Clinical, biochemical and molecular characterization of 12 patients with pyruvate carboxylase deficiency treated with triheptanoin.

Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive mitochondrial neurometabolic disorder of energy deficit resulting in high morbidity and mortality, with limited therapeutic options. The PC homotetramer has a critical role in gluconeogenesis, anaplerosis, neurotransmitter synthesis, and lipogenesis. The main biochemical and clinical findings in PC deficiency (PCD) include lactic acidosis, ketonuria, failure to thrive, and neurological dysfunction. Use of the anaplerotic agent triheptanoin on a limited number of individuals with PCD has had mixed results. We expand on the potential utility of triheptanoin in PCD by examining the clinical, biochemical, molecular, and health-related quality-of-life (HRQoL) findings in a cohort of 12 individuals with PCD (eight with Type A and two each with Types B and C) treated with triheptanoin ranging for 6 days to about 7 years. The main endpoints were changes in blood lactate and HRQoL scores, but collection of useful data was limited to about half of subjects. An overall trend of lactate reduction with time on triheptanoin was noted, but with significant variability among subjects and only one subject reaching close to statistical significance for this endpoint. Parent reported HRQoL assessments with treatment showed mixed results, with some subjects showing no change, some improvement, and some worsening of overall scores. Subjects with buried amino acids in the pyruvate carboxyltransferase domain of PC that undergo destabilizing replacements may be more likely to respond (with lactate reduction or HRQoL improvement) to triheptanoin compared to those with replacements that disrupt tetramerization or subunit-subunit interface contacts. The reason for this difference is unclear and requires further validation. We observed significant variability but an overall trend of lactate reduction with time on triheptanoin and mixed parent reported outcome changes by HRQoL assessments for subjects with PCD on long-term triheptanoin. The mixed results noted with triheptanoin therapy in this study could be due to endpoint data limitation, variability of disease severity between subjects, limitation of the parent reported HRQoL tool, or subject genotype variability. Alternative designed trials and more study subjects with PCD will be needed to validate important observations from this work.

[Analysis of child with pyruvate carboxylase deficiency type A due to compound heterozygous variants of the PC gene].

OBJECTIVE: To analyze the clinical features and genetic basis for a child with pyruvate carboxylase deficiency type A (PCD-A). METHODS: Clinical data of the child was retrospectively analyzed. The child and his parents were subjected to trio-whole exome sequencing, and candidate variants were verified by bioinformatics analysis. RESULTS: The child was admitted due to fever with vomiting and disturbance of consciousness. His clinical manifestations included severe decompensated acidosis, hypotension and intractable shock. Cranial MRI showed abnormal signal in the brain, and chest X-ray revealed acute pulmonary edema. DNA sequencing revealed that he has harbored compound heterozygous variants of the PC gene, namely c.182T>C (p.I61T) and c.2581G>A (p.V861M), which were respectively inherited from his father and mother. Neither variant was retrievable in the ClinVar and HGMD databases. Through prediction of protein structure, both variants may affect the functional stability of the protein product. CONCLUSION: The compound heterozygous variants of the PC gene probably underlay the PCD-A in this child. Combined with the clinical features, the child was ultimately diagnosed as PCD-A. Above finding has enriched the spectrum of PC gene variation underlying PCD-A.

Pyruvate carboxylase deficiency type A and type C: Characterization of five novel pathogenic variants in PC and analysis of the genotype-phenotype correlation.

Pyruvate carboxylase deficiency (PCD) is caused by biallelic mutations of the PC gene. The reported clinical spectrum includes a neonatal form with early death (type B), an infantile fatal form (type A), and a late-onset form with isolated mild intellectual delay (type C). Apart from homozygous stop-codon mutations leading to type B PCD, a genotype-phenotype correlation has not otherwise been discernible. Indeed, patients harboring biallelic heterozygous variants leading to PC activity near zero can present either with a fatal infantile type A or with a benign late onset type C form. In this study, we analyzed six novel patients with type A (three) and type C (three) PCD, and compared them with previously reported cases. First, we observed that type C PCD is not associated to homozygous variants in PC. In silico modeling was used to map former and novel variants associated to type A and C PCD, and to predict their potential effects on the enzyme structure and function. We found that variants lead to type A or type C phenotype based on the destabilization between the two major enzyme conformers. In general, our study on novel and previously reported patients improves the overall understanding on type A and C PCD.

