Hypoglycin A concentrations in seeds of Acer pseudoplatanus trees growing on atypical myopathy-affected and control pastures.

BACKGROUND: Hypoglycin A, found in seeds of Acer negundo, appears to cause seasonal pasture myopathy (SPM) in North America and is implicated in atypical myopathy (AM) in Europe. Acer negundo is uncommon in Europe. Thus, the potential source of hypoglycin A in Europe is unknown. HYPOTHESIS AND OBJECTIVES: We hypothesized that seeds of Acer pseudoplatanus were the source of hypoglycin A in Europe. Our objective was to determine the concentration of hypoglycin A in seeds of A. pseudoplatanus trees located in pastures where previous cases of AM had occurred. ANIMALS: None. METHODS: University of Berne records were searched to retrospectively identify 6 farms with 10 AM cases and 11 suspected AM deaths between 2007 and 2011. During October 2012, A. pseudoplatanus seeds were collected from 2 to 6 trees per pasture on 6 AM farms (7 pastures) from trees in or close to 2 pastures on 2 control farms where AM had not been previously reported. Hypoglycin A in seeds was analyzed by GC-MS. RESULTS: Acer pseudoplatanus trees were identified on all AM pastures. Hypoglycin A was detected in all A. pseudoplatanus seeds in highly variable concentrations ranging from 0.04 to 2.81 µg/mg (mean 0.69) on AM farms and 0.10 to 9.12 µg/mg (mean 1.59) on control farms. CONCLUSION AND CLINICAL IMPORTANCE: Preventing horses from grazing pastures containing A. pseudoplatanus seeds during late fall and early spring might be the best means to prevent AM.

Identification of methylenecyclopropyl acetic acid in serum of European horses with atypical myopathy.

REASONS FOR PERFORMING STUDY: It is hypothesised that European atypical myopathy (AM) has a similar basis as seasonal pasture myopathy in North America, which is now known to be caused by ingestion of hypoglycin A contained in seeds from the tree Acer negundo. Serum from horses with seasonal pasture myopathy contained the conjugated toxic metabolite of hypoglycin A, methylenecyclopropyl acetic acid (MCPA). STUDY DESIGN: Retrospective study on archived samples. OBJECTIVES: 1) To determine whether MCPA-carnitine was present in serum of European horses confirmed to have AM; 2) to determine whether Acer negundo or related Acer species were present on AM pastures in Europe. METHODS: Concentrations of MCPA-carnitine were analysed in banked serum samples of 17 AM horses from Europe and 3 diseased controls (tetanus, neoplasia and exertional rhabdomyolysis) using tandem mass spectrometry. Atypical myopathy was diagnosed by characteristic serum acylcarnitine profiles. Pastures of 12 AM farms were visited by experienced botanists and plant species were documented. RESULTS: Methylenecyclopropyl acetic acid-carnitine at high concentrations (20.39 ± 17.24 nmol/l; range 0.95-57.63 nmol/l; reference: <0.01 nmol/l) was identified in serum of AM but not disease controls (0.00 ± 0.00 nmol/l). Acer pseudoplatanus but not Acer negundo was present on all AM farms. CONCLUSIONS: Atypical myopathy in Europe, like seasonal pasture myopathy in North America, is highly associated with the toxic metabolite of hypoglycin A, MCPA-carnitine. This finding coupled with the presence of a tree of which seeds are known to also contain hypoglycin A indicates that ingestion of Acer pseudoplatanus is the probable cause of AM. This finding has major implications for the prevention of AM.

Seasonal pasture myopathy/atypical myopathy in North America associated with ingestion of hypoglycin A within seeds of the box elder tree.

