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 variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1–4]), 40 -phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 40 -phosphopantothenoylcysteine to 40 -phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDCConsejería de Educaci on, Juventud y Deporte, Comunidad de Madrid, Grant/Award Number: B2017/BMD3721; Instituto de Salud Carlos III, Grant/Award Number: PI19/01155; Ministerio de Economía, Industria y Competitividad, Grant/Award Number: BFU2017-82574-

Pathogenic variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy

12 páginas, 6 figurasCoenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.This work was funded by the Instituto de Salud Carlos (ISCIII), the European Regional Development Fund [PI19/01155], the Ministerio de Economía, Industria y Competitividad, Spain (BFU2017-82574-P), and the Consejería de Educacion, Juventud y Deporte, Comunidad de Madrid [B2017/BMD3721].Peer reviewe

Implicaciones del metabolismo de la glicina en la salud y la enfermedad humana: Hiperglicinemia no Cetósica, una enfermedad con fenotipo clínico neurológico

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 19-06-2023Esta tesis tiene embargado el acceso al texto completo hasta el 19-12-2024Una de las reacciones químicas del organismo que aporta unidades de carbono para sostener, entre otros, el crecimiento y la proliferación celular es el sistema de escisión de glicina (GCS). En este trabajo hemos abordado el estudio de mutaciones en los genes GLDC, AMT y GCSH codificantes de las proteínas del complejo GCS, identificadas en una batería de pacientes con diagnóstico genético de Hiperglicinemia No Cetósica (NKH), una patología catalogada como rara y con una expresión fenotípica eminentemente neurológica. Dentro de la cohorte inicial se identificó el primer caso portador de mutaciones bialélicas en GCSH. El diagnóstico molecular de este paciente fue el primer paso de un trabajo colaborativo a nivel mundial en el que finalmente se reunió un total de seis pacientes NKH con mutaciones bialélicas en dicho gen, entre ellas, 4 variantes de cambio de sentido. El modelado funcional de estas variantes implicó la generación de un sistema knockdown del gen de interés. El silenciamiento en la expresión de GCSH y la sobreexpresión de las variantes descritas en dicho modelo permitió reproducir las principales alteraciones bioquímicas y metabólicas acontecidas en estos pacientes y confirmar el doble papel propuesto para la proteína GCSH en la homeostasis de glicina y el metabolismo de ácido lipoico dentro de la célula. El resto de los pacientes de la cohorte portaba mutaciones en AMT y GLDC. Doce de las variantes en GLDC fueron clasificadas como posibles causantes de alteraciones en la estabilidad y plegamiento proteicos. La sobreexpresión de estos mutantes en condiciones favorecedoras de estabilidad incrementó no solo su presencia sino, en algunos casos, su actividad. Sin embargo, el uso de inhibidores de la proteostasis rindió resultados poco reproducibles en el rescate de proteína GCSP, aunque sí fue efectivo en el caso de GCST en fibroblastos de pacientes AMT. Estos resultados allanan el camino hacia el estudio de su uso como diana terapéutica para tratar la enfermedad NKH causada por mutaciones en el gen AMT. La identificación de nuevas causas de patología en pacientes sin diagnóstico y la implementación de nuevos modelos celulares son esenciales para identificar nuevos enfoques terapéuticos. En este trabajo hemos generado una línea de Células Madre Pluripotentes Inducidas (iPSCs) a partir de fibroblastos de paciente con variantes en GLDC, teóricamente modulables por estrategias potenciadoras del plegamiento proteico, similares a las células características de los primeros estadios del desarrollo. En este modelo, la deficiencia en GLDC se asociaba a una disminución en la proliferación celular y una consecuente adaptación a nivel metabólico o resiliencia celular en comparación con lo observado en una línea de iPSCs control, aparentemente relacionada con la implicación de GCS en el metabolismo de unidades de un carbono. Como la NKH es una patología eminentemente neurológica, hemos avanzado en la diferenciación de las iPSCs hasta células de linaje astrocitario, donde GLDC se ha descrito como marcador metabólico. Nuestro análisis detecta aumentos significativos en la expresión de algunos genes marcadores de glía radial (incluidos GFAP o VIM) así como de otros relacionados con el metabolismo de glicinaserina (como SRR) que si se verifican en estudios posteriores, supondrían un gran avance en la comprensión de la patología y por tanto en la identificación de opciones terapéuticas dirigidas. Nuestros resultados, aunque preliminares, resaltan la importancia del equilibrio en la regulación del metabolismo de glicina. La identificación de mecanismos fisiopatológicos que subyacen a la enfermedad, así como del efecto de mutaciones sobre la estructura y funcionamiento de proteínas es esencial para comprender la patología y plantear nuevos enfoques terapéuticos viable

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

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

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine–glycine–one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options