Inherited metabolic disorders presenting as hypoxic ischaemic encephalopathy: A case series of patients presenting at a tertiary care hospital in Pakistan.

In spite of the efforts and interventions by the Government of Pakistan and The World Health Organization, the neonatal mortality in Pakistan has declined by only 0.9% as compared to the global average decline of 2.1% between 2000 and 2010. This has resulted in failure to achieve the global Millennium Development Goal 4. Hypoxic-ischaemic encephalopathy, still birth, sepsis, pneumonia, diarrhoea and birth defects are commonly attributed as leading causes of neonatal mortality in Pakistan. Inherited metabolic disorders often present at the time of birth or the first few days of life. The clinical presentation of the inherited metabolic disorders including hypotonia, seizure and lactic acidosis overlap with clinical features of hypoxic-ischaemic encephalopathy and sepsis. Thus, these disorders are often either missed or wrongly diagnosed as hypoxicischaemic encephalopathy or sepsis unless the physicians actively investigate for the underlying inherited metabolic disorders. We present 4 neonates who had received the diagnosis of hypoxic-ischaemic encephalopathy and eventually were diagnosed to have various inherited metabolic disorders. Neonates with sepsis and hypoxic-ischaemic encephalopathy-like clinical presentation should be evaluated for inherited metabolic disorders.

Pyruvate Carboxylase Deficiency Type C: A Rare Cause of Acute Transient Flaccid Paralysis with Ketoacidosis.

Pyruvate carboxylase (PC) is a biotin-containing enzyme that is responsible for the adenosine triphosphate-dependent carboxylation of pyruvate to oxaloacetate, a key intermediate in the tricarboxylic acid cycle. PC deficiency (OMIM 266150) is a rare autosomal recessive metabolic disease, causing elevation of pyruvate, lactate, and alanine. Three types of PC deficiency have been described in the literature; A, B, and C. Type A PC deficiency, also called infantile or North American type, is characterized by infantile onset acidosis, failure to thrive, and developmental delay. The second subtype or type B, the neonatal or French form, presents usually in the neonatal period, mostly in the first 72 hours of life with severe lactic acidosis, truncal hypotonia, and seizures. The third type is called type C, is extremely rare with few cases published in the literature. In this case report, we present an 11-month-old girl who presented with acute flaccid paralysis, lethargy, and constipation with elevated ketones and lactate. She was confirmed genetically and biochemically to have PC deficiency type C. The patient's unusual presentation expands the clinical phenotype of this extremely rare disease.

Astrocyte Dysfunction in Developmental Neurometabolic Diseases.

Astrocytes play crucial roles in maintaining brain homeostasis and in orchestrating neural development, all through tightly coordinated steps that cooperate to maintain the balance needed for normal development. Here, we review the alterations in astrocyte functions that contribute to a variety of developmental neurometabolic disorders and provide additional data on the predominant role of astrocyte dysfunction in the neurometabolic neurodegenerative disease glutaric acidemia type I. Finally, we describe some of the therapeutical approaches directed to neurometabolic diseases and discuss if astrocytes can be possible therapeutic targets for treating these disorders.

Unsuccessful treatment of severe pyruvate carboxylase deficiency with triheptanoin.

UNLABELLED: Pyruvate carboxylase (PC) deficiency (OMIM 266150) is an autosomal recessive disorder that usually presents with lactic acidaemia and severe neurological dysfunction, leading to death in infancy. Because the enzyme is involved in gluconeogenesis and anaplerosis of the Krebs cycle, therapeutic strategies have included avoiding fasting and attempts to correct the defect of anaplerosis. Triheptanoin is a triglyceride of C7 fatty acids. The oxidation of odd chain fatty acids leads to the production not only of acetyl-CoA but also of propionyl-CoA, which is an anaplerotic substrate for the Krebs cycle. One infant with PC deficiency has previously been treated with triheptanoin as well as citrate and 2-chloropropionate. We report two further patients with PC deficiency, who were treated with triheptanoin, continuously from 11 and 21 days of age. They were also given citrate, aspartate and dichloroacetate. Triheptanoin did not lead to any clinical or biochemical improvement. The plasma and CSF lactate concentrations remained high with episodes of severe ketoacidosis and lactic acidosis. Both patients had severe hearing loss, roving eye movements, seizures and very limited neurodevelopmental progress; they died at the ages of 7 and 8 months. CONCLUSION: Though triheptanoin did not alter the clinical course in our patients, it was well tolerated. It remains possible that less severely affected patients might benefit from this form of therapy.

