Carnitine-acylcarnitine Translocase Deficiency Research Articles

Functional analysis of mutations in the human carnitine/acylcarnitine translocase in Aspergillus nidulans

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid β-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans Δ acuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

Mutational spectrum and DNA-based prenatal diagnosis in carnitine–acylcarnitine translocase deficiency

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69–70insTGTGC, and 609−1g > a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

Aberrant mRNA Splicing Associated with Coding Region Mutations in Children with Carnitine-Acylcarnitine Translocase Deficiency

This report describes three infants with genetic defects of carnitine-acylcarnitine translocase (CACT), an inner mitochondrial membrane carrier that is essential for long-chain fatty acid oxidation. Two of the patients were of European and Chinese origin; the third was from consanguineous Turkish parents. CACT activity was totally deficient in cultured skin fibroblasts from all three patients. Patient 1 was heterozygous for a paternal frameshift mutation (120 del T in exon 1) and a maternal lariat branch point mutation (−10 T → G in intron 2). Patient 2 was heterozygous for the same lariat branch point (−10T → G intron 2) mutation, derived from the father, and a maternal frameshift mutation (362 del G in exon 3). Patient 3 was homozygous for a frameshift mutation (306 del C in exon 3). All of the three frameshift mutations give rise to the same stop codon at amino acid residue 127 which is predicted to cause premature protein truncation. In addition, cDNA transcript analysis showed that these coding sequence mutations also increase the amount of aberrant mRNA splicing and exon skipping at distances up to 7.7 kb nucleotides from mutation sites. The data suggest that the stability of mRNA transcripts is decreased or the frequency of aberrant splicing is increased in the presence of CACT coding sequence mutations. These results confirm that CACT is the genetic locus of the recessive mutations responsible for the fatal defects of fatty acid metabolism previously associated with deficiency of translocase activity in these three cases.

Carnitine/Acylcarnitine Translocase Deficiency (Neonatal Phenotype): Successful Prenatal and Postmortem Diagnosis Associated with a Novel Mutation in a Single Family

The neonatal phenotype of carnitine-acylcarnitine translocase (CACT) deficiency is one of the most severe and usually lethal mitochondrial fat oxidation disorders characterized by hypoketotic hypoglycemia, hyperammonemia, cardiac abnormalities, and early death. In this study, the proband was the daughter of consanguineous Hispanic parents. At 36 h of life, she had bradycardia and died at 4 days of age without a specific diagnosis. In a subsequent pregnancy, prenatal counseling and amniocentesis were provided. Incubation of the amniocytes from this pregnancy and fibroblasts (from the dead proband) with [16-2H3]palmitic acid and analysis by tandem mass spectrometry revealed an increasedconcentration of [16-2H3]palmitoylcarnitine, suggesting the diagnoses of either CACT or carnitine palmitoyltransferase II (CPT-II) deficiency. CACT enzyme activity was absent in both cell lines. Molecular investigation of cDNA from the dead proband and her affected sibling revealed aberrant CACT cDNA species, including exon 3 skipping, both exon 3 and 4 skipping, and a 13-bp insertion at cDNA position 388. Investigation of these cell lines for mutations affecting CACT RNA processing by analysis of CACT gene sequences, including intron and exon boundaries, revealed a single nucleotide G deletion at the donor site in intron 3 which resulted in exon skipping and a 13-bp insertion. The proband and her affected sibling were homozygous for this deletion.

Functional Analysis of Mutant Human Carnitine Acylcarnitine Translocases in Yeast

Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient ∼27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.

Carnitine-acylcarnitine translocase deficiency: metabolic consequences of an impaired mitochondrial carnitine cycle

We describe a patient with carnitine-acylcarnitine translocase deficiency (MIM 212138), who presented with neonatal generalized seizures, heart failure, and coma. Laboratory evaluation revealed hypoglycemia, hyperammonemia, lactic acidemia, hyperuricemia, and mild dicarboxylic aciduria. The fact that total plasma carnitine (7.1 μmol/l [20–30]) and free carnitine (1.9 μmol/l [12–18]) were low together with a high acylcarnitine/free carnitine ratio of 2.7 [0.4–1.0] prompted acylcarnitine analysis. This revealed the presence of large amounts of long-chain derivatives including C 16:0, C 16:1, C 18:1, C 18:2. Based on these findings carnitine-acylcarnitine translocase deficiency was suspected which was confirmed by enzyme studies in fibroblasts. The underlying complex metabolic consequences of this defect are reviewed. Prenatal diagnosis was performed in a subsequent pregnancy and a defect ruled out by measurement of carnitine-acylcarnitine translocase activity in cultured chorionic villi cells. As the clinical recognition of a life-threatening fatty acid oxidation disorder may be difficult, defects in this pathway should be considered in any child with coma, an episode of a Reye-like syndrome, and cardiomyopathy. Since routine laboratory tests often do not provide clues about potential disorders and profiles of urinary organic acids may not be characteristic, we recommend to measure free carnitine and acylcarnitines in plasma in any child with hyperammonemia, hypo/hyperketotic hypoglycemia or lactic acidemia for prompt treatment, proper genetic counseling, and potential prenatal diagnosis.