REASONS FOR PERFORMING STUDY: We hypothesised that seasonal pasture myopathy (SPM), which closely resembles atypical myopathy (AM), was caused by ingestion of a seed-bearing plant abundant in autumn pastures. OBJECTIVES: To identify a common seed-bearing plant among autumn pastures of horses with SPM, and to determine whether the toxic amino acid hypoglycin A was present in the seeds and whether hypoglycin metabolites were present in SPM horse serum or urine. METHODS: Twelve SPM cases, 11 SPM pastures and 23 control farms were visited to identify a plant common to all SPM farms in autumn. A common seed was analysed for amino acid composition (n = 7/7) by GC-MS and its toxic metabolite (n = 4/4) identified in conjugated form in serum [tandem mass spectrometry (MS/MS)] and urine [gas chromatography (GC) MS]. Serum acylcarnitines and urine organic acid profiles (n = 7) were determined for SPM horses. RESULTS: Seeds from box elder trees (Acer negundo) were present on all SPM and 61% of control pastures. Hypoglycin A, known to cause acquired multiple acyl-CoA dehydrogenase deficiency (MADD), was found in box elder seeds. Serum acylcarnitines and urine organic acid profiles in SPM horses were typical for MADD. The hypoglycin A metabolite methylenecyclopropylacetic acid (MCPA), known to be toxic in other species, was found in conjugated form in SPM horse serum and urine. Horses with SPM had longer turn-out, more overgrazed pastures, and less supplemental feeding than control horses. POTENTIAL RELEVANCE: For the first time, SPM has been linked to a toxin in seeds abundant on autumn pastures whose identified metabolite, MCPA, is known to cause acquired MADD, the pathological mechanism behind SPM and AM. Further research is required to determine the lethal dose of hypoglycin A in horses, as well as factors that affect annual seed burden and hypoglycin A content in Acer species in North America and Europe.

Guanidinoacetate methyltransferase deficiency: first steps to newborn screening for a treatable neurometabolic disease.

BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis resulting in severe neurological complications in untreated patients. Currently available treatment is only successful to stop disease progression, but is not sufficient to reverse neurological complications occurring prior to diagnosis. Normal neurodevelopmental outcome in a patient, treated in the newborn period, highlights the importance of early diagnosis. METHODS: Targeted mutation analysis (c.59G>C and c.327G>A) in the GAMT gene by the QIAxcel system and GAA measurement by a novel two-tier method were performed in 3000 anonymized newborn blood dot spot cards. RESULTS: None of the targeted mutations were detected in any newborn. Two novel heterozygous variants (c.283_285dupGTC; p.Val95dup and c.278_283delinsCTCGATGCAC; p.Asp93AlafsX35) were identified by coincidence. Carrier frequency for these insertion/deletion types of GAMT mutations was 1/1475 in this small cohort of newborns. GAA levels were at or above the 99th percentile (3.12 µmol/l) in 4 newborns. Second-tier testing showed normal results for 4 newborns revealing 0.1% false positive rate. No GAMT mutations were identified in 4 of the newborns with elevated GAA levels in the first tier testing. CONCLUSION: This is the first two-tier study to investigate carrier frequency of GAMT deficiency in the small cohort of newborn population to establish evidence base for the first steps toward newborn screening for this treatable neurometabolic disorder.

Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts.

The differentiation of carnitine-acylcarnitine translocase deficiency (CACT) from carnitine palmitoyltransferase type II deficiency (CPT-II) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency from mitochondrial trifunctional protein deficiency (MTP) continues to be ambiguous using current acylcarnitine profiling techniques either from plasma or blood spots, or in the intact cell system (fibroblasts/amniocytes). Currently, enzyme assays are required to unequivocally differentiate CACT from CPT-II, and LCHAD from MTP. Over the years we have studied the responses of numerous FOD deficient cell lines to both even and odd numbered fatty acids of various chain lengths as well as branched-chain amino acids. In doing so, we discovered diagnostic elevations of unlabeled butyrylcarnitine detected only in CACT deficient cell lines when incubated with a shorter chain fatty acid, [7-2H3]heptanoate plus l-carnitine compared to the routinely used long-chain fatty acid, [16-2H3]palmitate. In monitoring the unlabeled C4/C5 acylcarnitine ratio, further differentiation from ETF/ETF-DH is also achieved. Similarly, incubating LCHAD and MTP deficient cell lines with the long-chain branched fatty acid, pristanic acid, and monitoring the C11/C9 acylcarnitine ratio has allowed differentiation between these disorders. These methods may be considered useful alternatives to specific enzyme assays for differentiation between these long-chain fatty acid oxidation disorders, as well as provide insight into new treatment strategies.

Prenatal diagnosis of multiple acyl-CoA dehydrogenase deficiency: association with elevated alpha-fetoprotein and cystic renal changes.

