Establishment of a non-integrated iPSC line (SDQLCHi043-A) from a male infant with propionic acidemia carrying compound heterozygote mutations in PCCB gene.

In this study, peripheral blood mononuclear cells were contributed from a male infant with propionic acidemia (PA) verified by clinical and genetic diagnosis, who inherited compound heterozygous mutations in the propionyl-CoA carboxylase subunit beta (PCCB) gene. Here, this iPS was generated by non-integrated episomal vectors with SOX2, BCL-XL, OCT4, C-MYC and OCT4. Also, this iPSC line exhibited the morphology of pluripotent stem cells, upward mRNA and protein expression of pluripotency markers, conspicuous in vitro differentiation potency and regular karyotype, and carried PCCB gene mutations, which provided an excellent model for the research and drug screening of PA.

Pathophysiological mechanisms of complications associated with propionic acidemia.

Propionic acidemia (PA) is a genetic metabolic disorder caused by mutations in the mitochondrial enzyme, propionyl-CoA carboxylase (PCC), which is responsible for converting propionyl-CoA to methylmalonyl-CoA for further metabolism in the tricarboxylic acid cycle. When this process is disrupted, propionyl-CoA and its metabolites accumulate, leading to a variety of complications including life-threatening cardiac diseases and other metabolic strokes. While the clinical symptoms and diagnosis of PA are well established, the underlying pathophysiological mechanisms of PA-induced diseases are not fully understood. As a result, there are currently few effective therapies for PA beyond dietary restriction. This review focuses on the pathophysiological mechanisms of the various complications associated with PA, drawing on extensive research and clinical reports. Most research suggests that propionyl-CoA and its metabolites can impair mitochondrial energy metabolism and cause cellular damage by inducing oxidative stress. However, direct evidence from in vivo studies is still lacking. Additionally, elevated levels of ammonia can be toxic, although not all PA patients develop hyperammonemia. The discovery of pathophysiological mechanisms underlying various complications associated with PA can aid in the development of more effective therapeutic treatments. The consequences of elevated odd-chain fatty acids in lipid metabolism and potential gene expression changes mediated by histone propionylation also warrant further investigation.

Identification and characterization of the largest deletion in the PCCA gene causing severe acute early-onset form of propionic acidemia.

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM_000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in the PCCA gene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877 bp length, "NG_008768.1:g.185211_403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM_000060.4(BTD):c.1330G > C.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

Neuropathological report of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive inheritable metabolic disease caused by mutations in the propionyl CoA carboxylase gene (PCC) that affects multiple systems of the human body. Here, we report neuropathological findings of a PA patient. The patient was a male infant who presented with increasing lethargy and poor feeding from four days postpartum. He gradually became comatose and died from complications after liver transplantation at three months old. The results of laboratory examination were consistent with PA, and genetic analysis revealed compound heterozygous mutations in the gene for PCC subunit beta: c.838dupC (rs769968548) and c.1127G>T (rs142982097). Brain-restricted autopsy was performed 23 h after his death, and the neuropathological examination revealed distinct astrocytosis, oligodendrocytic loss, neuronal loss, and demyelination across the brainstem, motor cortex, basal ganglia, and thalamus. Spongiosis, vacuolization, and the appearance of Alzheimer type II astrocytes and activated microglia were observed as well. This is the first brain autopsy report of PA with a clear genetic cause.

[Phenotypes and genotypes of 78 patients with propionic acidemia].

