Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The gene Myh9 encodes non-muscle myosin heavy chain I lA, an essential podocyte cytoskeleton structural protein. Abnormalities in the MYH9 gene are associated with chronic kidney disease (common in people of African ancestry) and rare hereditary autosomal dominant syndromes. In the current study, preexisting heterozygous Myh9 knockout mice aged 9 and 17 months were investigated to assess the biological role of the gene Myh9 product, non-muscle myosin IIA, in kidney structure and function. The objective was to determine the effects of potentially decreased expression of Myh9 genetic alteration and the role of Myh9 in the pathogenesis of chronic kidney disease (especially focal segmental glomerulosclerosis), and to develop a model to further study Myh9- related kidney disorders. Our results demonstrated that deletion of one allele of Myh9 in 129/Sv mice had no effect on the kidney structure compared with wildtype mice despite a significant decrease in expression of Myh9 in the heterozygous mice. / 2031-01-01

Myh9 is a gene that encodes for non-muscle myosin IIA (NM-IIA), an actin cytoskeleton component and protein involved in cell movement and adhesion in most cells, including podocytes. Autosomal dominant mutations in NM-IIA have been associated with focal segmental glomerular sclerosis (FSGS) in patients with the mutation. Furthermore, a strong association has been discovered between genetic variation in the Myh9 region on chromosome 22, and the increased risk of chronic kidney disease in African Americans. Later studies by Johnstone et al. using podocyte specific, Myh9 KO in mice showed that knocking out this gene alone was not enough to cause proteinuria or glomerular sclerosis. However, in our own laboratory we have found that when these same mice are exposed to models of glomerular hypertension, glomerular damage is promoted. This damage was preceded by evidence of podocyte loss in urine and tissue. Podocyte loss is a hallmark of kidney disease, and while it is known to occur in vivo, the mechanisms behind this phenomenon are unknown. It is believed that increase in glomerular capillary blood pressure is likely to be a strong contributing factor. Based on these findings, we hypothesized that the mechanical strain in the form of stretch, occurring during hypertension, may act as a second hit to Myh9 ablation in podocytes, causing changes in podocytes ability to adhere to the glomerular basement membrane (GBM), ultimately leading to the pathology seen in patients. To test this, we knocked down Myh9 in immortalized mouse podocytes cultured on flexible silicon membranes and exposed them to mechanical stretch for 24hrs. Cell adhesion was evaluated via cell attachment assays. Cell morphology and focal adhesions (FA) were examined using immunofluorescence and quantified using imageJ. Our results showed a significant decrease in transduced cells attached to the membrane after stretch, as well as an increase in FA size and number in cells that underwent stretch, except in the Myh9 knockdown. Cells with Myh9 knockdown also showed marked increase in area, with a decrease in FA size and number after exposure to stretch. These results support our hypothesis that Myh9 mutations may be a contributing factor to podocyte loss in patients with hypertension and chronic kidney disease.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

People of African ancestry (AA) are at greater risk of developing chronic kidney disease than those of non-AA. Much of this risk has been linked to specific genetic haplotypes on chromosome 22, near the genes APOL1, encoding apolipoprotein L1, and MYH9, encoding non-muscle myosin heavy chain IIA (NMHCIIA). The mechanisms by which the disease-associated chromosome 22 haplotypes promote kidney damage are unknown. Apolipoprotein L1 is a circulating protein with no known role in kidney function. However, the kidney disease-associated chromosome 22 haplotypes are protective against trypanosome infection, resulting in positive selective pressure for these haplotypes in western Africa, where trypanosome infection is endemic. In contrast, NMHCIIA may have an important role in glomerular function, and mutations in MYH9 are associated with glomerular disease, yet the disease-associated chromosome 22 haplotypes do not involve coding sequence variations in MYH9. With no clear disease-causing role for genes near the chromosome 22 risk locus, it is plausible that indirect mechanisms of gene regulation may be responsible for the increased disease risk. This study examines several potential pathways for kidney injury, including altered glomerular gene expression in carriers of chromosome 22 risk haplotypes, and the role of altered expression of MYH9 in podocyte cell biology and kidney disease. We found that carriers of chromosome 22 risk variants exhibited differential glomerular gene expression in pathways promoting kidney injury. We also found decreased glomerular NMHCIIA expression in human FSGS kidney biopsies, and altered cell structure and mechanical function when Myh9 is ablated in murine podocytes. Further, Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by the DOCA-salt uninephrectomy model of hypertension. Taken together, these findings demonstrate direct and indirect effects of chromosome 22 risk variants on glomerular gene expression which promote kidney injury.

