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.

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.

A novel delins (c.773_819+47delinsAA) mutation of the PCCA gene associated with neonatal-onset propionic acidemia: a case report.

BACKGROUND: Propionic acidemia (PA)(OMIM#606054) is an inborn error of branched-chain amino acid metabolism, caused by defects in the propionyl-CoA carboxylase (PCC) enzyme which encoded by the PCCA and PCCB genes. CASE PRESENTATION: Here we report a Chinese neonate diagnosed with suspected PA based on the clinical symptoms, gas chromatography-mass spectrometry (GC/MS), and brain imaging tests. Targeted next-generation sequencing (NGS) was performed on the proband. We detected only one heterozygous recurrent nonsense variant (c.937C > T, p.Arg313Ter) in the PCCA gene. When we manually checked the binary alignment map (BAM) diagram of PCCA gene, we found a heterozygous deletion chr13:100915039-100915132delinsAA (c.773_819 + 47delinsAA) (GRCh37.p13) inside the exon 10 in the PCCA gene. The results were validated by Sanger sequencing and qPCR method in the family: the variant (c.937C > T, p.Arg313Ter) was in the maternal allele, and the delins was in the paternal allele. When the mother was pregnant again, prenatal diagnosis was carried out through amniocentesis at 18 weeks gestation, the fetus carried neither of the two mutations. After birth, newborn screening was undertaken, the result was negative. CONCLUSIONS: We identified a recurrent c.937C > T and a novel c.773_819 + 47delinsAA mutations in the PCCA gene, which may be the genetic cause of the phenotype of this patient. Our findings expanded the spectrum of causative genotype-phenotype of the PCCA gene. For the cases, the NGS results revealed only a heterozygous mutation in autosomal recessive disease when the gene is associated with phenotypes, it is necessary to manually check the BAM diagram to improve the detection rate. Targeted NGS is an effective technique to detect the various genetic lesions responsible for the PA in one step. Genetic testing is essential for genetic counselling and prenatal diagnosis in the family to avoid birth defects.

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.

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.

Propionyl-CoA carboxylase - A review.

Propionyl-CoA carboxylase (PCC) is the enzyme which catalyzes the carboxylation of propionyl-CoA to methylmalonyl-CoA and is encoded by the genes PCCA and PCCB to form a hetero-dodecamer. Dysfunction of PCC leads to the inherited metabolic disorder propionic acidemia, which can result in an affected individual presenting with metabolic acidosis, hyperammonemia, lethargy, vomiting and sometimes coma and death if not treated. Individuals with propionic acidemia also have a number of long term complications resulting from the dysfunction of the PCC enzyme. Here we present an overview of the current knowledge about the structure and function of PCC. We review an updated list of human variants which are published and provide an overview of the disease.

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.

Seventeen Novel Mutations in PCCA and PCCB Genes in Indian Propionic Acidemia Patients, and Their Outcomes.

AIMS: The goal of this study was to identify mutations in the propionyl-CoA carboxylase alpha subunit (PCCA) and propionyl-CoA carboxylase beta subunit (PCCB) genes, and to assess their effects on propionic academia (PA) patients. METHODOLOGY: Twenty-five Indian children with PA were enrolled in this study. Bidirectional Sanger sequencing was performed on both the coding and flanking regions of the PCCA and PCCB genes and the chromatograms were analyzed. Bioinformatic tools were used to classify novel variations into pathogenic or benign. RESULTS: The majority of the cases (19/25, 76%) were of the early-onset (<90 days of age) type and 5 were of the late-onset type. The majority of patients had mutations in the PCCA gene (18/25). A total of 26 mutations were noted: 20 in the PCCA gene and 6 in PCCB gene. Seventeen mutations were novel (14 in PCCA and 3 in PCCB). The SNP c.937C>T (p.Arg313Ter), was noted in 9/36 (25%) alleles in the PCCA gene. All of the children were symptomatic and only three survived who are doing well with no major disabilities. CONCLUSION: The spectrum of mutations in the PCCA and PCCB genes among Indians is distinct from other populations. The absence of a common mutation signifies the heterogeneity and admixture of various subpopulations. These findings also suggest that individuals of Indian origin may not benefit from the mutation-based "carrier screening panels" offered by many genetic laboratories.

Propionic acidemia in a previously healthy adolescent with acute onset of dilated cardiomyopathy.