Objective: Propionic acidemia is a rare inherited metabolic disorder caused by propionyl CoA carboxylase (PCC) deficiency. This study aims to analyze the clinical characteristics and gene variations of Chinese patients with propionic acidemia, and to explore the correlation between clinical phenotypes and genotypes. Methods: Single-center, retrospective and observational study. Seventy-eight patients of propionic acidemia (46 males and 32 females) from 20 provinces and autonomous regions were admitted from January 2007 to April 2022. Their age of initial diagnosis ranged from 7 days to 15 years. The clinical manifestations, biochemical and metabolic abnormalities, genetic variations, diagnosis, treatment and outcome were studied. Chi-Square test or Mann-Whitney U test were used for statistical analysis. Results: Among 78 cases, 6 (7.7%) were identified by newborn screening; 72 (92.3%) were clinically diagnosed after onset, and the age of onset was 2 hours after birth to 15 years old; 32 cases had early-onset disease and 40 cases had late-onset disease. The initial manifestations included lethargy, hypotonia, vomiting, feeding difficulties, developmental delay, epilepsy, and coma. Among the 74 cases who accepted gene analysis, 35 (47.3%) had PCCA variants and 39 (52.7%) had PCCB variants. A total of 39 PCCA variants and 32 PCCB variants were detected, among which c.2002G>A and c.229C>T in PCCA and c.838dupC and c.1087T>C in PCCB were the most common variants in this cohort. The variants c.1228C>T and c.1283C>T in PCCB may be related to early-onset type. The variants c.838dupC, c.1127G>T and c.1316A>G in PCCB, and c.2002G>A in PCCA may be related to late-onset disease. Six patients detected by newborn screening and treated at asymptomatic stage developed normal. The clinically diagnosed 72 cases had varied complications. 10 (12.8%) cases of them died. 62 patients improved after metabolic therapy by L-carnitine and diet. Six patients received liver transplantation because of recurrent metabolic crisis. Their clinical symptoms were markedly improved. Conclusion: The clinical manifestations of propionic acidemia are complex and lack of specificity. Newborn screening and high-risk screening are keys for early treatment and better outcome. The correlation between the genotype and phenotype of propionic acidemia is unclear, but certain variants may be associated with early-onset or late-onset propionic acidemia.

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans.

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

Propionic acidemia in mice: Liver acyl-CoA levels and clinical course.

Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of propionyl-CoA carboxylase (PCC). We studied PA transgenic (Pat) mice that lack endogenous PCC but express a hypoactive human PCCA cDNA, permitting their survival. Pat cohorts followed from 3 to 20 weeks of age showed growth failure and lethal crises of lethargy and hyperammonemia, commoner in males (27/50, 54%) than in females (11/52, 21%) and occurring mainly in Pat mice with the most severe growth deficiency. Groups of Pat mice were studied under basal conditions (P-Ba mice) and during acute crises (P-Ac). Plasma acylcarnitines in P-Ba mice, compared to controls, showed markedly elevated C3- and low C2-carnitine, with a further decrease in C2-carnitine in P-Ac mice. These clinical and biochemical findings resemble those of human PA patients. Liver acyl-CoA measurements showed that propionyl-CoA was a minor species in controls (propionyl-CoA/acetyl-CoA ratio, 0.09). In contrast, in P-Ba liver the ratio was 1.4 and in P-Ac liver, 13, with concurrent reductions of the levels of acetyl-CoA and other acyl-CoAs. Plasma ammonia levels in control, P-Ba and P-Ac mice were 109 ± 10, 311 ± 48 and 551 ± 61 µmol/L respectively. Four-week administration to Pat mice, of carglumate (N-carbamyl-L-glutamic acid), an analogue of N-carbamylglutamate, the product of the only acyl-CoA-requiring reaction directly related to the urea cycle, was associated with increased food consumption, improved growth and absence of fatal crises. Pat mice showed many similarities to human PA patients and provide a useful model for studying tissue pathophysiology and treatment outcomes.

Mitochondrial damage in renal epithelial cells is potentiated by protein exposure in propionic aciduria.

Propionic aciduria (PA) is caused by deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Due to inefficient propionate catabolism patients are endangered by life-threatening ketoacidotic crisis. Protein and amino acid restriction are major therapeutic pillars. However, long-term complications like neurological deterioration and cardiac abnormalities cannot be prevented. Chronic kidney disease (CKD), which is a well-known characteristic of methylmalonic aciduria two enzymatic steps downstream from PCC, has been recognized as a novel late-onset complication in PA. The pathophysiology of CKD in PA is unclear. We investigated mitochondrial structure and metabolism in human renal tubular cells of healthy controls and PA patients. The cells were exposed to either standard cell culture conditions (NT), high protein (HP) or high concentrations of isoleucine and valine (I/V). Mitochondrial morphology changed to condensed, fractured morphology in PA cells irrespective of the cell culture medium. HP and I/V exposure, however, potentiated oxidative stress in PA cells. Mitochondrial mass was enriched in PA cells, and further increased by HP and I/V exposure suggesting a need for compensation. Alterations in the tricarboxylic acid cycle intermediates and accumulation of medium- and long-chain acylcarnitines pointed to altered mitochondrial energy metabolism. Mitophagy was silenced while autophagy as cellular defense mechanisms was highly active in PA cells. The data demonstrate that PA is associated with renal mitochondrial damage which is aggravated by protein and I/V load. Preservation of mitochondrial energy homeostasis in renal cells may be a potential future therapeutic target.

