Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

Post-zygotic genetic variation has previously been shown in healthy individuals and linked to various disorders. The definition of post-zygotic or somatic variation is the existence of genetically distinct populations of cells in a subject derived from a single zygote. Structural changes in the human genome are a major type of inter-individual genetic variation and copy number variation (CNV), involving changes in the copy number of genes, are one of the best studied category of structural genetic changes. In paper I we reported a pair of healthy female monozygotic (MZ) twins discordant for aneuploidy of chromosomes X and Y, contributing to the delineation of the frequency of somatic variation in MZ twins. It also illustrates the plasticity of the genome for tolerating large aberrations in healthy subjects. In paper II we showed age-related accumulation of copy number variation in the nuclear genomes in vivo for both megabase- and kilobase-range variants. Using age-stratified MZ twins and single-born subjects, we detected megabase-range aberrations in 3.4% of people ≥60 years old but not in individuals younger than 55 years. Moreover, the longitudinal analysis of subjects with aberrations suggests that the aberrant cell clones are not immortalized and disappear from circulation. We also showed that sorted blood cells display different genomic profiles.  The detected recurrent rearrangements are candidates for common age-related defects in blood cells. This work might help to describe the cause of an age-related decline in the number of cell clones in the blood, which is one of the hallmarks of immunosenescence. In paper III we described a variable number tandem repeat (VNTR) ~4 kb upstream of the IFNAR1 gene, which was somatically variable.  We detected 14 alleles displaying inter- and intra-individual variation. Further analyses indicated strong clustering of transcription factor binding sites within this region, suggesting an enhancer. This putative VNTR-based enhancer might influence the transcriptional regulation of neighboring cytokine receptor genes and the pathways they are involved in. These three studies stress the importance of research on post-zygotic variation in genetics. Furthermore, they emphasize that biobanks should consider sampling of multiple tissues to better address this issue in the genetic studies.

Orientadores: Ivone Panhoca, Maria de Lurdes Zanolli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:54:08Z (GMT). No. of bitstreams: 1 Barbetta_NaraiLopez_D.pdf: 1072561 bytes, checksum: 97748c3ef22ded8023f4570dfec3f773 (MD5) Previous issue date: 2008 / Resumo: A família é o primeiro agrupamento a que a criança passa a fazer parte, incondicionalmente e a descoberta de uma gestação gemelar certamente abala o imaginário familiar e social. Nestas famílias, algumas práticas podem acompanhar o processo de gestação, nascimento e desenvolvimento dessas crianças. Incluem-se a escolha de roupas iguais ou com diferença apenas na cor, nomes com semelhanças fonéticas, rotinas parecidas quanto à alimentação e sono. Porém, estudos sobre gêmeos monozigóticos têm apontado, por diversas vezes, um questionamento sobre o lugar e a importância que ocupam fatores como carga genética e influência ambiental. Mas a ampliação de estudos voltados ao controle genético tem deixado claro que qualquer particularidade de comportamento decorre, ao mesmo tempo, tanto de efeitos dos genes, quanto de fatores ambientais. Os gêmeos monozigóticos podem apresentar um desenvolvimento normal, mas, também, podem mostrar um atraso na aquisição da linguagem oral como forma efetiva de comunicação. O objetivo, deste estudo, foi acompanhar as famílias longitudinalmente, analisando os pressupostos e práticas sociais manifestados discursivamente, a respeito da gemelaridade, a partir do diagnóstico da gestação gemelar de crianças monozigóticas, considerando-se, em especial, o desenvolvimento da linguagem e o processo de constituição da identidade das crianças, bem como subsidiar a contribuição de alguns profissionais da saúde neste desenvolvimento. O trabalho pauta-se na abordagem naturalista/observacional, na perspectiva histórico-cultural e no paradigma de natureza indiciária. Para a coleta de dados, foram realizadas oito entrevistas semi-estruturadas, videogravadas, com dez famílias que tinham em sua composição direta um par de gêmeos idênticos, em um período de trinta meses. O material foi analisado considerando-se: a descoberta da gestação gemelar, escolha de nomes, vestuário, rotina, interação, linguagem e identidade. As categorias analisadas apareceram em todos os discursos coletados e revelaram a dificuldade familiar em assimilar a presença de gêmeos, que acabam sendo ¿dois vistos como um¿. Quanto à linguagem, oito famílias relataram um desenvolvimento diferenciado, mas não foi ressaltado de maneira tão relevante a ponto de causar preocupação nas famílias. Os aspectos sociais envolvidos perante a semelhança são por demais marcantes, para vencer a possibilidade de alteração no processo de desenvolvimento da linguagem e identidade dessas crianças / Abstract: A família é o primeiro agrupamento a que a criança passa a fazer parte, incondicionalmente e a descoberta de uma gestação gemelar certamente abala o imaginário familiar e social. Nestas famílias, algumas práticas podem acompanhar o processo de gestação, nascimento e desenvolvimento dessas crianças. Incluem-se a escolha de roupas iguais ou com diferença apenas na cor, nomes com semelhanças fonéticas, rotinas parecidas quanto à alimentação e sono. Porém, estudos sobre gêmeos monozigóticos têm apontado, por diversas vezes, um questionamento sobre o lugar e a importância que ocupam fatores como carga genética e influência ambiental. Mas a ampliação de estudos voltados ao controle genético tem deixado claro que qualquer particularidade de comportamento decorre, ao mesmo tempo, tanto de efeitos dos genes, quanto de fatores ambientais. Os gêmeos monozigóticos podem apresentar um desenvolvimento normal, mas, também, podem mostrar um atraso na aquisição da linguagem oral como forma efetiva de comunicação. O objetivo, deste estudo, foi acompanhar as famílias longitudinalmente, analisando os pressupostos e práticas sociais manifestados discursivamente, a respeito da gemelaridade, a partir do diagnóstico da gestação gemelar de crianças monozigóticas, considerando-se, em especial, o desenvolvimento da linguagem e o processo de constituição da identidade das crianças, bem como subsidiar a contribuição de alguns profissionais da saúde neste desenvolvimento. O trabalho pauta-se na abordagem naturalista/observacional, na perspectiva histórico-cultural e no paradigma de natureza indiciária. Para a coleta de dados, foram realizadas oito entrevistas semi-estruturadas, videogravadas, com dez famílias que tinham em sua composição direta um par de gêmeos idênticos, em um período de trinta meses. O material foi analisado considerando-se: a descoberta da gestação gemelar, escolha de nomes, vestuário, rotina, interação, linguagem e identidade. As categorias analisadas apareceram em todos os discursos coletados e revelaram a dificuldade familiar em assimilar a presença de gêmeos, que acabam sendo ¿dois vistos como um¿. Quanto à linguagem, oito famílias relataram um desenvolvimento diferenciado, mas não foi ressaltado de maneira tão relevante a ponto de causar preocupação nas famílias. Os aspectos sociais envolvidos perante a semelhança são por demais marcantes, para vencer a possibilidade de alteração no processo de desenvolvimento da linguagem e identidade dessas crianças / Doutorado / Saude da Criança e do Adolescente / Doutor em Saude da Criança e do Adolescente

The schooling of twins can be daunting and challenging for twins, parents and educators. Separation or non-separation during the foundation phase cannot be left to chance. The process of deciding whether it is in both twins’ best interest to school them together or separately relies on various factors, which includes premature birth, individuality, dominance, relationship type and friendship-forming patterns. Parties involved in making the decision must consider each twin’s emotional, cognitive, behavioural, and social development. Premature separation of twins can lead to internal as well as external behaviour problems, while non-separation of mature twins may be detrimental to each twin’s individual progress and development. The study aimed to identify the determining factors when deciding upon schooling twins together or separately when they enter into the foundation phase. Existing school policies regarding classroom allocation of twins were considered. Three sets of twins, including teachers, parents, twins and other significant individuals involved in the process of separation or non-separation, were interviewed. Based on the findings, recommendations were made to assist parents and educators in making informed decisions about the schooling of twins. / Psychology of Education / M. Ed. (Guidance and Counselling)

The schooling of twins can be daunting and challenging for twins, parents and educators. Separation or non-separation during the foundation phase cannot be left to chance. The process of deciding whether it is in both twins’ best interest to school them together or separately relies on various factors, which includes premature birth, individuality, dominance, relationship type and friendship-forming patterns. Parties involved in making the decision must consider each twin’s emotional, cognitive, behavioural, and social development. Premature separation of twins can lead to internal as well as external behaviour problems, while non-separation of mature twins may be detrimental to each twin’s individual progress and development. The study aimed to identify the determining factors when deciding upon schooling twins together or separately when they enter into the foundation phase. Existing school policies regarding classroom allocation of twins were considered. Three sets of twins, including teachers, parents, twins and other significant individuals involved in the process of separation or non-separation, were interviewed. Based on the findings, recommendations were made to assist parents and educators in making informed decisions about the schooling of twins. / Psychology of Education / M. Ed. (Guidance and Counselling)

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