UNLABELLED: Propionic acidemia (PA) is a rare autosomal recessive organic aciduria resulting from defects in propionyl-CoA-carboxylase (PCC), a key enzyme of intermediate energy metabolism. PA mostly manifests during the neonatal period, when affected newborns develop severe metabolic acidosis and hyperammonemia. We present a previously healthy teenager, who suffered from acute fatigue and breathlessness. The patient was tachycardic, displayed a precordial heave and a systolic murmur. Cardiac investigations revealed severe dilated cardiomyopathy (DCM). Biochemical work up led to the diagnosis of PA. Remarkably, this patient of consanguineous Hispanic origin was in a good general health condition before the acute onset of DCM. Diagnosis of PA was confirmed by enzymatic and molecular genetic analysis, the latter revealing a novel homozygous mutation in the PCCB gene (c.1229G > A; p.R410Q). Residual PCC enzyme activity of approximately 14 % of normal was detected in patient's lymphocytes and fibroblasts, thereby providing a possible explanation for the hitherto asymptomatic phenotype. CONCLUSION: Isolated DCM, although rare, can be the leading and/or sole symptom of late-onset PA. Therefore, patients with DCM should receive a comprehensive diagnostic evaluation including selective screening for inborn errors of metabolism.

Two frequent mutations associated with the classic form of propionic acidemia in Taiwan.

Propionyl-CoA carboxylase (PCC) is involved in the catabolism of branched chain amino acids, odd-numbered fatty acids, cholesterol, and other metabolites. PCC consists of two subunits, α and ß, encoded by the PCCA and PCCB genes, respectively. Mutations in the PCCA or PCCB subunit gene may lead to propionic acidemia. In this study, we performed mutation analysis on ten propionic acidemia patients from eight unrelated and nonconsanguineous families in Taiwan. Two PCCA mutations, c.229C→T (p.R77W) and c.1262A→C (p.Q421P), were identified in a PCCA-deficient patient. Six mutations in the PCCB gene, including c.-4156_183+3713del, c.580T→C (p.S194P), c.838dup (p.L280Pfs 11), c.1301C→T (p.A434V), c.1316A→G (P.Y439C), and c.1534C→T (p.R512C), were identified in seven PCCB-deficient families. The c.-4156_183+3713del mutation is the first known large deletion that affects the PCCB gene functions. Furthermore, the c.1301C→T and c.-4156_183+3713del mutations in the PCCB gene have not been reported previously. Clinical features demonstrated that these two frequent mutations are associated with low enzyme activity and a classic propionic acidemia phenotype.

Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

The molecular landscape of propionic acidemia and methylmalonic aciduria in Latin America.

In this work, we review the clinical and genetic data in 14 Latin American propionic acidemia (PA) and 15 methylmalonic aciduria (MMAuria) patients. In the PA patients, we have identified four different changes in the PCCA gene, including one novel one (c.414+5G>A) affecting the splicing process. The PCCB mutational spectrum included two prevalent changes accounting for close to 60% of the mutant alleles studied and one novel change (c.494G>C) which by functional analysis is clearly pathogenic. We have also identified the deep intronic change c.654+462A>G, and the results of the antisense treatment in the patient's cell line confirmed the functional recovery of PCC activity. All PA patients bearing out-of-frame mutations presented the disease earlier while patients bearing in hemizygous fashion p.E168K and p.R165W presented the disease later. Regarding the MMAuria patients, we have found three novel mutations in the MUT gene (c.1068G>A, c.1587_1594del8 and c.593delA) and one in the MMAB gene (c.349-1 G>C). Two patients with MMAuria with homocystinuria cblC type are carriers of the frequent c.271dupA mutation. All mut(0), cblB and cblC patients presented the symptoms early and in general had more neurological complications, while cblA and mut(-) patients exhibited a late-onset presentation, and in general the long-term outcome was better. The results presented in this work emphasize the importance of the genetic analysis of the patients not only for diagnostic purposes but also to research into novel therapies based on the genotype.

Unusual presentation of propionic acidaemia as isolated cardiomyopathy.

Propionic acidaemia (PA) is an autosomal recessive disease that results from deficiency of propionyl-CoA carboxylase (PCC). In the majority of reported cases, the phenotype includes metabolic acidosis and/or neurological deficits. We report on a 14-year-old Asian-American male with PA who presented with isolated cardiomyopathy without any documented episodes of metabolic acidosis or evidence of any neurocognitive deficits. On routine metabolic screening, the patient was found to have urine organic acids suggestive of PA. Biochemical and genetic characterization confirmed a PCC deficiency with two novel mutations in PCCB: IVS7 + 2 T > G (c.763 + 2 T > G) and p.R410Q (c.1229 G > A). Residual enzyme activity likely explains our patient's mild phenotype. Splicing mutations tend to result in a milder phenotype as these mutations may still produce small amounts of normal enzyme. In addition, the similar p.R410W mutation has been shown to have partial residual activity. Moreover, this case illustrates that a thorough metabolic evaluation should be performed in both paediatric and adult patients with cardiomyopathy. Such an evaluation has important implications for clinical management and genetic counselling.

Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA.

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.