We report the occurrence of multiple acyl-CoA dehydrogenase deficiency (MADD) in two consecutive pregnancies in a young, Caucasian, non-consanguineous couple. In the first pregnancy, the maternal serum alpha-fetoprotein was elevated. A sonogram showed growth delay, cystic renal disease, and oligohydramnios; the parents decided to terminate the pregnancy. Postmortem examination confirmed the cystic renal disease and showed hepatic steatosis, raising the suspicion of a metabolic disorder. The diagnosis of MADD was made by immunoblot studies on cultured fibroblasts. In the subsequent pregnancy, a sonogram at 15 weeks' gestation showed an early growth delay but normal kidneys. The maternal serum and amniotic fluid concentrations of alpha-fetoprotein were elevated, and the amniotic fluid acylcarnitine profile was consistent with MADD. In vitro metabolic studies on cultured amniocytes confirmed the diagnosis. A follow-up sonogram showed cystic renal changes. These cases provide additional information regarding the evolution of renal changes in affected fetuses and show a relationship with elevated alpha-fetoprotein, which may be useful in counseling the couple at risk. MADD should be considered in the differential diagnosis of elevated alpha-fetoprotein and cystic renal disease. Early growth delay may be an additional feature.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

Identification of a novel mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency.

A novel mutation was identified in two unrelated patients with medium-chain acyl-CoA dehydrogenase deficiency. First, a 19-year-old Caucasian female presented with a devastating illness, resulting in sudden death in adulthood which is unusual. The second patient, now a 3.5-year-old male, presented at 17 months of age with a hypoglycemic seizure and dehydration. Sequence analysis revealed a novel mutation G617T in exon 8 resulting in an arginine to leucine substitution at codon 206 (R206L). Both patients were compound heterozygous for this G617T and the common mutation A985G.

Death caused by perioperative fasting and sedation in a child with unrecognized very long chain acyl-coenzyme A dehydrogenase deficiency.

An adopted 3(1/2)-year-old girl with no prior medical problems died after a routine dental procedure. More than 2 years later, acylcarnitine analysis of dried blood found on her bedding revealed she had very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. Perioperative oral fasting, without intravenous administration of glucose, may be detrimental to children with certain metabolic and endocrine disorders. Newborn screening by tandem mass spectrometry will detect disorders of fatty acid oxidation such as VLCAD and allow early and preventive treatment.

Evidence for a short-chain carnitine-acylcarnitine translocase in mitochondria specifically related to the metabolism of branched-chain amino acids.

Carnitine-acylcarnitine translocase (CATR) deficiency is a severe defect in fatty acid oxidation which presents early in life most frequently with hypoglycemia, hyperammonemia, and severe cardiac abnormalities. CATR exchanges acylcarnitines of various chain lengths for free carnitine across the mitochondrial membrane. In vitro studies in intact fibroblasts from patients with documented deficiency of CATR were probed with stable-isotope-labeled precursors and the resulting acylcarnitines were analyzed by tandem mass spectrometry. After a 72-h incubation with l-[(2)H(3)]carnitine the translocase-deficient cells produced acylcarnitines in which the deuterium was incorporated into short-chain acylcarnitines, C2-C5. Experiments with simultaneous incubation of l-[(2)H(3)]carnitine and l-[(13)C(6)]isoleucine produced [(13)C(5)]2-methylbutyryl-[(2)H(3)]carnitine and [(13)C(3)]propionyl-[(2)H(3)]carnitine indicating exchange of labeled acylcarnitine from inside the mitochondrial matrix with labeled free carnitine. These studies support the possible existence of a "branched-chain" carnitine-acylcarnitine translocator in mitochondria.

Deficiency of biotinyl-AMP synthetase activity in fibroblasts of patients with holocarboxylase synthetase deficiency.

