Clinical manifestations

Presentation and course

The various clinical features and treatments of individuals with pyruvate carboxylase have been reviewed (78). Approximately half of the patients with pyruvate carboxylase deficiency were severely ill in the neonatal period and died within the first 4 months of life (French phenotype or Type A) (61; 16; 76; 07; 57; 59; 27; 54; 83; 45; 60; 19). The other half of the patients had clinical symptoms during infancy (North American phenotype or Type B) (11; 41; 20; 73; 66; 05; 04; 58; 57; 29; 49; 68; 52). Several children with a mild or benign variant of pyruvate carboxylase deficiency have been reported (74; 28; 32; 03). These children have mild clinical and metabolic problems, and one child even had normal development.

Patients with the Type B or French phenotype are usually normal at birth. After 3 to 48 hours they become acutely ill with hypothermia, hypotonia, lethargy, and vomiting. The majority of them die in the neonatal period. Some survive for several weeks. They are, however, unresponsive and severely hypotonic and die before the age of 5 months, usually from respiratory abnormalities. However, one child who survived the neonatal period with lactic acidemia is now 9 years old and has only mild developmental delay (62). One affected child exhibited infantile parkinsonism and GABAergic hypotransmission (50). An individual with type B disease was under poor control, and his ammonia increased to 860 µmol/L (36). He was homozygous for a novel 12-base pair deletion in exon 8.

Patients with the North American phenotype become severely ill between 2 and 5 months of age. In some of these patients, symptoms are noticed at birth and include hypotonia, feeding difficulties, failure to thrive, convulsions, and respiratory difficulties. Initially, development is normal, but delays soon become obvious. Patients become progressively hypotonic, unaware of their surroundings, and unable to smile or support their head. They consistently have numerous episodes of acute vomiting, dehydration, tachypnea, facial pallor, and cold cyanotic extremities, characteristically precipitated by metabolic acidosis or infectious stress. Pyramidal tract signs, ataxia, and nystagmus are observed. Mental retardation is evident in all patients. Convulsions complicate the clinical picture in several patients. Funduscopic examination reveals no abnormalities. The course of the disease is generally progressive, and most patients die in infancy.

Several children with pyruvate carboxylase deficiency have had milder clinical courses (74; 65; 28; 32; 03). The oldest of these children is about 10 years old. Some have exhibited neurologic symptoms, whereas others have shown normal development with few metabolic abnormalities. These patients will probably be found to have missense gene mutations.

In the largest series of nine children with pyruvate carboxylase deficiency (Type B), all exhibited axial hypotonia and tachypnea at birth. Tremors and hypokinesis were observed in most children, whereas seizures were uncommon. Hypoglycemia, lactic acidosis, hyperammonemia, hypernatremia, and elevated concentrations of citrulline, proline, and lysine were common (77). These children had cystic periventricular leukomalacia. Most of these children died despite rigorous therapy (26). A child who was initially diagnosed as having hypoxic-ischemic encephalopathy in the newborn period was subsequently diagnosed as having pyruvate carboxylase deficiency (86).

Individuals with pyruvate carboxylase deficiency were classified by their clinical phenotypes as having the infantile form (Type A), the neonatal form (Type B), and the benign form (Type C) (79; 78). These patients, one with Type A, five with Type B, and two with Type C, had a variety of mutations, including eight novel complex mutations. These phenotypic designations correlated poorly with outcomes. Moreover, mosaicism was found in five of these patients; four of them had prolonged survival, and the fifth died of medical complications. This report further emphasizes that the clinical phenotype classifications are probably of little practical value. The clinical characteristics of these various types of pyruvate carboxylase deficiencies have been reviewed (42). Novel variants in individuals with type A and type C, together with previously reported individuals, were studied to evaluate genotype-phenotype correlations and found some possible explanations for their differences (14). A 4-year-old female presented with what appeared to be diabetic ketoacidosis with metabolic acidosis, Kussmaul breathing, hyperglycemia, and ketonemia and was subsequently shown to have pyruvate carboxylase deficiency type C (21). An infant with Type B pyruvate carboxylase deficiency was delivered after exhibiting widened posterior horns of lateral ventricles, huge subependymal cysts, and increased biparietal diameter and head circumference prenatally (85).