Arrhythmias and conduction defects as presenting symptoms of fatty acid oxidation disorders in children.

The clinical manifestations of inherited disorders of fatty acid oxidation vary according to the enzymatic defect. They may present as isolated cardiomyopathy, sudden death, progressive skeletal myopathy, or hepatic failure. Arrhythmia is an unusual presenting symptom of fatty acid oxidation deficiencies. Over a period of 25 years, 107 patients were diagnosed with an inherited fatty acid oxidation disorder. Arrhythmia was the predominant presenting symptom in 24 cases. These 24 cases included 15 ventricular tachycardias, 4 atrial tachycardias, 4 sinus node dysfunctions with episodes of atrial tachycardia, 6 atrioventricular blocks, and 4 left bundle-branch blocks in newborn infants. Conduction disorders and atrial tachycardias were observed in patients with defects of long-chain fatty acid transport across the inner mitochondrial membrane (carnitine palmitoyl transferase type II deficiency and carnitine acylcarnitine translocase deficiency) and in patients with trifunctional protein deficiency. Ventricular tachycardias were observed in patients with any type of fatty acid oxidation deficiency. Arrhythmias were absent in patients with primary carnitine carrier, carnitine palmitoyl transferase I, and medium chain acyl coenzyme A dehydrogenase deficiencies. The accumulation of arrhythmogenic intermediary metabolites of fatty acids, such as long-chain acylcarnitines, may be responsible for arrhythmias. Inborn errors of fatty acid oxidation should be considered in unexplained sudden death or near-miss in infants and in infants with conduction defects or ventricular tachycardia. Diagnosis can be easily ascertained by an acylcarnitine profile from blood spots on filter paper.

Carnitine-Acylcarnitine Translocase Deficiency

Carnitine-acylcarnitine translocase deficiency, like other defects of mitochondrial fatty acid oxidation, is an autosomal, recessively inherited disorder. When the deficiency is near total, it is usually fatal, affects life soon after birth, and constitutes one of the causes of skeletal muscle myopathy, cardiac and liver abnormalities, and childhood sudden death. The presenting features have included neonatal distress, convulsions, hypoglycemia, hyperammonemia, hypoketonemia, intermittent dicarboxyluria, hypothermia, apnea, neurological deterioration, and hypocarnitinemia with grossly elevated acylcarnitines. Two cases of partial translocase deficiency (4-6% residual activity) with milder symptoms and without cardiac involvement have also been identified. Evidence so far indicates that the translocase protein is the product of a single gene. In two cases of translocase deficiency, the accompanying mutations have been identified. The benefits of prenatal diagnosis have been provided to the affected families by assays of the translocase and/or fatty acid oxidation in cultured amniotic/villous cells. In one such case genetic counseling was made possible even when the only specimen available from a deceased sibling was the Guthrie card.

Carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase deficiency, like other defects of mitochondrial fatty acid oxidation, is an autosomal, recessively inherited disorder. When the deficiency is near total, it is usually fatal, affects life soon after birth, and constitutes one of the causes of skeletal muscle myopathy, cardiac and liver abnormalities, and childhood sudden death. The presenting features have included neonatal distress, convulsions, hypoglycemia, hyperammonemia, hypoketonemia, intermittent dicarboxyluria, hypothermia, apnea, neurological deterioration, and hypocarnitinemia with grossly elevated acylcarnitines. Two cases of partial translocase deficiency (4-6% residual activity) with milder symptoms and without cardiac involvement have also been identified. Evidence so far indicates that the translocase protein is the product of a single gene. In two cases of translocase deficiency, the accompanying mutations have been identified. The benefits of prenatal diagnosis have been provided to the affected families by assays of the translocase and/or fatty acid oxidation in cultured amniotic/villous cells. In one such case genetic counseling was made possible even when the only specimen available from a deceased sibling was the Guthrie card.

