Algumas síndromes genéticas monogênicas apresentam fenótipos considerados progeróides, ou seja, desenvolvem precocemente características clínicas semelhantes às observadas no envelhecimento humano normal. A relação fisiopatológica entre essas doenças e o processo de envelhecimento vem sendo estudada, sendo que o entendimento dos mecanismos moleculares em um campo contribui para a compreensão do outro. As síndromes de Hutchinson-Gilford e Rothmund-Thomson são duas condições progeróides raras, já bem caracterizadas clinicamente, que são causadas por alterações nos genes LMNA (em um alelo) e RECQL4 (nos dois alelos), respectivamente. No entanto, em cerca de 40% a 60% dos indivíduos com a síndrome de Rothmund-Thomson, não são encontradas mutações em RECQL4, constituindo um subgrupo chamado de tipo I; desse modo, os casos com alteração no gene constituem o tipo II da síndrome. Indivíduos com o tipo II apresentam um risco aumentado para o desenvolvimento de câncer, particularmente o osteossarcoma. Neste trabalho, nove pacientes com sinais progeróides foram avaliados clinicamente e tiveram o DNA sequenciado. Um paciente recebeu o diagnóstico clínico de síndrome de Hutchinson-Gilford, que foi confirmado pela mutação patogênica mais frequente encontrada no gene LMNA (p.Gly608Gly). Oito pacientes jovens, com mediana de idade de 2 anos e 2 meses, foram diagnosticados clinicamente como afetados pela síndrome de Rothmund-Thomson, cujas características clínicas mais comuns incluíam déficit pôndero-estatural, cabelos e sobrancelhas/cílios esparsos, fronte ampla, lesão cutânea (eritema em face e lesão poiquilodérmica) e anomalias ósseas, alterações típicas da síndrome. Catarata estava presente em 50% dos indivíduos. Nenhum dos pacientes desenvolveu algum tipo de tumor até o momento. O sequenciamento do gene RECQL4 mostrou a presença de três variantes patogênicas diferentes, em três probandos (37,5%), sendo dois em homozigose e um em heterozigose composta, todas já descritas previamente na literatura. Em busca de alterações em outro gene que pudesse explicar o quadro apresentado pelos pacientes sem mutação, três dos probandos, incluindo os genitores de um deles, tiveram o exoma sequenciado. No entanto, não foram encontradas, nessa etapa, variantes adicionais que explicassem na totalidade os fenótipos apresentados. Comparando os achados clínicos dos pacientes com a síndrome de Rothmund-Thomson tipo I e tipo II, foi observada diferença estatisticamente significante na incidência de catarata subcapsular nos pacientes sem mutação (p<0,05), semelhante ao que é descrito na literatura. Diante dos achados clínicos e moleculares obtidos, foi realizado o aconselhamento genético para todos os indivíduos, enfatizando a evolução, os cuidados e acompanhamentos necessários para as duas doenças em questão e fornecendo informações aos genitores dos probandos sobre o risco de recorrência para a prole futura / Some monogenic disorders exhibit progeroid phenotypes, in other words, they develop premature characteristics similar to those observed in normal human aging. The physiopathological correlation between these diseases and the aging process has being studied, and the understanding of the molecular mechanisms in one field contributes to the understanding of the other. The Hutchinson-Gilford and Rothmund-Thomson syndromes are two clinically well-characterized rare progerioid conditions that are caused by changes in the LMNA (in one allele) and RECQL4 (in both alleles) genes, respectively. However, in about 40% to 60% of individuals with Rothmund-Thomson syndrome, no mutations are found in RECQL4, constituting a subgroup called type I; thus, cases with mutations in the gene constitute the type II group of the syndrome. Individuals with type II have an increased risk for the development of cancer, particularly osteosarcoma. In this study, nine patients with progerioid signs were clinically evaluated and had the DNA sequenced. One patient was clinically diagnosed with Hutchinson-Gilford syndrome, which was confirmed by the most frequent pathogenic mutation found in the LMNA gene (p.Gly608Gly). Eight young patients with median age of 2 years and 2 months were clinically diagnosed as affected by Rothmund-Thomson syndrome, whose most common clinical features included: short stature; sparse hair, eyebrows and eyelashes; erythematous skin lesions and poikiloderma; and bone abnormalities; all typical of the syndrome. Cataract was present in 50% of individuals. None of the patients has developed any type of tumor at this time. Sequencing of the RECQL4 gene showed the presence of three different pathogenic variants in three probands (37.5%), two in homozygous and one in compound heterozygosity, all previously described in the literature. In search of alterations in another gene that could explain the phenotype presented by the patients without mutation, three of the probands, including the parents of one of them, had the exoma sequenced. However, there were no additional variants at this stage that fully explained the phenotypes presented by these individuals. Comparing the clinical findings of patients with Rothmund-Thomson syndrome type I and type II, a statistically significant difference was observed in the incidence of subcapsular cataract in patients without mutation (p <0.05), similar to that described in the literature. In light of the clinical and molecular findings, genetic counseling was performed for all individuals, emphasizing the evolution, care and follow-up needed for the two diseases in question and providing information to the parents of the probands on the risk of recurrence for future offspring

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC), also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells. This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions. Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation relative to the nucleus under shear stress.

<p>Cellular structural mechanics play a key role in homeostasis by transducing mechanical signals to regulate gene expression and by providing adaptive structural stability for the cell. The alteration of nuclear mechanics in various laminopathies and in natural aging can damage these key functions. Arterial endothelial cells appear to be especially vulnerable due to the importance of shear force mechanotransduction to structure and gene regulation as is made evident by the prominent role of atherosclerosis in Hutchinson-Gilford progeria syndrome (HGPS) and in natural aging. Computational models of cellular mechanics may provide a useful tool for exploring the structural hypothesis of laminopathy at the intracellular level. This thesis explores this topic by introducing the biological background of cellular mechanics and lamin proteins in arterial endothelial cells, investigating disease states related to aberrant lamin proteins, and exploring computational models of the cell structure. It then presents a finite element model designed specifically for investigation of nuclear shear forces in arterial endothelial cells. Model results demonstrate that changes in nuclear material properties consistent with those observed in progerin-expressing cells may result in substantial increases in stress concentrations on the nuclear membrane. This supports the hypothesis that progerin disrupts homeostatic regulation of gene expression in response to hemodynamic shear by altering the mechanical properties of the nucleus.</p>

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.