Eur J Pediatr (2014) 173:361–366 DOI 10.1007/s00431-013-2166-5

ORIGINAL ARTICLE

Unsuccessful treatment of severe pyruvate carboxylase deficiency with triheptanoin C. Breen & F. J. White & C. A. B. Scott & L. Heptinstall & J. H. Walter & S. A. Jones & A. A. M. Morris

Received: 7 July 2013 / Revised: 19 September 2013 / Accepted: 25 September 2013 / Published online: 10 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract 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 2chloropropionate. 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.

Keywords Anaplerotic therapy . Pyruvate carboxylase deficiency . Triheptanoin C. Breen : F. J. White : C. A. B. Scott : L. Heptinstall : J. H. Walter : S. A. Jones : A. A. M. Morris (*) Willink Unit, Genetic Medicine, Manchester Academic Health Sciences Centre, Central Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9WL, UK e-mail: [email protected]

Abbreviations CSF Cerebrospinal Fluid DNA Deoxyribonucleic acid ERG Electroretinography LCT Long chain triglycerides MRI Magnetic resonance imaging PC Pyruvate carboxylase PDHc Pyruvate dehydrogenase complex

Introduction Pyruvate carboxylase (PC, E.C. 6.4.1.1) catalyses the conversion of pyruvate to oxaloacetate. It is expressed at high levels in liver and kidney and at low levels in many tissues, including brain, muscle, adipocytes and fibroblasts. PC has several metabolic roles. First, PC is essential for anaplerosis of the Krebs cycle; Krebs cycle intermediates are used by many other pathways, and the continued function of the Krebs cycle requires PC to replenish intramitochondrial oxaloacetate. PC is also needed for gluconeogenesis because the last step of glycolysis (conversion of phosphoenolpyruvate to pyruvate) is irreversible. Third, PC has a role in the export of acetyl-CoA out of mitochondria via the pyruvate/malate shuttle; this is particularly important for the synthesis of fatty acids. PC deficiency (OMIM 266150) is a rare, autosomal recessive inborn error of metabolism. There are two main clinical phenotypes. Patients with the most severe, ‘French’ phenotype (Group B) present soon after birth with hypotonia, severe ketoacidosis and lactic acidosis, liver impairment and hyperammonaemia. Plasma citrulline concentrations are high, and glutamine concentrations are low. There is severe neurological dysfunction and most patients die by 6 months of age [4]. Children with the slightly less severe ‘North American’ phenotype (Group A) usually present before 6 months of age

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with chronic lactic acidosis, hypotonia and very poor psychomotor development; death occurs in infancy. Typically, there is no hyperammonaemia and citrulline levels are normal. Convulsions occur in both French and North American phenotypes. Brain imaging often shows periventricular cysts and delayed myelination. Renal tubular acidosis has been described in several patients [3, 6, 8]. A rare, milder form of PC deficiency is also recognised: patients have episodes of acidosis but relatively normal psychomotor development. These patients probably have higher residual enzyme activity in vivo, though it has not been possible to distinguish the groups of patients according to enzyme activity in vitro [9]. The lactic acidosis and ketoacidosis seen in PC deficiency can be explained by the deficiency of oxaloacetate and the inability to incorporate acetyl-CoA into the Krebs cycle. The lactic acidosis and ketoacidosis both worsen during illness or fasting. The Krebs cycle dysfunction also impairs energy production and this is thought to be primarily responsible for the neurological problems. Decreased fatty acid synthesis may cause the defective myelination. Depletion of oxaloacetate leads to low aspartate levels, which may interfere with the urea cycle, explaining the hyperammonaemia. Therapeutic strategies have included avoiding fasting and attempts to correct the defect of anaplerosis. Various anaplerotic substrates have been tried, including citrate, which can be converted to oxaloacetate by citrate lyase, and aspartate, which can be transaminated to oxaloacetate [1, 8]. The accompanying electrolyte load, however, limits the amount of these chemicals that can be given. In one patient, treatment with citrate and aspartate reduced the blood lactate concentration and ketonuria; there was, however, no change in the CSF metabolites and, though the child was still alive at 3.5 years, the neurological outcome was very poor [1]. 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. The latter can be converted to succinyl-CoA, which is an anaplerotic substrate for the Krebs cycle. Triheptanoin is a particularly good anaplerotic agent as it can be given in large quantities without accompanying electrolytes. Moreover, on first pass through the liver, triheptanoin is largely converted to C5 ketone bodies, which can cross the blood–brain barrier and might, potentially, correct the metabolic disorder in the brain. One infant with the ‘French’ form of PC deficiency has previously been treated with triheptanoin as well as citrate and 2-chloropropionate [6]. Treatment led to an improvement in liver function and other biochemical parameters, including the CSF glutamine concentration, but she died at the age of 6 months. We report two patients with the ‘French’ form of PC deficiency. Despite treatment with triheptanoin, citrate and aspartate, they had severe neurodevelopmental problems and they died at the ages of 7 and 8 months.