Case Report: Prenatal neurological injury in a neonate with pyruvate carboxylase deficiency type B.

Background: Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis. PC deficiency (PCD) is an extremely rare autosomal recessive metabolic disease and is divided into three types. Type B PCD is clinically featured by lactic acidosis, hyperammonemia, hypercitrullinemia, hypotonia, abnormal movement, and seizures. Case presentation: Here, we report the first case of type B PCD in China, presenting with intractable lactic acidosis shortly after birth. A compound heterozygous mutation in the PC gene was identified by whole-exome sequencing, NM_001040716.2: c.1154_1155del and c.152G>A, which were inherited from her asymptomatic parents, respectively. Furthermore, prenatal neuroradiological presentations including widened posterior horns of lateral ventricles, huge subependymal cysts, and increased biparietal diameter and head circumference were concerned. Symptomatic treatment was taken and the infant died at 26 days. Conclusion: To our knowledge, this is the minimum gestational age (22w5d) that's when the prenatal onset of the neuroradiologic phenotype of PCD was observed. PCD has a poor prognosis and lacks an effective treatment, so this paper is shared to highlight the importance of PCD prenatal diagnosis in the absence of family history.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

Generation of an induced pluripotent stem cell line (SHCDNi007-A) from a patient with pyruvate carboxylase deficiency carrying compound heterozygous (c.182 T > C/ c.2581G > A) variants in PC.

Pyruvate carboxylase (PC) deficiency (PCD), due to biallelic PC variants, is a rare inherited metabolic disease, which is characterized by seizures, global developmental delay, as well as lactic acidosis, and elevated plasma pyruvate and alanine levels in affected individuals. In the present study, a new induced pluripotent stem cell line (SHCDNi007-A) was generated from the peripheral blood mononuclear cells of a 2-month-old male infant with biallelic PC mutations c.(182 T > C;2581G > A), i.e. p.(Ile61Thr;Val861Met). This cell line is expected to facilitate the in vitro modeling of the disease pathophysiology and the development of future therapeutics for PCD.

Pyruvate carboxylase deficiency type C as a differential diagnosis of diabetic ketoacidosis.

OBJECTIVES: Type C pyruvate carboxylase (PC) deficiency is extremely rare, and has been described in only a few patients in literature to date. Herein, we present the case of a four-year-old patient admitted with diabetic ketoacidosis and diagnosed with type C PC deficiency based on clinical and biochemical findings. CASE PRESENTATION: A Turkish girl was referred to the intensive care unit at the age of three-years with a three-day history of vomiting and abdominal pain. Upon physical examination, the patient was found to be experiencing lethargy, dehydration, and Kussmaul breathing. Hyperglycemia, metabolic acidosis, and ketonemia were detected. Clinical and laboratory findings pointed to a prediagnosis of diabetic ketoacidosis. Intravenous fluid, bicarbonate, and insulin treatments were initiated. Elevated alanine and proline levels were recorded in plasma amino acid analysis, while urinary organic acid level analysis revealed increased lactate, pyruvate, 3-OH-butyrate, and acetoacetate levels. Whole exome sequencing revealed homozygous c.584C>T (p.Ala195Val) mutation in the PC gene. CONCLUSIONS: To date, there have been no reports in literature of type C phenotype patients manifesting with DKA. Our case is the first case with the type C phenotype to be admitted with clinical and laboratory findings of DKA.

Pyruvate carboxylase deficiency: mechanisms, mimics and anaplerosis.

Pyruvate carboxylase (PC) is a regulated mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a critical transition that replenishes citric acid cycle intermediates and facilitates other biosynthetic reactions that drive anabolism. Its deficiency causes multiorgan metabolic imbalance that predominantly manifests with lactic acidemia and neurological dysfunction at an early age. Three clinical forms of PC deficiency have been identified: an infantile form (Type A), a severe neonatal form (Type B), and a benign form (Type C), all of which exhibit clinical or biochemical correlates of impaired anaplerosis. There is no effective treatment for these patients and most, except those affected by the benign form, die in early life. We review the physiology of this enzyme and dissect the major clinical, biochemical, and genetic aspects of its dysfunction, emphasizing features that distinguish PC deficiency from other causes of lactic acidemia that render PC deficiency potentially treatable using novel interventions capable of enhancing anaplerosis.

Challenges in the management of an ignored cause of hyperammonemic encephalopathy: pyruvate carboxylase deficiency.

Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive disease and provides clinics in three essential phenotypes. Type B PC deficiency is characterized by lactic acidosis and hyperammonemia. We report a Turkish patient who was diagnosed with type B PC deficiency. Despite the application of anaplerotic treatment with biotin, citrate and arginine-aspartate, continuous veno-venous hemodialysis (CVVHD) treatments were applied due to the failure to keep hyperammonemia and lactic acidosis under control. Ammonia values increasing to 860 µmol/L were observed. A homozygous novel variant was detected in PC gene analyses containing a 12-base pair deletion on exon 8. Although the mutation found was not reported previously, it was accepted as a pathogenic variant due to its presence in a functional region of the protein. In type B PC deficiency, although a high level of ammonia is expected, it rarely exceeds 200 µmol/L. As far as we know, the present case has the highest ammonia values in the literature. This paper has been shared to highlight to keep PC deficiency in mind regarding the differential diagnosis of hyperammonemia, particularly in the presence of lactic acidosis, and to serve as a model for the use of different modalities in the management process of PC deficiency.

Structural insights on pathogenic effects of novel mutations causing pyruvate carboxylase deficiency.

Pyruvate carboxylase (PC), a key enzyme for gluconeogenesis and anaplerotic pathways, consists of four domains, namely, biotin carboxylase (BC), carboxyltransferase (CT), pyruvate carboxylase tetramerization (PT), and biotin carboxyl carrier protein (BCCP). PC deficiency is a rare metabolic disorder inherited in an autosomal recessive way. The most severe form (form B) is characterized by neonatal lethal lactic acidosis, whereas patients with form A suffer chronic lactic acidosis with psychomotor retardation. Diagnosis of PC deficiency relies on enzymatic assay and identification of the PC gene mutations. To date, six mutations of the PC gene have been identified. We report nine novel mutations of the PC gene, in five unrelated patients: three being affected with form B, and the others with form A. Three of them were frameshift mutations predicted to introduce a premature termination codon, the remaining ones being five nucleotide substitutions and one in frame deletion. Impact of these mutations on mRNA was assessed by RT-PCR. Evidence for a deleterious effect of the missense mutations was achieved using protein alignments and three-dimensional structural prediction, thanks to our modeling of the human PC structure. Altogether, our data and those previously reported indicate that form B is consistently associated with at least one truncating mutation, mostly lying in CT (C-terminal part) or BCCP domains, whereas form A always results from association of two missense mutations located in BC or CT (N-terminal part) domains. Finally, although most PC mutations are suggested to interfere with biotin metabolism, none of the PC-deficient patients was biotin-responsive.

The molecular basis of pyruvate carboxylase deficiency: mosaicism correlates with prolonged survival.

Pyruvate carboxylase (PC) deficiency (OMIM, 266150) is a rare autosomal recessive disease. The revised PC gene structure described in this report consists of 20 coding exons and four non-coding exons at the 5'-untranslated region (5'-UTR). The gene codes for three transcripts due to alternative splicing: variant 1 (NM_000920.3), variant 2 (NM_022172.2) and variant 3 (BC011617.2). PC deficiency is manifested by three clinical phenotypes-an infantile form (Type A), a neonatal form (Type B), and a benign form (Type C). We report the molecular basis for eight cases (one Type A, five Type B and two Type C) of PC deficiency. Eight novel complex mutations were identified representing different combinations of missense mutations, deletions, a splice site substitution and a nonsense mutation. The classical phenotypes (A, B and C) correlated poorly with clinical outcomes. Mosaicism was found in five cases (one Type A, three Type B and one Type C) and four of these cases had prolonged survival. Death in the fifth case resulted from unrelated medical complications. The discrepancy between the current findings and the existing classification system should be addressed to accommodate these new observations.

Biotin-response organicaciduria. Multiple carboxylase defects and complementation studies with propionicacidemia in cultured fibroblasts.