The Structure and Organization of the Human Carnitine/Acylcarnitine Translocase (CACT) Gene

The carnitine/acylcarnitine translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine and it is, therefore, essential for the fatty acid β-oxidation pathway. We have determined the exon–intron structure of the human CACT gene, which is responsible for a genetic disorder of fatty acid oxidation called CACT deficiency. The gene spans about 16.5 kb and consists of nine exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rules. All the introns except one are located at the level of the sequences coding for the extramembranous loops of CACT. We have designed a series of intronic oligonucleotide primers for amplifying each of the CACT exons together with their flanking intronic sequences, in segments well suited to detect mutations that would affect splicing of mRNA as well as the coding sequence itself.

A patient with carnitine-acylcarnitine translocase deficiency with a mild phenotype

Carnitine-acylcarnitine translocase deficiency, a rare β-oxidation defect, is manifest in most cases by cardiomyopathy and death in early childhood. We report an affected patient, 3 years of age, who has had no serious complications. The residual enzyme activity in fibroblasts was higher than in previously reported patients, which may explain the benign clinical course. (J Pediatr 1998;132:514-6)

Mitochondrial carnitine-acylcarnitine translocase deficiency presenting as sudden neonatal death: Postmortem diagnosis, prenatal exclusion in subsequent pregnancies, and biochemical specificity of the mitochondrial translocase

A breast-fed female infant died suddenly in the neonatal period at 31 hours of age with profound macrovesicular fatty infiltration of liver, kidney, and muscle on postmortem examination, suggestive of a defect in fatty acid β-oxidation. Fatty acid and palmitoylcarnitine oxidation studies and direct enzyme study of cultured skin fibroblasts suggested a deficiency in the oxidation of long-chain fatty acids distal to carnitine palmitoyltransferase I and before long-chain acyl–coenzyme A dehydrogenases. Deficient activity of carnitine-acylcarnitine translocase was demonstrated with intermediate levels of activity in the infant’s parents, consistent with autosomal recessive inheritance. Fatty acid oxidation studies showed deficient oxidation of fatty acids at all chain lengths from C 10:0 to C 24:0 , with partially reduced oxidation of C 26:0 fatty acid, indicating the occurrence of a single mitochondrial carnitine-acylcarnitine translocase and demonstrating the requirement in vivo for l -carnitine for mitochondrial transport of all medium- and long-chain fatty acyl moieties. The disorder may have been precipitated in this breast-fed infant by poor initial feeding, fasting stress, and the long-chain triglycerides of human milk. The severity of the disorder prompted prenatal diagnosis, and affected siblings were excluded in two subsequent pregnancies by fatty acid oxidation in cultured chorionic villus cells and amniocytes. (J Pediatr 1997;131:220-5)

Neonatal hyperammonemia caused by a defect of carnitine-acylcarnitine translocase

Carnitine-acylcarnitine translocase deficiency is a newly recognized inborn error of metabolism that involves transport of long-chain fatty acids into mitochondria, which in turn impairs mitochondrial β-oxidation, and ketogenesis. We report a new familial example; the affected twins had neonatal distress, hyperammonemia, and transient intracardiac conduction defects.Clinical and biochemical analysis of both our patients and the two previously reported patients revealed that this inherited defect could be manifested during the neonatal period without any of the signs classically associated with fatty oxidation defects. In contrast, all four patients had sustained and “isolated” hyperammonemia, which could be misinterpreted as being caused by urea cycle defects. We conclude that carnitine-acylcarnitine translocase deficiency is a potential differential diagnosis in neonates with unexplained neonatal hyperammonemia. Cardiac and muscle involvement may represent further early pivotal symptoms. (J P EDIATR 1995; 127:723-8)

Carnitine-acylcarnitine translocase deficiency with severe hypoglycemia and auriculo ventricular block. Translocase assay in permeabilized fibroblasts.

Deficiency of the enzymes of mitochondrial fatty acid oxidation and related carnitine dependent steps have been shown to be one of the causes of the fasting-induced hypoketotic hypoglycemia. We describe here carnitine-acylcarnitine translocase deficiency in a neonate who died eight days after birth. The proband showed severe fasting-induced hypoketotic hypoglycemia, high plasma creatine kinase, heartbeat disorder, hypothermia, and hyperammonemia. The plasma-free carnitine on day three was only 3 microM, and 92% of the total carnitine (37 microM) was present as acylcarnitine. Treatments with intravenous glucose, carnitine, and medium-chain triglycerides had been tried without improvements. Measurements in fibroblasts confirmed deficient oxidation of palmitate and showed normal activities of the carnitine palmitoyltransferases I and II and of the three acyl-CoA dehydrogenases. A total deficiency of the carnitine-acyl-carnitine translocase was found in fibroblasts using the carnitine acetylation assay (1986. Biochem. J. 236:143-148). This assay has been further simplified by seeking conditions permitting application to permeabilized fibroblasts and lymphocytes.