Dual mRNA therapy restores metabolic function in long-term studies in mice with propionic acidemia.

Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccBBs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccBBs activity improvement, and a double mutant of PccBBs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by kcat/Km. Moreover, this PccBBs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO2 molecules, which was confirmed by isotope labeling experiment with NaH13CO3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO2 fixation reactions.

Biochemical and anaplerotic applications of in vitro models of propionic acidemia and methylmalonic acidemia using patient-derived primary hepatocytes.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, which are caused by a deficiency in the enzyme propionyl-CoA carboxylase or the enzyme methylmalonyl-CoA (MM-CoA) mutase, respectively. The functional consequence of PA or MMA is the inability to catabolize P-CoA to MM-CoA or MM-CoA to succinyl-CoA, resulting in the accumulation of P-CoA and other metabolic intermediates, such as propionylcarnitine (C3), 3-hydroxypropionic acid, methylcitric acid (MCA), and methylmalonic acid (only in MMA). P-CoA and its metabolic intermediates, at high concentrations found in PA and MMA, inhibit enzymes in the first steps of the urea cycle as well as enzymes in the tricarboxylic acid (TCA) cycle, causing a reduction in mitochondrial energy production. We previously showed that metabolic defects of PA could be recapitulated using PA patient-derived primary hepatocytes in a novel organotypic system. Here, we sought to investigate whether treatment of normal human primary hepatocytes with propionate would recapitulate some of the biochemical features of PA and MMA in the same platform. We found that high levels of propionate resulted in high levels of intracellular P-CoA in normal hepatocytes. Analysis of TCA cycle intermediates by GC-MS/MS indicated that propionate may inhibit enzymes of the TCA cycle as shown in PA, but is also incorporated in the TCA cycle, which does not occur in PA. To better recapitulate the disease phenotype, we obtained hepatocytes derived from livers of PA and MMA patients. We characterized the PA and MMA donors by measuring key proximal biomarkers, including P-CoA, MM-CoA, as well as clinical biomarkers propionylcarnitine-to-acetylcarnitine ratios (C3/C2), MCA, and methylmalonic acid. Additionally, we used isotopically-labeled amino acids to investigate the contribution of relevant amino acids to production of P-CoA in models of metabolic stability or acute metabolic crisis. As observed clinically, we demonstrated that the isoleucine and valine catabolism pathways are the greatest sources of P-CoA in PA and MMA donor cells and that each donor showed differential sensitivity to isoleucine and valine. We also studied the effects of disodium citrate, an anaplerotic therapy, which resulted in a significant increase in the absolute concentration of TCA cycle intermediates, which is in agreement with the benefit observed clinically. Our human cell-based PA and MMA disease models can inform preclinical drug discovery and development where mouse models of these diseases are inaccurate, particularly in well-described species differences in branched-chain amino acid catabolism.

Combinations of exonic deletions and rare mutations lead to misdiagnosis of propionic acidemia.

Propionic acidemia (PA) is an inborn metabolic error characterized by the accumulation of propionic acid due to deficiency of propionyl-CoA carboxylase (PCC). In this study, we present an intractable case with PCC activity defects. Although next-generation sequencing was applied twice to test genetic defects of the patients, no pathogenic mutations of a metabolic disease gene were identified. Mutations related to the disease were screened in prenatal diagnosis, but the mother still gave birth to an unhealthy neonate. We analyzed the second sequencing data and found that a novel synonymous PCCA mutation c.1746 G>C (p.S582S), which leads to an exon 19 skip, was screened out. Furthermore, a deletion mutation covering exon 3 and exon 4 of the PCCA gene was identified using q-PCR and DNA breakpoint test. Both of these can result in the loss of PCCA protein function. The finding expands the mutation spectrum of the PCCA gene and indicates that another technology such as cDNA analysis, multiplex ligation-dependent probe amplification (MLPA), or long-read whole-genome sequencing should be considered to improve the detection rates of special cases.

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.

Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to Methylmalonyl-CoA Decarboxylase Structure-Function Studies.

Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and ß-ketoacylsynthases.

Propionate enters GABAergic neurons, inhibits GABA transaminase, causes GABA accumulation and lethargy in a model of propionic acidemia.

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.

Propionyl-CoA carboxylase pcca-1 and pccb-1 gene deletions in Caenorhabditis elegans globally impair mitochondrial energy metabolism.

