PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

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.

Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy.

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

Cardiomyocytes Derived from Induced Pluripotent Stem Cells as a Disease Model for Propionic Acidemia.

Propionic acidemia (PA), one of the most frequent life-threatening organic acidemias, is caused by mutations in either the PCCA or PCCB genes encoding both subunits of the mitochondrial propionyl-CoA carboxylase (PCC) enzyme. Cardiac alterations (hypertrophy, dilated cardiomyopathy, long QT) are one of the major causes of mortality in patients surviving the neonatal period. To overcome limitations of current cellular models of PA, we generated induced pluripotent stem cells (iPSCs) from a PA patient with defects in the PCCA gene, and successfully differentiated them into cardiomyocytes. PCCA iPSC-derived cardiomyocytes exhibited reduced oxygen consumption, an accumulation of residual bodies and lipid droplets, and increased ribosomal biogenesis. Furthermore, we found increased protein levels of HERP, GRP78, GRP75, SIG-1R and MFN2, suggesting endoplasmic reticulum stress and calcium perturbations in these cells. We also analyzed a series of heart-enriched miRNAs previously found deregulated in the heart tissue of a PA murine model and confirmed their altered expression. Our novel results show that PA iPSC-cardiomyocytes represent a promising model for investigating the pathological mechanisms underlying PA cardiomyopathies, also serving as an ex vivo platform for therapeutic evaluation.

Identification of 34 novel mutations in propionic acidemia: Functional characterization of missense variants and phenotype associations.

Propionic acidemia (PA) is caused by mutations in the PCCA and PCCB genes, encoding α and ß subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Up to date, >200 pathogenic mutations have been identified, mostly missense defects. Genetic analysis in PA patients referred to the laboratory for the past 15 years identified 20 novel variants in the PCCA gene and 14 in the PCCB gene. 21 missense variants were predicted as probably disease-causing by different bioinformatics algorithms. Structural analysis in the available 3D model of the PCC enzyme indicated potential instability for most of them. Functional analysis in a eukaryotic system confirmed the pathogenic effect for the missense variants and for one amino acid deletion, as they all exhibited reduced or null PCC activity and protein levels compared to wild-type constructs. PCCB variants p.E168del, p.Q58P and p.I460T resulted in medium-high protein levels and no activity. Variants p.R230C and p.C712S in PCCA, and p.G188A, p.R272W and p.H534R in PCCB retained both partial PCC activity and medium-high protein levels. Available patients-derived fibroblasts carriers of some of these mutations were grown at 28 °C or 37 °C and a slight increase in PCC activity or protein could be detected in some cases at the folding-permissive conditions. Examination of available clinical data showed correlation of the results of the functional analysis with disease severity for most mutations, with some notable exceptions, confirming the notion that the final phenotypic outcome in PA is not easily predicted.

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.

Role of miRNAs in human disease and inborn errors of metabolism.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression posttranscriptionally by base pairing with target messenger RNAs (mRNAs). They are estimated to target ∼60% of all human protein-coding genes and are involved in regulating key physiological processes and intracellular signaling pathways. They also exhibit tissue specificity, and their dysregulation is linked to the progression of pathology. Identifying disease associated miRNAs and their respective targets provides novel molecular insight into disease, enabling the design of new therapeutic strategies. Notably, miRNAs are present in stable form in biological fluids, making them amenable to routine clinical processing and analysis, which has paved the way for their use as novel biomarkers of disease and response to therapy. One of the most relevant findings in miRNA research concerns the therapeutic modulation of specific miRNA levels in vitro and in vivo, which has led to miRNA-based drugs entering clinical trials. Most studies relative to miRNA profiling, association with pathology, and therapeutical modulation have been conducted for cancer, cardiovascular and neurodegenerative diseases. However, for different monogenic diseases, including inborn errors of metabolism (IEM), research contributing to alterations to physiopathology caused by miRNAs is steadily increasing. Herein, we review the biogenesis pathway and mode of miRNA action, their known roles in disease states, and use of circulating miRNAs as biomarkers, describing the available research tools for basic and clinical studies. In addition, we summarize recent literature on miRNA studies in inherited metabolic diseases.

Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients' cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls' fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.

Functional analysis of novel variants identified in cis in the PCCB gene in a patient with propionic acidemia.

Next-generation sequencing has improved the diagnosis of inborn errors of metabolism, allowing rapid confirmation of cases detected by clinical/biochemical studies or newborn screening. The challenge, however, remains for establishing the pathogenicity of the identified variants, especially for novel missense changes or small in-frame deletions. In this work we report a propionic acidemia patient exhibiting a severe neonatal form with coma and hyperammonaemia. Genetic analysis identified the previously described pathogenic PCCB variant p.R512C in the maternal allele and two novel PCCB variants in cis in the paternal allele, p.G246del and p.S322F. Expression analysis in a eukaryotic system confirmed the deleterious effect of the novel missense variant and of the one amino acid deletion, as they both exhibited reduced protein levels and reduced or null PCC activity compared to the wild-type construct. Accordingly, the double mutant resulted in no residual activity. This study increases the knowledge of the genotype-phenotype correlations in the rare disease propionic acidemia and highlights the necessity of functional analysis of novel variants to understand their contribution to disease severity and to accurately classify their pathogenic status. In conclusion, two novel PCCB pathogenic variants have been identified, expanding the current mutational spectrum of propionic acidemia.

Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder propionic acidemia by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. Interestingly, the PCCA pseudoexon offers an unexploited potential to upregulate gene expression because healthy tissues show relatively high inclusion levels. By blocking inclusion of the nonactivated wild-type pseudoexon, we can increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels in not only patient fibroblasts harboring PCCA missense variants but also those harboring PCCB missense variants. This is a potential treatment strategy for propionic acidemia.

Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects.

Oxidative stress has been described as a putative disease mechanism in pathologies associated with an elevation of homocysteine. An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders of cobalamin metabolism, particularly with methylmalonic aciduria (MMA) combined with homocystinuria cblC type. In this work, we have evaluated several parameters related to oxidative stress and apoptosis in fibroblasts from patients with homocystinuria due to defects in the MTR, MTRR, and MTHFR genes involved in the remethylation pathway of homocysteine. We have also evaluated these processes by knocking down the MTRR gene in cellular models, and complementation by transducing the wild-type gene in cblE mutant fibroblasts. All cell lines showed a significant increase in ROS content and in MnSOD expression level, and also a higher rate of apoptosis with similar levels to the ones in cblC fibroblasts. The amount of the active phosphorylated forms of p38 and JNK stress-kinases was also increased. ROS content and apoptosis rate increased in control fibroblasts and in a glioblastoma cell line by shRNA-mediated silencing of MTRR gene expression. In contrast, wild-type MTRR gene corrected mutant cell lines showed a decrease in ROS and apoptosis levels. To the best of our knowledge, this study provides the first evidence that an impaired remethylation capacity due to low MTRR and MTR activity might be partially responsible for stress response.

Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. PA is caused by mutations in either the PCCA or PCCB genes encoding the α- and ß-subunits of the PCC enzyme which are assembled as an α6ß6 dodecamer. In this study we have investigated the molecular basis of the defect in ten fibroblast samples from PA patients. Using homology modeling with the recently solved crystal structure of the PCC holoenzyme and a eukaryotic expression system we have analyzed the structural and functional effect of novel point mutations, also revealing a novel splice defect by minigene analysis. In addition, we have investigated the contribution of oxidative stress to cellular damage measuring reactive oxygen species (ROS) levels and apoptosis parameters in patient fibroblasts, as recent studies point to a secondary mitochondrial dysfunction as pathophysiological mechanism in this disorder. The results show an increase in intracellular ROS content compared to controls, correlating with the activation of the JNK and p38 signaling pathways. Highest ROS levels were present in cells harboring functionally null mutations, including one severe missense mutation. This work provides molecular insight into the pathogenicity of PA variants and indicates that oxidative stress may be a major contributing factor to the cellular damage, supporting the proposal of antioxidant strategies as novel supplementary therapy in this rare disease.

Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells.

The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defect.

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.

Defining the pathogenicity of creatine deficiency syndrome.

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense-mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1-10, 2011. © 2011 Wiley-Liss, Inc.

Different altered pattern expression of genes related to apoptosis in isolated methylmalonic aciduria cblB type and combined with homocystinuria cblC type.

An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders involved in cobalamin metabolism, including isolated methylmalonic aciduria (MMA) cblB type and MMA combined with homocystinuria (MMAHC) cblC type. Given the relevance of p38 and JNK kinases in stress-response, their activation in fibroblasts from a spectrum of patients (mut, cblA, cblB, cblC and cblE) was analyzed revealing an increased expression of the phosphorylated-forms, specially in cblB and cblC cell lines that presented the highest ROS and apoptosis levels. To gain further insight into the molecular mechanisms responsible for the enhanced apoptotic process observed in cblB and cblC fibroblasts, we evaluated the expression pattern of 84 apoptosis-related genes by quantitative real-time PCR. An elevated number of pro-apoptotic genes were overexpressed in cblC cells showing a higher rate of apoptosis compared to cblB and control samples. Additionally, apoptosis appears to be mainly triggered through the extrinsic pathway in cblC, while the intrinsic pathway was primarily activated in cblB cells. The differences observed regarding the apoptosis rate and preferred pathway between cblB and cblC patients, who both built up methylmalonic acid, might be explained by the accumulated homocysteine in the cblC group. The loss of MMACHC function in cblC patients might be partially responsible for the oxidative stress and apoptosis processes observed in these cell lines. Our results suggest that ROS production may represent a genetic modifier of the phenotype and support the potential of using antioxidants as a novel therapeutic strategy to improve the severe neurological outcome of these rare diseases.

Functional and structural analysis of five mutations identified in methylmalonic aciduria cblB type.

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K(M) for ATP and K(D) for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations.

Cardiac Complications of Propionic and Other Inherited Organic Acidemias.

Clinical observations and experimental studies have determined that systemic acid-base disturbances can profoundly affect the heart. A wealth of information is available on the effects of altered pH on cardiac function but, by comparison, much less is known about the actions of the organic anions that accumulate alongside H+ ions in acidosis. In the blood and other body fluids, these organic chemical species can collectively reach concentrations of several millimolar in severe metabolic acidoses, as in the case of inherited organic acidemias, and exert powerful biological actions on the heart that are not intuitive to predict. Indeed, cardiac pathologies, such as cardiomyopathy and arrhythmia, are frequently reported in organic acidemia patients, but the underlying pathophysiological mechanisms are not well established. Research efforts in the area of organic anion physiology have increased dramatically in recent years, particularly for propionate, which accumulates in propionic acidemia, one of the commonest organic acidemias characterized by a high incidence of cardiac disease. This Review provides a comprehensive historical overview of all known organic acidemias that feature cardiac complications and a state-of-the-art overview of the cardiac sequelae observed in propionic acidemia. The article identifies the most promising candidates for molecular mechanisms that become aberrantly engaged by propionate anions (and its metabolites), and discusses how these may result in cardiac derangements in propionic acidemia. Key clinical and experimental findings are considered in the context of potential therapies in the near future.

Generation of a gene-corrected human isogenic line (UAMi006-A) from propionic acidemia patient iPSC with an homozygous mutation in the PCCB gene using CRISPR/Cas9 technology.

Propionic acidemia (PA) is an inherited metabolic disease caused by mutations in the PCCA and PCCB genes. We have previously generated an induced pluripotent stem cell (iPSC) line (UAMi004-A) from a PA patient with the c.1218_1231del14ins12 (p.Gly407Argfs*14) homozygous mutation in the PCCB gene. Here, we report the generation of the isogenic control in which the mutation was genetically corrected using CRISPR/Cas9 technology. Off-target editing presence was excluded and the iPSCs had typical embryonic stem cell-like morphology and normal karyotype that expressed pluripotency markers and maintained their in vitro differentiation potential.