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Materials and methods Case reports Patient 1 This girl was the first child to consanguineous parents of Pakistani extraction. After a normal pregnancy and birth (birth weight 3.26 kg), she presented with tachypnoea at 8 h of age. Investigations revealed a metabolic acidosis (pH 7.18, pCO2 1.6 kPa, bicarbonate 4.8 mmol/L) with a blood lactate of 19.5 mmol/L, ketonuria and normoglycaemia. There was hyperammonaemia (maximum 330 μmol/L on day 7 of life, normal <50) and mild liver dysfunction. Organic acid analysis showed increased excretion of lactate, ketone bodies and phenolic acids. There were elevated plasma concentrations of alanine and citrulline, with a low glutamine concentration (Table 1). The CSF lactate was 4.7 mmol/L (normal <2.6). Initial cranial ultrasound examination showed a left periventricular cyst. Magnetic resonance imaging (MRI) of the brain at 2 months of age showed cystic dilatation of the frontal horn of the left lateral ventricle. There was increased signal on T2-weighted images in the unmyelinated white matter of the cerebral and cerebellar hemispheres and also in the posterior limbs of the internal capsule. The cerebellum, pons and medulla were hypoplastic. The patient showed no response to thiamine, biotin or sodium dichloroacetate (50 mg/kg/day, started for 1 month after diagnosis, restarted from 2 to 5 months of age; see Fig. 1). At 15 days of age, we started treatment with citrate (5 mmol/kg/day, given as Albright’s solution) and aspartate (4 mmol/kg/day). The latter was given as arginine aspartate, partly to minimise the sodium load and partly because a previous patient required arginine supplements [1]. These measures reduced the requirement for sodium bicarbonate but led to no other biochemical changes. The patient had a persistent hyperlactataemia (usually 8– 12 mmol/L) and a chronic bicarbonate requirement of 8– 10 mmol/kg/day. She also had recurrent episodes of more severe acidosis. During these episodes, she became acidotic over a few hours without any obvious precipitant and presented with a Kussmaul respiratory pattern. The plasma lactate concentration changed little, but there was ketosis (3hydroxybutyrate up to 4.1 mmol/L) and evidence of renal tubular acidosis (urine pH 6.2). At these times, she required up to 50 mmol/kg/day of intravenous sodium bicarbonate. Surprisingly, on none of these occasions did she develop hypernatraemia or fluid overload. The episodes resolved after 3–4 days. Venous access was difficult and several central venous lines had to be removed because of thrombosis.

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Table 1 Plasma and CSF concentrations of selected amino acids before and during treatment with triheptanoin Plasma Patient Age Aspartate Glutamate Glutamine Alanine Citrulline