INTRODUÇÃO: A nefrite lúpica é uma complicação frequente e de alta morbimortalidade do lúpus eritematoso sistêmico (LES). A evolução para insuficiência renal crônica terminal varia entre 8 e 15% dos casos, após um período de 5 anos. A fase inicial da nefrite se deve a uma atividade imunológica exacerbada que leva a sequelas renais, como a fibrose intersticial, sinéquias glomerulares, e glomeruloesclerose. Uma vez instalada, vários fatores aceleram a velocidade de progressão da insuficiência renal, como a presença de proteinúria residual, hipertensão arterial sistêmica e a etnia do paciente. Estudos recentes mostraram que a presença de polimorfismos do MYH9 são altamente prevalentes em pacientes com GESF (glomeruloesclerose focal e segmentar), nefropatia do HIV e em pacientes com doença renal crônica não diabética. Os polimorfismos do MYH9 mais relacionados com essas doenças são os do haplótipo E1, causados pelos polimorfismos rs4821480, rs2032487, rs4821481 e rs3752462, presentes principalmente na população negra e de hispano-americanos. No Brasil não há estudos sobre a prevalência desse gene. MÉTODOS: Nosso estudo analisou retrospectivamente 196 pacientes com nefrite lúpica, acompanhadas no ambulatório de glomerulopatias do Hospital das Clínicas da USP. Foram recuperados os dados clínicos e laboratoriais dos pacientes de janeiro de 1999 a dezembro de 2010. Foi feita análise dos polimorfismos do haplótipo E1 do gene do MYH9 (rs4821480, rs2032487, rs4821481 e rs3752462) e correlacionados com suas características clínicas e laboratoriais, apresentando como desfecho a duplicação da creatinina ou a evolução para doença renal crônica terminal. RESULTADOS: O tempo de seguimento médio dos pacientes foi de 6,1 anos, com a creatinina inicial média de 1,6 g/dL e proteinúria média de 3,9 g/dia. Dezenove pacientes não recuperaram função renal, mantendo-se em diálise. Dos 177 pacientes restantes 43 (24%) apresentaram o desfecho de duplicação (DC) da creatinina, ou necessidade de diálise (DRCT). Pacientes progressores eram tinham maior SLEDAI renal (10 vs 8,9 p=0,04), maior índice de cronicidade renal à biópsia (5 vs 2, p<0,001) e maior frequência de reativações da doença renal (flare renal) (82,9% x 53,8%, p=0,002), assim menores índices de remissão completa ou parcial (p<0,0001). Os 4 polimorfismos se segregam em conjunto, ou seja, como um haplótipo, pelo modelo de Hardy-Weinberg. Analisando separadamente cada polimorfismo, apenas o rs3752462, apresenta associação com o desfecho DC/DRCT, na análise por genótipo (CC/CT/TT, p=0,03) e quando feita análise TT/CT vs CC (p=0,02). Não houve relação dos polimorfismos com a etnia negra ou parda. Pacientes com haplótipo E1 eram progressores em 28% dos casos, conferindo um OR de 1,79 (IC 1,02 a 3,0) de DC/DRC. DISCUSSÃO: A presença do haplótipo E1 têm alta prevalência em pacientes portadores de nefrite lúpica no Brasil, sendo fator de risco para progressão da doença renal crônica / BACKGROUND: Lupus nephritis (LN) is a frequent complication with high morbidity and mortality of systemic lupus erythematosus (SLE). Chronic renal failure is observed in 8 to 15% of the patients after 5 years of follow up. LN is an inflammatory disease after a systemic autoimmune activation. Once inflammation is shutdown several renal and nonrenal factors, such as residual proteinuria, hypertension and ethnicity of the patient, may emerge and impose to the kidney a chronic phenotype (interstitial fibrosis, glomerular adhesions and glomerulosclerosis. Recently E1 haplotype (rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms) of the MYH9 gene was associated to progressive kidney diseases in patients with FSGS (focal segmental glomerulosclerosis), HIV nephropathy and non-diabetic chronic kidney disease, in african american and spanic american patients. In Brazil there is no data on this subject. METHODS: Retrospective analysis of 196 patients with LN followed in our outpatient glomerular disease ward were enrolled glomerulopathies. Patients clinical data from January 1999 to December 2010 were retrieved and MYH9 rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms were genotyped. Outcome was defined as doubling of serum creatinine, or end stage renal disease (ESRD). RESULTS: The mean follow-up of patients was 6.1 years, with an initial mean creatinine of 1.6 g/dL and mean proteinuria 3.9 g/day. On enrollment nineteen patients were on dialysis and did not recover renal function, they were withdraw from analyses of progressive kidney disease. On follow up, from 177 remaining patients, 43 (24%) showed the composite outcome: dialysis, or doubling creatinine. Progressors had higher renal SLEDAI (10 vs 8.9, p = 0.04), higher chronicity index at biopsy (5 vs 2, p <0.001) and more frequently renal flares (82, 9% vs. 53.8%, p=0.002), as well as lower rates of complete or partial remission (p <0.0001). The four polymorphisms segregate as a haplotype, according the Hardy-Weinberg model. Analysing each polymorphism, only TT/CT genotype from rs3752462 polymorphism was associated with the outcome of DC/ESRD (p = 0.02). E1 haplotype were associated with progression with an OR of 1.79 (CI 1.02 to 3.0). DISCUSSION: The presence of the E1 haplotype is associated with worse prognosis of chronic renal failure in lupus nephritis patients

INTRODUÇÃO: A nefrite lúpica é uma complicação frequente e de alta morbimortalidade do lúpus eritematoso sistêmico (LES). A evolução para insuficiência renal crônica terminal varia entre 8 e 15% dos casos, após um período de 5 anos. A fase inicial da nefrite se deve a uma atividade imunológica exacerbada que leva a sequelas renais, como a fibrose intersticial, sinéquias glomerulares, e glomeruloesclerose. Uma vez instalada, vários fatores aceleram a velocidade de progressão da insuficiência renal, como a presença de proteinúria residual, hipertensão arterial sistêmica e a etnia do paciente. Estudos recentes mostraram que a presença de polimorfismos do MYH9 são altamente prevalentes em pacientes com GESF (glomeruloesclerose focal e segmentar), nefropatia do HIV e em pacientes com doença renal crônica não diabética. Os polimorfismos do MYH9 mais relacionados com essas doenças são os do haplótipo E1, causados pelos polimorfismos rs4821480, rs2032487, rs4821481 e rs3752462, presentes principalmente na população negra e de hispano-americanos. No Brasil não há estudos sobre a prevalência desse gene. MÉTODOS: Nosso estudo analisou retrospectivamente 196 pacientes com nefrite lúpica, acompanhadas no ambulatório de glomerulopatias do Hospital das Clínicas da USP. Foram recuperados os dados clínicos e laboratoriais dos pacientes de janeiro de 1999 a dezembro de 2010. Foi feita análise dos polimorfismos do haplótipo E1 do gene do MYH9 (rs4821480, rs2032487, rs4821481 e rs3752462) e correlacionados com suas características clínicas e laboratoriais, apresentando como desfecho a duplicação da creatinina ou a evolução para doença renal crônica terminal. RESULTADOS: O tempo de seguimento médio dos pacientes foi de 6,1 anos, com a creatinina inicial média de 1,6 g/dL e proteinúria média de 3,9 