Propionic acidemia (PA) is a classical inborn error of metabolism with high morbidity that results from the inability of the propionyl-CoA carboxylase (PCC) enzyme to convert propionyl-CoA to methylmalonyl-CoA. PA is inherited in an autosomal recessive fashion due to functional loss of both alleles of either PCCA or PCCB. These genes are highly conserved across evolutionarily diverse species and share extensive similarity with pcca-1 and pccb-1 in the nematode, Caenorhabditis elegans. Here, we report the global metabolic effects of deletion in a single PCC gene, either pcca-1 or pccb-1, in C. elegans. Animal lifespan was significantly reduced relative to wild-type worms in both mutant strains, although to a greater degree in pcca-1. Mitochondrial oxidative phosphorylation (OXPHOS) capacity and efficiency as determined by direct polarography of isolated mitochondria were also significantly reduced in both mutant strains. While in vivo quantitation of mitochondrial physiology was normal in pccb-1 mutants, pcca-1 deletion mutants had significantly increased mitochondrial matrix oxidant burden as well as significantly decreased mitochondrial membrane potential and mitochondrial content. Whole worm steady-state free amino acid profiling by UPLC revealed reduced levels in both mutant strains of the glutathione precursor cysteine, possibly suggestive of increased oxidative stress. Intermediary metabolic flux analysis by GC/MS with 1,6-13C2-glucose further showed both PCC deletion strains had decreased accumulation of a distal tricarboxylic acid (TCA) cycle metabolic intermediate (+1 malate), isotopic enrichment in a proximal TCA cycle intermediate (+1 citrate), and increased +1 lactate accumulation. GC/MS analysis further revealed accumulation in the PCC mutants of a small amount of 3-hydroxypropionate, which appeared to be metabolized in C. elegans to oxalate through a unique metabolic pathway. Collectively, these detailed metabolic investigations in translational PA model animals with genetic-based PCC deficiency reveal their significantly dysregulated energy metabolism at multiple levels, including reduced mitochondrial OXPHOS capacity, increased oxidative stress, and inhibition of distal TCA cycle flux, culminating in reduced animal lifespan. These findings demonstrate that the pathophysiology of PA extends well beyond what has classically been understood as a single PCC enzyme deficiency with toxic precursor accumulation, and suggest that therapeutically targeting the globally disrupted energy metabolism may offer novel treatment opportunities for PA. SUMMARY: Two C. elegans model animals of propionic acidemia with single-gene pcca-1 or pccb-1 deletions have reduced lifespan with significantly reduced mitochondrial energy metabolism and increased oxidative stress, reflecting the disease's broader pathophysiology beyond a single enzyme deficiency with toxic precursor accumulation.

Propionic acidemia as a cause of adult-onset dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is extremely heterogeneous with a large proportion due to dominantly inherited disease-causing variants in sarcomeric genes. Recessive metabolic diseases may cause DCM, usually with onset in childhood, and in the context of systemic disease. Whether metabolic defects can also cause adult-onset DCM is currently unknown. Therefore, we performed an extensive metabolic screening in 36 consecutive adult-onset DCM patients. Diagnoses were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Measurement of propionyl-CoA carboxylase (PCC) activity was done in fibroblasts. Whole exome sequencing (WES) data of 157 additional DCM patients were analyzed for genetic defects. We found a metabolic profile characteristic for propionic acidemia in a patient with severe DCM from 55 years of age. Genetic analysis demonstrated compound heterozygous variants in PCCA. Enzymatic activity of PCC in fibroblasts was markedly reduced. A targeted analysis of the PCCA and PCCB genes using available WES data from 157 further DCM patients subsequently identified another patient with propionic acidemia. This patient had compound heterozygous variants in PCCB, and developed severe DCM from 42 years of age. Adult-onset DCM can be caused by propionic acidemia, an autosomal recessive inheritable metabolic disorder usually presenting as neonatal or childhood disease. Current guidelines advise a low-protein diet to ameliorate or prevent detrimental aspects of the disease. Long-term follow-up of a larger group of patients may show whether this diet would also ameliorate DCM. Our results suggest that diagnostic metabolic screening to identify propionic acidemia and related disorders in DCM patients is justified.

Determination of methylmalonyl coenzyme A by ultra high-performance liquid chromatography tandem mass spectrometry for measuring propionyl coenzyme A carboxylase activity in patients with propionic acidemia.

Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5µmol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3-7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan.