Generation and characterization of a human iPSC line from a patient with propionic acidemia due to defects in the PCCA gene

Human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia carrying mutations in the PCCA gene: c.1899+4_1899+7delAGTA; p.(Cys616_Val633del) and c.1430 −−?_1643+?del; p.(Gly477Glufs*9). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stabilityThis work was supported by Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (grant numbers SAF2013-43005-R and SAF2016-76004-R). The authors thank INDEPF (Instituto de investigación y desarrollo social de enfermedades poco frecuentes), and E.Mansilla for her excellent assistance in the karyotype analysis (Instituto de Genética Médica y Molecular del Hospital Universitario de La Paz, Madrid, Spain). Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces (grant number CNXVII

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discoveryResearch reported in this work was funded by Grants of Spanish Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER) PI16/00573, and Fundación Isabel Gemio- Fundación La Caixa (LCF/PR/PR16/11110018). The authors thank the Cytogenetic unit from Centro Nacional de Investigaciones Oncológicas (CNIO) for its excellent technical assistance. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces. LAC is a PhD student funded by the Asociación Española para el Estudio de Metabolopatías Congénitas (AEPMEC). ALM is a postdoctoral researcher of Comunidad Autónoma de Madrid (PEJD-2017-POST/BMD-3671). EAB is a PhD student funded by the FPU program of the Spanish Ministry of Science, Innovation and Universities (FPU15/02923

Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy

Cardiac alterations (hypertrophic/dilated cardiomyopathy, and rhythm alterations) are one of the major causes of mortality and morbidity in propionic acidemia (PA), caused by the deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC), involved in the catabolism of branched-chain amino acids, cholesterol, and odd-chain fatty acids. Impaired mitochondrial oxidative phosphorylation has been documented in heart biopsies of PA patients, as well as in the hypomorphic Pcca−/−(A138T) mouse model, in the latter correlating with increased oxidative damage and elevated expression of cardiac dysfunction biomarkers atrial and brain natriuretic peptides (ANP and BNP) and beta-myosin heavy chain (β-MHC). Here we characterize the cardiac phenotype in the PA mouse model by histological and echocardiography studies and identify a series of upregulated cardiac-enriched microRNAs (miRNAs) in the PA mouse heart, some of them also altered as circulating miRNAs in PA patients’ plasma samples. In PA mice hearts, we show alterations in signaling pathways regulated by the identified miRNAs, which could be contributing to cardiac remodeling and dysfunction; notably, an activation of the mammalian target of rapamycin (mTOR) pathway and a decrease in autophagy, which are reverted by rapamycin treatment. In vitro studies in HL-1 cardiomyocytes indicate that propionate, the major toxic metabolite accumulating in the disease, triggers the increase in expression levels of miRNAs, BNP, and β-MHC, concomitant with an increase in reactive oxygen species. Our results highlight miRNAs and signaling alterations in the PCC-deficient heart which may contribute to the development of PA-associated cardiomyopathy and provide a basis to identify new targets for therapeutic interventionThis work was supported by Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (grant number SAF2016-76004-R) and by Fundación Isabel Gemio and Fundación La Caixa (LCF/PR/PR16/ 11110018). AFG is funded by the FPI-UAM program, EAB and ARB by the Spanish Ministry of Science, Innovation and Universities (predoctoral fellowships FPU15/02923 and BES-2014-069420, respectively

Integrative epigenomics in Sjögren´s syndrome reveals novel pathways and a strong interaction between the HLA, autoantibodies and the interferon signature

Primary Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population

Estudios fisiopatológicos para la búsqueda de nuevas dianas terapéuticas en acidemia propiónica mediante la caracterización del modelo murino y el desarrollo de nuevos modelos celulares humanos basados en iPSCs

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-10-2020Esta tesis tiene embargado el acceso al texto completo hasta el 29-04-2022La acidemia propiónica (AP) es una enfermedad neurometabólica hereditaria causada por defectos en los genes PCCA y PCCB que codifican, respectivamente, para las subunidades α y β de la enzima mitocondrial propionil-CoA carboxilasa (PCC). La AP suele presentarse de forma neonatal severa, aunque hay presentaciones más tardías y con un fenotipo más suave. Las complicaciones a largo plazo más frecuentes son las relacionadas con los sistemas neurológico, principalmente en el estriado, y cardiovascular, especialmente hipertrofia cardíaca, cardiomiopatía dilatada y arritmias. Actualmente, no se conocen con exactitud los mecanismos moleculares implicados en el desarrollo de estas alteraciones y aún no existe un tratamiento efectivo para esta enfermedad. Estudios previos han mostrado la implicación de la disfunción mitocondrial y la alteración de la homeostasis redox en el desarrollo de la AP, tanto en muestras de pacientes como en el modelo murino hipomorfo Pcca-/-(A138T). Los estudios realizados en este trabajo para completar la caracterización del modelo murino de AP han puesto de manifiesto la desregulación de la biogénesis mitocondrial y los procesos de muerte celular y la alteración de la expresión de enzimas antioxidantes de forma tejido-específica. Además, se ha observado una alteración en el proceso autofágico, y en algunas de sus vías reguladoras, que podría representar uno de los mecanismos implicados en el desarrollo de las complicaciones cardíacas en el modelo murino de AP. La evaluación del tratamiento con rapamicina ha mostrado efectos beneficiosos restaurando la autofagia, lo que pone de manifiesto la importancia de la investigación de este compuesto, o sus derivados, como terapia adyuvante en pacientes AP que presenten cardiomiopatías. Por otro lado, la necesidad de obtener nuevos modelos celulares humanos que nos permitan estudiar en mayor profundidad la fisiopatología de la AP y evaluar el efecto de nuevos compuestos terapéuticos nos ha llevado a la generación y caracterización de células madre pluripotentes inducidas (iPSCs) a partir de fibroblastos de un paciente con mutaciones en el gen PCCA y otro, en PCCB. Estas líneas de iPSCs han supuesto una herramienta con un gran potencial para modelar la enfermedad ya que se han diferenciado a neuronas (iNs) GABAérgicas, astrocitos (iAs) y cardiomiocitos (iCMs) inducidos. Estudios preliminares han mostrado que las iNs PCCA poseen, aparentemente, una menor capacidad de formación de sinapsis que se correlaciona con defectos en el metabolismo energético mitocondrial. Asimismo, la caracterización de los iCMs PCCA ha revelado una disminución del metabolismo energético mitocondrial que se relaciona con una alteración en la expresión de genes de la cadena de transporte de electrones y de la biogénesis mitocondrial. Además, presentan alteraciones en la biogénesis ribosomal, en la composición y la respuesta a estrés del retículo endoplásmico y en la expresión de microRNAs relacionados con daño cardíaco. Estas vías alteradas, que pueden contribuir a la fisiopatología de la enfermedad, suponen un punto de partida para la identificación de nuevas dianas terapéuticas en la APEste trabajo ha sido realizado en el Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (CSIC-UAM) y en una estancia corta en el Lund Stem Cell Center (Lund, Sweden). Este trabajo ha sido posible gracias a una ayuda para la formación del profesorado universitario (FPU15/02923) y a la ayuda a la movilidad para estancias breves y traslados temporales para beneficiarios del programa FPU (EST17/00224

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.Economia y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER) PI16/00573, and Fundacion Isabel Gemio- Fundacion La Caixa (LCF/PR/PR16/11110018) Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces. LAC is a PhD student funded by the Asociacion Espanola para el Estudio de Metabolopatias Congenitas (AEPMEC). ALM is a postdoctoral researcher of Comunidad Autonoma de Madrid (PEJD-2017-POST/BMD-367

Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia.

Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA

Generation and characterization of a human iPSC line (UAMi004-A) from a patient with propionic acidemia due to defects in the PCCB gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.Spanish Ministry of Economy and Competitiveness and European Regional Development Fund. The authors thank INDEPF (Instituto de investigación y desarrollo social de enfermedades poco frecuentes), the Cytogenetic unit from Centro Nacional de Investigaciones Oncológicas (CNIO) and Mar Álvarez for their excellent technical assistance. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces

Generation and characterization of a human iPSC line (UAMi004-A) from a patient with propionic acidemia due to defects in the PCCB gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiencyResearch reported in this work was funded by Grant PAF107 from the Propionic Acidemia Foundation and by grant SAF2016-76004-R from Spanish Ministry of Economy and Competitiveness and European Regional Development Fund. The authors thank INDEPF (Instituto de investigación y desarrollo social de enfermedades poco frecuentes), the Cytogenetic unit from Centro Nacional de Investigaciones Oncológicas(CNIO) and Mar Álvarez for their excellent technical assistance. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces. ALM is a postdoctoral researcher of Comunidad Autónoma de Madrid (PEJD-2017-POST/BMD-3671). EABis a PhD student funded by the FPU program of the Spanish Ministry ofScience, Innovation and Universities (FPU15/02923