g/dia. Dezenove pacientes não recuperaram função renal, mantendo-se em diálise. Dos 177 pacientes restantes 43 (24%) apresentaram o desfecho de duplicação (DC) da creatinina, ou necessidade de diálise (DRCT). Pacientes progressores eram tinham maior SLEDAI renal (10 vs 8,9 p=0,04), maior índice de cronicidade renal à biópsia (5 vs 2, p<0,001) e maior frequência de reativações da doença renal (flare renal) (82,9% x 53,8%, p=0,002), assim menores índices de remissão completa ou parcial (p<0,0001). Os 4 polimorfismos se segregam em conjunto, ou seja, como um haplótipo, pelo modelo de Hardy-Weinberg. Analisando separadamente cada polimorfismo, apenas o rs3752462, apresenta associação com o desfecho DC/DRCT, na análise por genótipo (CC/CT/TT, p=0,03) e quando feita análise TT/CT vs CC (p=0,02). Não houve relação dos polimorfismos com a etnia negra ou parda. Pacientes com haplótipo E1 eram progressores em 28% dos casos, conferindo um OR de 1,79 (IC 1,02 a 3,0) de DC/DRC. DISCUSSÃO: A presença do haplótipo E1 têm alta prevalência em pacientes portadores de nefrite lúpica no Brasil, sendo fator de risco para progressão da doença renal crônica / BACKGROUND: Lupus nephritis (LN) is a frequent complication with high morbidity and mortality of systemic lupus erythematosus (SLE). Chronic renal failure is observed in 8 to 15% of the patients after 5 years of follow up. LN is an inflammatory disease after a systemic autoimmune activation. Once inflammation is shutdown several renal and nonrenal factors, such as residual proteinuria, hypertension and ethnicity of the patient, may emerge and impose to the kidney a chronic phenotype (interstitial fibrosis, glomerular adhesions and glomerulosclerosis. Recently E1 haplotype (rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms) of the MYH9 gene was associated to progressive kidney diseases in patients with FSGS (focal segmental glomerulosclerosis), HIV nephropathy and non-diabetic chronic kidney disease, in african american and spanic american patients. In Brazil there is no data on this subject. METHODS: Retrospective analysis of 196 patients with LN followed in our outpatient glomerular disease ward were enrolled glomerulopathies. Patients clinical data from January 1999 to December 2010 were retrieved and MYH9 rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms were genotyped. Outcome was defined as doubling of serum creatinine, or end stage renal disease (ESRD). RESULTS: The mean follow-up of patients was 6.1 years, with an initial mean creatinine of 1.6 g/dL and mean proteinuria 3.9 g/day. On enrollment nineteen patients were on dialysis and did not recover renal function, they were withdraw from analyses of progressive kidney disease. On follow up, from 177 remaining patients, 43 (24%) showed the composite outcome: dialysis, or doubling creatinine. Progressors had higher renal SLEDAI (10 vs 8.9, p = 0.04), higher chronicity index at biopsy (5 vs 2, p <0.001) and more frequently renal flares (82, 9% vs. 53.8%, p=0.002), as well as lower rates of complete or partial remission (p <0.0001). The four polymorphisms segregate as a haplotype, according the Hardy-Weinberg model. Analysing each polymorphism, only TT/CT genotype from rs3752462 polymorphism was associated with the outcome of DC/ESRD (p = 0.02). E1 haplotype were associated with progression with an OR of 1.79 (CI 1.02 to 3.0). DISCUSSION: The presence of the E1 haplotype is associated with worse prognosis of chronic renal failure in lupus nephritis patients

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.