Fibroblast cultures from two individuals with biotin-responsive organicacidemia were found to have a pleiotropic deficiency of propionyl-CoA carboxylase, beta-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase activities after growth in biotin limited culture medium, conditions which do not affect the carboxylase activities of normal cells. All three enzyme activities were restored to normal levels after transferring the mutant strains to biotin-rich medium. Both patients excreted abnormal levels of an array of metabolic intermediates, including beta-methylcrotonate, beta-hydroxyisovalerate, beta-hydroxypropionate, and lactate, which reflect metabolic blocks at all three carboxylase sites.14 mutants deficient in only propionyl-CoA carboxylase activity from patients with propionicacidemia and the two biotin-responsive strains were examined for complementation with seven previously mapped pcc mutants. No new pcc complementation groups were identified. Nine of the mutants were mapped to group pccA. The remaining 12 mutants mapped to pccBC or its B or C subgroups, confirming the complex nature of this group. The biotin-responsive mutants failed to complement each other but did complement mutants from all the pcc groups. Thus biotin-responsive organicacidemia is defined by a new complementation group, bio. The results obtained in this study suggest that the bio mutants have a defect of either biotin transport or a common holocarboxylase synthetase required for the biotin activation of all three mitochondrial carboxylases.

Mutant holocarboxylase synthetase: evidence for the enzyme defect in early infantile biotin-responsive multiple carboxylase deficiency.

Biotin-responsive multiple carboxylase deficiency is an inherited disorder of organic acid metabolism in man in which there are deficiencies of propionyl-coenzyme A (CoA), 3-methylcrotonyl-CoA, and pyruvate carboxylases that can be corrected with large doses of biotin. It has been proposed that the basic defect in patients with the early infantile form of the disease is in holocarboxylase synthetase, the enzyme that covalently attaches biotin to the inactive apocarboxylases to form active holocarboxylases. We have developed an assay for holocarboxylase synthetase in extracts of human fibroblasts using as substrate apopropionyl-CoA carboxylase partially purified from livers of biotin-deficient rats. Fibroblasts from the initial patient with the infantile form of biotin-responsive multiple carboxylase deficiency were shown to have abnormal holocarboxylase synthetase activity with a maximum velocity about 30-40% of normal, a Km for ATP of 0.3 mM similar to the normal Km of 0.2 mM, and a highly elevated Km for biotin of 126 ng/ml, about 60 times the normal Km of 2 ng/ml. These results show that the primary defect in this patient is a mutation affecting holocarboxylase synthetase activity, and thus a genetic defect of the metabolism of biotin.

Anaplerotic diet therapy in inherited metabolic disease: therapeutic potential.

Beginning with phenylketonuria, dietary therapy for inborn errors has focused primarily on the restriction of the precursor to an affected catabolic pathway in an attempt to limit the production of potential toxins. Anaplerotic therapy is based on the concept that there may exist an energy deficit in these diseases that might be improved by providing alternative substrate for both the citric acid cycle (CAC) and the electron transport chain for enhanced ATP production. This article focuses on this basic problem, as it may relate to most catabolic disorders, and provides our current experience involving inherited diseases of mitochondrial fat oxidation, glycogen storage, and pyruvate metabolism using the anaplerotic compound triheptanoin. The observations have led to a realization that 'inter-organ' signalling and 'nutrient sensors' such as adenylate monophosphate mediated-protein kinase (AMPK) and mTOR (mammalian target of rapamycin) appear to play a significant role in the intermediary metabolism of these diseases. Activated AMPK turns on catabolic pathways to augment ATP production while turning off synthetic pathways that consume ATP. Information is provided regarding the inter-organ requirements for more normal metabolic function during crisis and how anaplerotic therapy using triheptanoin, as a direct source of substrate to the CAC for energy production, appears to be a more successful approach to an improved quality of life for these patients.

Primary amino acid sequence and structure of human pyruvate carboxylase.

Pyruvate carboxylase (PC) (pyruvate:carbon dioxide ligase (ADP-forming), EC 6.4.1.1.), a nuclear-encoded mitochondrial enzyme, catalyzes the conversion of pyruvate to oxaloacetate. We have isolated and characterized cDNAs spanning the entire coding region of human PC. The sequence of human PC has an open reading frame of 3537 nucleotides which encodes for a polypeptide with a length of 1178 amino acids. The identity of the cDNA as PC is confirmed by comparison to PC cDNAs of other species and sequenced peptide fragments of mammalian PC. The M(r) of the full length precursor protein is 129,576 and that of the mature apoprotein is 127,370. RNA blot analysis from a variety of human tissues demonstrates that the highest level of PC mRNA is found in liver corresponding to this tissue's high level of PC activity. Based on homology with other biotin-containing proteins, the ATP, pyruvate, and biotin-binding sites can be identified. One of two patients with documented PC deficiency was found to be missing PC mRNA, further confirming the identity of this cDNA.