos de mosaicismo foram detectadas linhagens celulares adicionais. Também não houve diferença significativa entre a frequência de núcleos contendo apenas o sinal do X nos dois tecidos desses pacientes (p=0,3750). Estes resultados indicam que a pesquisa de mosaicismo com linhagem 45,X em indivíduos com DDS ou esterilidade e cariótipo 46,XY pode ser feita por meio de FISH em mucosa oral, com vantagens evidentes em termos de custo e rapidez, além de ser feita a partir de tecido obtido de modo não invasivo / Abstract: Ambiguous genitalia and sterility with a 46,XY karyotype may be due to mosaicism with a 45, X karyotype not detectable in peripheral blood lymphocytes. When this cell line is not detected, these individuals fail to be investigated over a range of clinical problems. This study aimed to verify whether fluorescence in situ hybridization (FISH) in cells from buccal smear could be employed to detect cryptomosaicism with a 45,X cell line in individuals with a 46,XY karyotype. The sample consisted of 19 healthy men aged 20 to 30 years and five patients with disorders of sex development (DSD) aged 5 to 23 years, four with mosaicism 45,X/46,XY and one with testicular dysgenesis 46, XY associated with growth deficiency. After confirming that the healthy young men had a 46,XY karyotype in 50 metaphases from peripheral blood lymphocytes, FISH analysis with probes specific for chromosomes X and Y was done in 1,000 nuclei from peripheral blood lymphocytes and 1,000 from buccal smear, followed by comparison of the proportion of nuclei containing only the signal of the X chromosome in these tissues. The same analysis was performed in five patients with DDS. The distribution of the proportion of interphase nuclei of lymphocytes and buccal smear containing only the X signal in healthy young was consistent with normal distribution; a number greater than 12:1,000 in lymphocytes and 13:1,000 in buccal smear should be considered indicative of mosaicism in our laboratory. The frequency of these nuclei in both tissues did not differ significantly (p = 0.6855). In patients with DDS the frequency of nuclei containing only the X signal differed significantly from that observed in normal individuals both in lymphocytes (p = 0.0008) and buccal smear (p = 0.0008). In the patient with a prior 46,XY karyotype, a 45,X cell line was confirmed by FISH in metaphases, and in one case of mosaicism additional cell lines were detected. There was also no significant difference between the frequency of nuclei containing only the X signal in the two tissues of these patients (p = 0.3750). These results indicate that investigation of mosaicism with 45,X cell line in individuals with 46,XY DSD or sterility can be done by FISH in cells from buccal smear, with obvious advantages in terms of cost and speed, using a tissue obtained noninvasively / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson–Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).