A simple, rapid assay was developed to diagnose holocarboxylase synthetase deficiency. Holocarboxylase synthetase first catalyzes the formation of biotinyl-AMP from biotin and ATP, an activity designated as biotinyl-AMP synthetase. In the second step of the reaction, biotin is transferred from biotinyl-AMP to the enzymatically inactive apocarboxylase to form an active holocarboxylase. The assay for holocarboxylase synthetase activity therefore requires a protein apocarboxylase substrate which is not readily available. In the assay for biotinyl-AMP synthetase, hydroxylamine reacts nonenzymatically with the product of the enzymatic reaction, biotinyl-AMP, to form biotinylhydroxamate. At the end of the reaction, unreacted radioactive biotin substrate, which is negatively charged at neutral pH, is bound to an anion-exchange resin and a neutral radioactive biotinylhydroxamate product in the supernatant is counted. In fibroblasts from 11 patients with proven holocarboxylase synthetase deficiency, the mean biotinyl-AMP synthetase activity at 25 nM biotin was 4% of the control mean with a range of 0.2 to 8%. This is an improved assay because it does not require preparation of an apocarboxylase substrate and is suitable for the diagnosis of patients with holocarboxylase synthetase deficiency.

Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized defect in human valine metabolism.

A 2-year-old female was well until 12 months of age when she was found to be anemic and had dilated cardiomyopathy. Total plasma carnitine was 6 microM and acylcarnitine analysis while receiving carnitine supplement revealed an increase in the four-carbon species. Urine organic acids were normal. In vitro analysis of the mitochondrial pathways for beta oxidation, and leucine, valine, and isoleucine metabolism was performed in fibroblasts using stable isotope-labeled precursors to these pathways followed by acylcarnitine analysis by tandem mass spectrometry. 16-2H3-palmitate was metabolized normally down to the level of butyryl-CoA thus excluding SCAD deficiency. 13C6-leucine and 13C6-isoleucine were also metabolized normally. 13C5-valine incubation revealed a significant increase in 13C4-isobutyrylcarnitine without any incorporation into propionylcarnitine as is observed normally. These same precursors were also evaluated in fibroblasts with proven ETF-QO deficiency in which acyl-CoA dehydrogenase deficiencies in each of these pathways was clearly identified. These results indicate that in the human, there is an isobutyryl-CoA dehydrogenase which exists as a separate enzyme serving only the valine pathway in addition to the 2-methyl branched-chain dehydrogenase which serves both the valine and the isoleucine pathways in both rat and human.

A new case of malonyl coenzyme A decarboxylase deficiency presenting with cardiomyopathy.

UNLABELLED: A new case of mitochondrial malonyl coenzyme A decarboxylase deficiency is described. The patient presented with an initial episode of metabolic acidosis, seizures, hypoglycemia, and cardiac failure at 2 months of age which slowly resolved. Subsequent evaluations at 4 years of age for developmental delay revealed a prominent elevation of malonic acid in urine. Malonyl carnitine was also elevated. The activity of Malonyl CoA decarboxylase in cultured fibroblasts was 7% of normal. CONCLUSION: Malonyl CoA decarboxylase deficiency may result in inhibition of fatty acid oxidation, which may account for the cardiomyopathy.

Methylmalonic acidemia, cobalamin C type, presenting with cutaneous manifestations.

BACKGROUND: Erosive dermatitis resembling the skin lesions of acrodermatitis enteropathica has been described in a number of aminoacidopathies and organic acidemias. In some, the dermatitis is a manifestation of untreated disease, while in others, including methylmalonic acidemia, skin lesions have been ascribed to nutritional deficiency due to therapeutic amino acid restrictions. OBSERVATIONS: We report 2 cases of methylmalonic acidemia presenting with cutaneous manifestations in the perinatal period before restrictive nutritional interventions. The cutaneous involvement consisted of cheilitis and diffuse erythema with erosions and desquamation. Methylmalonic acidemia, cobalamin C type, was subsequently diagnosed in both cases. CONCLUSIONS: An erosive, desquamating dermatitis with histopathologic characteristics resembling acrodermatitis enteropathica may be a presenting sign in cobalamin C methylmalonic acidemia, even in the absence of long-standing nutritional restrictions or deficiency.

Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency (MCD). We have identified six different point mutations in the HCS gene in nine patients with MCD. Two of the mutations are frequent among the MCD patients analyzed. Four of the mutations cluster in the putative biotin-binding domain as deduced from the corresponding Escherichia coli enzyme and consistent with an explanation for biotin-responsiveness based on altered affinity for biotin. The two others may define an additional domain involved in biotin-binding or biotin-mediated stabilization of the protein.