Prognosis and complications

Pyruvate carboxylase deficiency is associated with invariably fatal outcomes in patients with the French phenotype and the North American phenotype. In the former, patients usually die in the neonatal period, whereas in the latter, during infancy. Two patients, initially symptomatic at 2 and 5 months, survived until 5 years of age (57), and another was 7 years and 9 months old when he died (66). A patient with a benign variant had normal mental and motor development. Despite several episodes of metabolic acidosis, she continues to be in good general health at the age of 7 years. The outcome of children with pyruvate carboxylase deficiency is variable and may positively correlate with the severity of the initial clinical presentation (17). A patient with Type C exhibited transient flaccid paralysis with ketoacidosis (02).

Clinical vignette

A 3-day-old male infant exhibited respiratory distress, lethargy, hypotonia, and myoclonic seizures. He had severe ketolactic acidosis, hyperammonemia, and hypoglycemia. He also had a markedly elevated plasma lactate and lactate-pyruvate ratio. Urinary organic acids showed elevated lactate, pyruvate, and 3-OH-butyrate as well as increased excretion of alpha-ketoglutarate. Plasma 3-OH-butyrate was increased, and the ratio of 3-OH-butyrate to acetoacetate was decreased. Plasma amino acid analysis was normal except for a mild elevation of alanine. Based on these results, a diagnosis of pyruvate carboxylase deficiency was considered, and the child was treated with biotin and glucose infusions. However, he continued to deteriorate rapidly and exhibit seizures that failed to respond to anticonvulsive medications. He died at 10 days of age. A liver sample obtained at autopsy had undetectable pyruvate carboxylase activity, thereby confirming the diagnosis of pyruvate carboxylase deficiency.

Biological basis

Etiology and pathogenesis

The disease is caused by defects in pyruvate carboxylase. The disorder is inherited as an autosomal recessive trait.

Pyruvate carboxylase (pyruvate:CO² ligase; E.C. 6.4.1.1) catalyzes the formation of oxaloacetate from pyruvate and bicarbonate at the expense of ATP (71). The molecular mechanism of this multifunctional enzyme has been elucidated (44). Pyruvate carboxylase is localized in the mitochondrial matrix (82; 08). Pyruvate carboxylase and the other mitochondrial carboxylases are rapidly biotinylated in the cytosol before transfer into mitochondria (34). It has an important gluconeogenic, anaplerotic, and lipogenetic role. In the last step of glycolysis, phosphoenolpyruvate is converted to pyruvate, but the reverse reaction cannot be used for gluconeogenesis. Pyruvate must first be carboxylated with the formation of oxaloacetate. Because the latter cannot diffuse freely out of the mitochondrion, oxaloacetate is reduced to malate, which reaches the cytoplasm using the malate/aspartate shuttle. Malate is reoxidized to oxaloacetate, which now can be converted to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase and can be used for further gluconeogenesis. Pyruvate carboxylase controlling the first step of gluconeogenesis has an important regulating role (23). Important organs for gluconeogenesis are the liver and kidney. The anaplerotic role of pyruvate carboxylase is even more important than the gluconeogenic role. The cell can increase the capacity of the Krebs cycle by increasing the concentration of Krebs cycle intermediates that function as carrier molecules. By converting pyruvate, an easily available substrate, to oxaloacetate, pyruvate carboxylase becomes the most important anaplerotic enzyme. Acetyl-CoA functions as the signal. When Krebs cycle capacity is too low, acetyl-CoA will accumulate and activate pyruvate carboxylase, which increases production of oxaloacetate. In case of severe pyruvate carboxylase deficiency, there will be a lack of Krebs cycle intermediates, resulting in a low concentration of reducing equivalents in the mitochondrial matrix. This will cause a shift of the redox equilibrium between 3-OH-butyrate and acetoacetate into the direction of the latter, and the ratio of 3-OH-butyrate to acetoacetate will be decreased (20). There also will be a shortage of aspartate because aspartate is formed in the mitochondrial matrix from oxaloacetate by a transamination reaction. As a consequence, the malate/aspartate shuttle, which normally serves as an import mechanism of reducing equivalents from the cytoplasm into the mitochondrial matrix, will be impaired. The result is that in the cytoplasm the redox equilibrium between lactate and pyruvate will be driven into the direction of the former. The ratio of lactate to pyruvate will be increased.

Pyruvate carboxylase is also important for lipogenesis (39). Fatty acids are synthesized in the cytoplasm from acetyl-CoA. Intramitochondrially produced acetyl-CoA cannot freely diffuse to the cytoplasm. By the action of citrate synthase, acetyl-CoA is ligated to oxaloacetate with formation of citrate that can be translocated to the cytoplasm where it is cleaved to oxaloacetate and acetyl-CoA. Intramitochondrially formed oxaloacetate can also be converted to malate, which shuttles to the cytoplasm where it generates reducing equivalents necessary for lipid synthesis. The lipogenetic role of pyruvate carboxylase, thus, consists in producing oxaloacetate. The increased demand of lipogenesis is associated with increasing concentration of pyruvate carboxylase mRNA (25). Important lipogenetic tissues are liver, adipose tissue, lactating mammary gland, adrenal gland, and brain.

Pyruvate carboxylase has been purified from liver (63). The complex has a molecular weight of 500 kDa. It is a tetramer composed of four identical subunits (71). The molecular weight of the subunit is 130 kDa (06). The subunit protein is encoded in the nucleus. Following its synthesis in the cytoplasm it is imported into the mitochondrial matrix. To be catalytically active the pyruvate carboxylase subunits must form a tetramer. More than half of the patients with the French phenotype had absence of pyruvate carboxylase protein and of the corresponding mRNA. Patients with the North American phenotype generally had cross-reacting material (CRM-positive) (57; 56), as did the patient with the benign variant of pyruvate carboxylase deficiency (74).

The full-length cDNA of human pyruvate carboxylase has been cloned and sequenced by two groups (40; 80). The coding sequence is 3534 bp and is flanked by an 82 bp 5’ untranslated sequence and a 389 bp 3’ untranslated sequence excluding the poly-A tail. The translated amino acid sequence is 1178 residues and contains a biotin attachment site and ATP and pyruvate binding sites. The full-length mature protein is 127,370 Da. The first 20 residues constitute the mitochondrial signal peptide sequence that is cleaved off after translocation of the protein into the mitochondrial matrix. The sequence 198–203 is probably part of the ATP-binding site and the 618-626 sequence is located at or near the pyruvate-binding site of the enzyme. Biotin is covalently attached to an epsilon amino group of a lysine located 35 residues from the carboxyl terminus of the protein (24; 37). Twenty-six amino acids more proximal from this lysine residue is a "Pro-Met-Pro" sequence, important for the "flip-flop" movement of the biotin-bearing arm, necessary for transferring the CO2-group toward pyruvate (84). The highest level of carboxylase mRNA is in liver (80). The gene for pyruvate carboxylase is located on the long arm of human chromosome 11 (24).

Multiple different mutations have been identified in the pyruvate carboxylase gene that causes pyruvate carboxylase deficiency (56; 80; 81; 13; 12; 47; 51; 30; 64; 67; 87). Mutations in the Type A form appear to be more severe, with deletions resulting essentially in no enzyme activity (81), and those in the Type B form are missense mutations resulting in residual enzyme activity (13; 12). There is a diverse spectrum of variants, including intronic variants, that have been found to cause pyruvate carboxylase deficiency (69). However, a study described an individual homozygous for a missense mutation that causes a severe form of type B deficiency (51). In addition, their studies did not find recurrent mutations among individuals from Turkey where there is a high degree of consanguinity.

An individual with pyruvate carboxylase deficiency was shown to have nemaline rods on muscle biopsy (70). This was speculated to be due to cellular energy shortage and altered energy metabolism of pyruvate.

A report summarizes the neurologic findings in pyruvate carboxylase deficiency and compares them to those of pyruvate dehydrogenase deficiency and disorders of the Krebs cycle (18). Only a few detailed neuropathological descriptions were reported in the literature. The brain of one patient with the French phenotype was examined by G Lyon (61). Widespread diffuse and symmetrical demyelination of the cerebral and cerebellar white matter and symmetrical periventricular cavities around the frontal and temporal horns of the lateral ventricles and between the dentate nuclei and the fourth ventricle were found. Gray structures were normal. One patient with the North American phenotype had mildly enlarged lateral ventricles and a reduction in the amount of the cerebral white matter, particularly in the corpus callosum (04). Microscopic examination disclosed a severe depletion of neurons in the cerebral cortex, numerous ectopic neurons in the white matter, and ectopic glia in the subarachnoid space. Myelin was poorly formed in a bilaterally symmetrical distribution in the cerebral and cerebellar hemisphere. In addition, there were accumulations of macrophages in the perivascular spaces of white matter, subependymal gliosis, and hyperplasia of protoplasmatic astrocytes in cortical structures. Microscopic examination of the liver revealed steatosis with preserved hepatic lobular architecture (04). Examination of the kidney showed diffuse vacuolation of the distal renal tubules.