1 1 daya ND 48 356 492 54

CSF

4 wks 10 211 250 550 10

20 wks 25 226 315 924 19

2 4 daysa 4 ND 185 402 28

6 wks 22 261 238 1,450 31

18 wks 7 32 228 926 32

Normal

1

Normal

4–12 27–77 520–742 274–384 8–36

3 7 279 83 ND

2–8 0–117 160–535 13–37 0–8

ND not detected, wks weeks. All concentrations are in micromol/litre a

Before starting triheptanoin

Patient 2 This girl was the fifth child of healthy, first cousin parents of Pakistani origin. One previous child is healthy but three had PC deficiency. The latter patients were managed symptomatically; one died at 5 weeks of age and two died aged 5 months. For patient 2, the pregnancy and delivery were normal (birth weight 3.11 kg). She developed a severe metabolic acidosis within a few hours of birth (maximum plasma lactate concentration 32 mmol/L). The blood glucose concentration remained normal but the plasma ammonia concentration rose to 260 μmol/L. Organic acid analysis only showed increased lactate and ketone bodies. The plasma alanine concentration was raised, with a normal citrulline concentration and a low glutamine concentration (Table 1). Cranial ultrasound examination showed paraventricular cysts adjacent to the frontal horns of the lateral ventricles and clusters of cysts at the caudothalamic notch bilaterally. Cranial MRI at 2 months of age confirmed the presence of cysts adjacent to the frontal horns of the lateral ventricles and the anterior aspect of the left lateral ventricle; it also showed abnormal myelination in the cerebral hemispheres (especially the frontal and parietal lobes), the posterior limbs of the internal capsules, cerebral peduncles, midbrain and cerebellum. The patient was treated with sodium bicarbonate, biotin and aspartate (2.6 mmol/kg/day given as arginine aspartate), following which the plasma ammonia concentration fell to normal and the lactate fell to 10.4 mmol/L. Subsequently, citrate was added (2.6 mmol/kg/day given as Albright’s solution); this allowed the dose of sodium bicarbonate to be reduced but did not lead to any other biochemical improvement. Though she had a chronic lactic acidosis (usually 9–13 mmol/L), this patient had only one episode of severe acidosis at the age of 2 months, associated with an acute illness. Methods PC activity was assayed in cultured skin fibroblasts at the biochemical genetics laboratory, Willink Unit, by measuring the ATP-dependent carboxylation of pyruvate using 14C-

NaHCO3. The assay was based on the method reported by Gompertz and colleagues [5] with the adaption of substituting propionyl-CoA with pyruvate as the substrate and with the addition of using individual patient blanks. DNA extracted from cultured fibroblasts was screened for the presence of mutations in all exons of the PC gene using bidirectional automated Sanger sequencing analysis (Applied Biosystems BigDye Terminator v1.1 cycle sequencing kit). Both patients were treated w ith Triheptanoin (Oleochemicals, Germany) with written informed consent in view of the experimental nature of this treatment. Triheptanoin was commenced at 3 weeks of age in patient 1 and at 11 days of age in patient 2. Initially, triheptanoin was given in addition to the usual intake of normal infant formula; it was started at 18–23 % energy intake and, as there was no gastrointestinal disturbance, the dose was increased to 36.5 % energy in patient 1 and 32 % energy in patient 2. The total feed provided 156 kcal/kg/day for patient 1 and 180 kcal/kg/day for patient 2, with approximately 7 % energy as protein, 28 % as carbohydrate and 30 % as long chain triglycerides (LCT). On this regimen, both patients gained weight rapidly. An optimal rate of weight gain was achieved by changing to a modular feed with a lower energy content. The feed was based on normal infant formula, with supplements of protein, glucose polymer, long chain fat emulsion, vitamins and minerals. The modular feed of patient 1 provided 110 kcal/kg/day, 2.2 g protein/kg/day with 8 % energy from protein, 24 % from LCT, 24 % from carbohydrate and 44 % from triheptanoin. The feed of patient 2 provided 100 kcal/kg/day, 2 g protein/kg/day with 9 % energy as protein, 27 % as LCT, 28 % as carbohydrate and 36 % as triheptanoin. The higher dose of triheptanoin was used in patient 1 to ensure that the lack of clinical response was not due to inadequate intake. Both patients were fed regularly throughout the 24-h period to minimise gluconeogenesis. They were fed every 3–4 h throughout life, but after 3 months of age, they sometimes went for a maximum of 6 h between feeds overnight. Patient 1 had a continuous nasogastric feed during periods of acute illness.

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Fig. 1 Plasma lactate concentration over time in patient 1 and treatment; treatment bar height represents dose change over time in relation to initial treatdose (millimoles per kilogram per day). treatment with ment with triheptanoin, treatment with sodium dichloroacetate,

bicarbonate,

Treatment with triheptanoin, aspartate and citrate was continued throughout life in both patients. The doses of triheptanoin and citrate were adjusted regularly as the patients grew to maintain the same dose per kilogram; the dose of aspartate was not increased after the first 2 months in either patient as the plasma aspartate and glutamate concentrations were maintained within or above the normal range.

two base pair deletion in exon 10 (c.1154_1155delGC). The resulting frameshift creates a stop codon at codon 394 p.(Arg385GlnfsTer10); the truncated protein would lack both the pyruvate and biotin binding domains.