Magnetic resonance imaging of a 20-month-old boy revealed leukodystrophy of the brainstem and the subcortical white matter (31). A brother and sister with the disorder were both born with macrocephaly and severe ischemic-like lesions of the brain (10). Periventricular leukomalacia was found on fetal ultrasonography. Both died in the first week of life. Another child with the French-type disease had periventricular cysts and diffuse hypomyelination (53). He died at 6 months of age. In a subsequent pregnancy, periventricular cysts were seen on fetal ultrasonography. Finding periventricular cysts on fetal ultrasonography of at-risk pregnancy may be a useful prenatal indicator of an affected fetus.

Epidemiology

Pyruvate carboxylase deficiency is a rare disorder. Over 30 children with pyruvate carboxylase deficiency have been reported.

Prevention

Prenatal diagnosis of pyruvate carboxylase deficiency is possible in amniotic fluid cells (43) and chorionic villi biopsies (27). Successful prenatal diagnosis of pyruvate carboxylase deficiency has been performed in an at-risk pregnancy by finding low enzyme activity in a chorionic villi sample (75). The pregnancy was terminated, and no enzymatic activity was found in the postmortem fetal liver. In a second pregnancy to this same woman, there was normal enzyme activity in a chorionic villi sample, and the pregnancy resulted in a normal male infant. See the Pathogenesis and pathophysiology section for a discussion of periventricular cysts in affected fetuses.

Differential diagnosis

Hyperlactacidemia can be caused by defects of several other enzymes including pyruvate dehydrogenase complex, enzyme complexes of respiratory chain, enzymes of gluconeogenesis (glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-diphosphatase), Krebs cycle enzymes (fumarase), enzymes involved in mitochondrial beta-oxidation of fatty acids, and enzymes involved in metabolism of several organic acids.

Diagnostic workup

Blood analyses of individuals with the French phenotype of pyruvate carboxylase deficiency showed metabolic acidosis, hyperlactacidemia, and hypoglycemia in some patients. Serum lactate concentrations varied between 10 and 20 mmol (normal less than 2.2), with a ratio of lactate to pyruvate between 50 and 100 (normal less than 28). Blood 3-OH-butyrate was increased (0.5 to 2.7 mmol, normal less than 0.1). The ratio of 3-OH-butyrate to acetoacetate was decreased (less than 2, normal 2.5 to 3). Hyperammonemia was a constant finding (100 to 600 mg/dL, normal less than 60) as was a striking increase of plasma citrulline (300 to 400 mmol, normal less than 40). Plasma amino acid profiles showed an increase of lysine and proline, which is a nonspecific finding. Hyperalaninemia, usually accompanying hyperlactacidemia, was not found in several patients with the French phenotype of pyruvate carboxylase deficiency. Plasma aspartate was normal. Only one patient had a significant decrease in plasma aspartate (76).

Serum lactate in patients with the North American phenotype was increased but less than in patients with the French phenotype. It varied between 2 and 10 mmol. Lactate-to-pyruvate ratios were normal or moderately increased (less than 50, normal less than 28). Plasma ammonia concentration was normal as was citrulline concentration. Plasma alanine concentration was increased in all patients (0.5 to 1.4 mmol, normal less than 0.455). An increase of plasma 3-OH-butyrate and free fatty acids was found and the ratio of 3-OH-butyrate to acetoacetate was normal or decreased. Serum lactate concentration, lactate-to-pyruvate ratio, and plasma alanine concentrations were increased in cerebrospinal fluid. Glutamine was decreased in cerebrospinal fluid and in postmortem brain (52). Urinary organic acids showed elevated lactate, pyruvate, and 3-OH-butyrate as well as increased excretion of alpha-ketoglutarate. Overnight serum glucose concentrations usually were normal but decreased after a 24-hour fast (04). Hypoglycemia was present especially during acute episodes of metabolic acidosis. During these episodes, serum lactate concentrations generally were above 10 mmol.