Results Enzymology and molecular biology The diagnosis of PC deficiency was confirmed on cultured fibroblasts. In patient 1, the pyruvate carboxylase activity was 0.11 nmol/h per mg protein, and it was 0.08 nmol/h per mg protein (reference range 6–40) in patient 2. Propionyl-CoA carboxylase was measured as a reference enzyme, and its activity was normal in each patient. Mutation analysis revealed homozygous novel mutations in both patients. Patient 1 was homozygous for the sequence variation c.506 G>A in exon 6 of the PC gene (NM_000920.3). Both parents are heterozygous for this variant. There is little effect on splicing, but the resulting amino acid change, (p.Gly169Asp), affects a highly conserved glycine residue and is predicted to be pathogenic. Patient 2 was homozygous for a

treatment with citrate, treatment with arginine,

admission to hos-

pital with acute acidosis

Response to treatment with triheptanoin There was no improvement in clinical or biochemical parameters in response to the treatment. In particular, the plasma lactate concentrations were consistently high (Figs. 1 and 2), apart from two normal plasma values early in the course in patient 1. The CSF lactate remained elevated in patient 1 (8.5 mmol/L at 5 months of age); it was not measured in patient 2. On triheptanoin, the plasma 3-hydroxybutyrate concentration ranged from 0.89 to 4.10 mmol/L in patient 1 (the higher values occurring episodes of acidosis), and the concentration was 0.61 mmol/ L in patient 2 (aged 1 month). Treatment had no effect on the chronic bicarbonate requirement in either patient. Patient 1 continued to have episodes of severe acidosis. Triheptanoin treatment was never interrupted prior to admission with these episodes and neither was there any evidence to suggest poor absorption. Patient 2 only had one episode of severe acidosis; this occurred whilst she was receiving triheptanoin and, again, there was nothing at the time to suggest poor absorption. The plasma glutamine concentrations remained low in both patients (Table 1); indeed, triheptanoin treatment led to an

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Fig. 2 Plasma lactate concentration over time in patient 2 and treatment; treatment bar height represents dose change over time in relation to initial dose (millimoles per kilogram per day). treatment with triheptanoin, treatment with sodium bicarbonate, treatment with citrate, treatment with arginine

initial fall in plasma glutamine in patient 1. The CSF glutamine concentration was normal on triheptanoin in patient 1 (Table 1) but there were no measurements before treatment for comparison. Both patients had normal plasma citrulline concentrations before and during triheptanoin treatment (the initial high value in patient 1 had normalised before the oil was started). Aspartic acid and glutamic acid levels in plasma were undetectable or in the lower part of the normal range before treatment. On treatment with aspartate, citrate and triheptanoin, the concentrations rose and remained within or above the normal range (Table 1). Organic acid analysis in patient 1 demonstrated an initial reduction in lactate excretion following treatment, reflecting the plasma findings, but lactate excretion increased again after 2 weeks to pre-treatment levels. Excretion of 3hydroxybutyrate increased when triheptanoin was introduced and remained elevated thereafter. In patient 2, lactate and 3hydroxybutyrate excretion were elevated at similar levels prior to and throughout treatment. Both patients showed increased excretion of derivatives of heptanoate oxidation, with small but significant amounts of pimelate, 3-hydroxypentanoate, 3ketopentanoate and 3-hydroxypropionate. Following treatment, excretion of the Krebs cycle intermediates, fumarate, succinate and malate, increased to normal levels in both patients. Psychomotor development for both patients was very abnormal. Patient 1 started smiling and had some vocalisation at the age of 7 months, but had severe truncal hypotonia, with no head control. She was able to breast or bottle feed initially, but progressively required more tube feeding. Patient 2 had normal limb tone and severe truncal hypotonia. She made no developmental progress. She also fed from a bottle initially, but this was changed to nasogastric tube feeding at the age of 6 months when a feeding assessment showed a high risk of aspiration. In both patients, auditory brainstem response testing showed

severe hearing loss (threshold 90–110 dB at 1–4 kHz); transient-evoked otoacoustic emissions were recorded in patient 2 suggesting auditory neuropathy or dys-synchrony. Both patients had roving eye movements but normal pupil responses and slightly pale optic discs on fundoscopy; patient 1 had a divergent squint. In patient 2, the ERG responses were of low amplitude and visual evoked potentials were absent, suggesting cortical visual impairment and mild optic atrophy. The head circumference of patient 1 was on the 25th centile at birth and remained on this centile, whereas the head circumference of patient 2 was above the 50th centile at birth and subsequently fell to below the 0.4th centile. Both patients gained weight satisfactorily. A mixture of focal and generalised seizures started at the age of 2 weeks in patient 2 and 8 weeks in patient 1; both patients were treated with phenobarbital but continued to have several brief seizures (mostly eye twitching) each day. Patient 1 died at 7 months of age from respiratory failure secondary to pneumonia. Patient 2 died at 8 months of age after developing extensive left lower lobe collapse and consolidation and severe acidosis. Neither family gave permission for autopsy examination.

Discussion Our patients demonstrate the severe clinical course of PC deficiency, following most closely the French subtype of this disease (though patient 2 was found to have a normal plasma citrulline). In both patients, the hyperammonaemia appeared to respond to treatment with aspartate but, in other respects, anaplerotic therapy did not appear to alter the course of the disease. Despite treatment, our patients had a persistent hyperlactataemia, with episodes of severe ketoacidosis and renal tubular acidosis, particularly in patient 1, who had

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multiple prolonged admissions to hospital with acidosis. Both of our patients also had profound neurological handicap with little, if any, psychomotor progress; neuroimaging showed various abnormalities, including impaired myelination. Patient 2 did survive for 3 months longer than her siblings with the same condition, but this was probably due to other aspects of her management rather than the triheptanoin. The siblings were kept comfortable but did not receive active management during acute illnesses because of the poor prognosis; in contrast, patient 2 received active management, including antibiotics and intravenous sodium bicarbonate during an acute illness at the age of 2 months, because it was hoped that triheptanoin might improve the long-term outlook. There has been one previous report of triheptanoin therapy in a patient with PC deficiency of the French phenotype [6]. Treatment with triheptanoin at 7 days of age led to normalisation of the plasma ammonia concentrations and improved liver function. The plasma lactate concentration also fell, though it remained moderately elevated (4–5 mmol/L in the long term). Plasma glutamine concentrations increased, as did the urinary excretion of citric acid cycle intermediates. Raised concentrations of C5-ketone bodies were demonstrated in plasma and CSF. Treatment with citrate and 2chloroproprionate was added at 4 months of age following an episode of ketoacidosis and lactic acidosis. Psychomotor development was said to be normal at 3 months of age, apart from mild axial hypotonia and myelination appeared normal on MRI and, subsequently, at autopsy. The patient died at the age of 6 months secondary to infection and ketoacidosis. Our patients differed from this one most importantly in their failure to make neurodevelopmental progress. The reason for this difference is not clear. We did not measure C5-ketone body concentrations in our patients but we would expect them to have been similar to those in the patient reported by Mochel and colleagues [6]; our patients, like theirs, excreted small but significant amounts of 3-hydroxypentanoate and 3ketopentanoate. The triheptanoin doses in our patients were at least as high as in their patient, and the dietary composition was similar in other respects (providing 35–44 % energy as triheptanoin and 24–30 % as LCT). Mochel and colleagues [6] gave 2-chloropropionate from 4 months of age to activate the pyruvate dehydrogenase complex (PDHc) and reduce lactate production; we treated patient 1 with dichloroacetate to activate PDHc. All three patients also received other anaplerotic substrates: our two patients were given citrate at 2.6–5 mmol/ kg/day and aspartate at 2.6–4 mmol/kg/day, starting within the first month; Mochel and colleagues [6] gave their patient citrate at 7.5 mmol/kg/day from 4 months of age. Ahmad and colleagues [1] gave a patient higher doses of aspartate (10 mmol/kg/day) and citrate (7.5 mmol/kg/day) but these were their only anaplerotic substrates; we would expect the triheptanoin used in our patients to generate far more oxaloacetate. It is conceivable that our patients might have profited from

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higher doses of aspartate and citrate but this seems unlikely, given the raised plasma aspartate and glutamate concentrations and the normal excretion of Krebs cycle intermediates. Our experience suggests that triheptanoin may not be a very effective treatment for patients with severe PC deficiency. Unfortunately, other forms of treatment, including liver transplantation, have also been of limited benefit [7]. This is partly because neurological damage in severe PC deficiency starts prenatally. Our patients had abnormalities on neuroimaging in the newborn period (as did the patient of Mochel and colleagues, [6]), and prenatal abnormalities have been reported in other patients [2]. Though triheptanoin did not alter the clinical course in our patients, it was well tolerated and neither patient had significant gastrointestinal side effects. The administration of the treatment was also acceptable to the families in both cases. It remains possible that patients with other phenotypes might make a better response to this form of therapy. Acknowledgments The authors are grateful to Dr Guy Besley for the enzyme assays and helpful discussion. Conflict of interest The authors have no conflicts of interest to declare.

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