Fetuses of six pregnancies with pyruvate carboxylase deficiency revealed ventriculomegaly, frontal horn impairment associated with subependymal, and paraventricular cysts (22). These findings may provide a clue that an at-risk pregnancy has the disorder.

The patient with the benign variant had several acute episodes of metabolic acidosis with elevated blood lactate, pyruvate, alanine, 3-OH-butyrate, acetoacetate, and lysine and undetectable low aspartate.

Cultured skin fibroblasts are used preferentially to assay catalytic activity of pyruvate carboxylase (20). A radiometric method provides the most reliable results (72). The activity is 10-fold higher in liver than in cultured skin fibroblasts, but the need for a liver biopsy limits the use of this tissue. Enzyme activity can be determined in postmortem liver, although results have to be interpreted with caution because of rapid postmortem degradation of the enzyme. Kerr and colleagues updated a method for assaying pyruvate carboxylase activity in crude homogenates of cultured skin fibroblasts, lymphocytes, and frozen liver (35).

Pyruvate carboxylase has low activity in skeletal muscle, which makes this tissue unsuitable for assay. Pyruvate carboxylase activity was severely decreased in all patients with the French phenotype (less than 5% of normal). In patients with the North American phenotype, it varied from less than 5% to 23% of controls. A patient with the benign variant had less than 2% of activity in her cultured skin fibroblasts as compared with controls.

Pyruvate carboxylase deficiency should be considered in the differential diagnosis of fetal abortions or terminated pregnancies when an inherited metabolic disorder is suspected (15).

Management

Patients with isolated pyruvate carboxylase deficiency did not respond to biotin therapy (61; 20; 04; 07). Daily administration of thiamine was used to lower the concentration of lactate by stimulating pyruvate dehydrogenase complex, but this was without significant clinical effect. Oral administration of other cofactors and medications (eg, dichloroacetate) did not influence the lactic acidemia or improve the clinical condition. A comatose child with severe ketolactic acidemia with low aspartate and glutamine and elevated citrulline and proline was treated with high doses of citrate and aspartate to provide oxaloacetate (01). The ketolactic acidosis improved, and the plasma amino acids became normal, with the exception of arginine. Glutamine continued to be low in the cerebrospinal fluid. Although the child has experienced milder metabolic compromise during infections or fasting, she is profoundly retarded, has spasticity, and only partially controlled myoclonic seizures. Treatment with adrenocorticotropic hormone in two patients with pyruvate carboxylase deficiency and infantile spasms induced severe metabolic acidosis with greatly increased blood lactate, pyruvate, and alanine concentrations (60). Neither high-fat nor high-carbohydrate diets were beneficial. Administration of substrates, including aspartic and glutamic acids, had no consistent effect on blood lactate concentrations. Acute metabolic crises can be detrimental both physically and mentally. Prompt treatment with oral or intravenous glucose-containing fluids and bicarbonate is advised during these episodes. Orthotopic liver transplantation has been performed in one child with pyruvate carboxylase deficiency (48). Ketolactic acidosis markedly improved, but the low glutamine concentration in the cerebrospinal fluid did not correct following transplantation. Oral triheptanoin, an odd carbon triglyceride that is a source of acetyl CoA and anaplerotic propionyl CoA, has been used to treat an infant with the disorder (46). The child exhibited rapid reversal of hepatic failure but died of at 6 months of age from sepsis. Further trials of this therapy are necessary to draw any conclusions about its efficacy. Various novel interventions that enhance anaplerosis or replenish the citric acid intermediates for the treatment of the disorder have been reviewed (42).

It is important to avoid fasting and a ketogenic diet in individuals with pyruvate carboxylase deficiency (78).

Several children with pyruvate carboxylase deficiency have been treated with triheptanoin, a triglyceride that provides anaplerotic substrates for the TCA cycle, in addition to citrate and 2-chloropropionate (09). Although the medication was well tolerated, it did not alter the clinical course. In another study, triheptanion was used in the treatment of 12 individuals with pyruvate carboxylase deficiency with mixed results; some exhibited no change, some improved, and some became worse (38). The authors suggested alternatively designed trials were needed to determine the efficacy of this anaplerotic agent in individuals with pyruvate carboxylase deficiency.

Special considerations

Anesthesia

Surgeons and anesthesiologists must have a thorough understanding of the disorder when performing surgery, particularly liver transplantation, in individuals with this volatile disorder (55).