ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.

BACKGROUND:Acyl-CoA thioesterases are enzymes that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A (CoASH). These enzymes have been identified in several cellular compartments and are thought to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. However, to date no patients deficient in acyl-CoA thioesterases have been identified.DESIGN:Acyl-CoA thioesterase activity was measured in human skin fibroblasts. Western-blot analysis was used to determine Type-II acyl-CoA thioesterase protein levels in patients.RESULTS:Acyl-CoA thioesterase activity was found in human fibroblasts with all saturated acyl-CoAs from C4-CoA to C18-CoA, with highest activity detected with lauroyl-CoA and myristoyl-CoA (C12-CoA and C14-CoA). An antibody that recognizes the major isoforms of Type-II acyl-CoA thioesterases precipitated the majority of acyl-CoA thioesterase activity in fibroblasts, showing that the main thioesterase activity detected in fibroblasts is catalyzed by Type-II thioesterases. Measurement of acyl-CoA thioesterase activity from fibroblasts of 34 patients with putative fatty acid oxidation disorders resulted in the identification of three patients with lowered Type-II acyl-CoA thioesterase activity in fibroblasts. These patients also had lowered expression of Type-II acyl-CoA thioesterase protein in fibroblasts as judged by Western-blot analysis. However, mutation analysis failed to identify any mutation in the coding sequences for the mitochondrial acyl-CoA thioesterase II (MTE-II) or the cytosolic acyl-CoA thioesterase II (CTE-II).CONCLUSIONS:We have described three patients with lowered Type-II acyl-CoA thioesterase protein and activity in human skin fibroblasts, which is the first description of patients with a putative defect in acyl-CoA thioesterases.

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders.

Hydrops-ectopic calcification-"moth-eaten" (HEM) or Greenberg skeletal dysplasia is an autosomal recessive chondrodystrophy with a lethal course, characterized by fetal hydrops, short limbs, and abnormal chondro-osseous calcification. We found elevated levels of cholesta-8,14-dien-3beta-ol in cultured skin fibroblasts of an 18-wk-old fetus with HEM, compatible with a deficiency of the cholesterol biosynthetic enzyme 3beta-hydroxysterol delta(14)-reductase. Sequence analysis of two candidate genes encoding putative human sterol delta(14)-reductases (TM7SF2 and LBR) identified a homozygous 1599-1605TCTTCTA-->CTAGAAG substitution in exon 13 of the LBR gene encoding the lamin B receptor, which results in a truncated protein. Functional complementation of the HEM cells by transfection with control LBR cDNA confirmed that LBR encoded the defective sterol delta(14)-reductase. Mutations in LBR recently have been reported also to cause Pelger-Huët anomaly, an autosomal dominant trait characterized by hypolobulated nuclei and abnormal chromatin structure in granulocytes. The fact that the healthy mother of the fetus showed hypolobulated nuclei in 60% of her granulocytes confirms that classic Pelger-Huët anomaly represents the heterozygous state of 3beta-hydroxysterol delta(14)-reductase deficiency.

A biochemical variant of argininosuccinate lyase deficiency, found in five individuals, is introduced. In comparison to classical patients, the variant cases of argininosuccinate lyase deficiency were characterized by residual enzyme activity as measured by the incorporation of [ 14 C]citrulline into proteins. The five patients of different ethnic backgrounds presented with relatively mild clinical symptoms, variable age of onset, marked argininosuccinic aciduria and severe, but not complete, deficiency of argininosuccinate lyase. [ 14 C]Citrulline incorporation into proteins, which is completely blocked in classical argininosuccinic aciduria, was only partially reduced in fibroblasts of these patients. Further investigation showed that previous standard conditions of the assay were not optimal. Higher concentrations of citrulline in the incubation medium strongly stimulated 14 C incorporation in normal cells, but not in the patients; as a result, the relative incorporation level in the patients dropped to 6–28% compared to 18–75% of normal in the original procedure. Prenatal diagnosis was successfully performed in three of the families. Affected pregnancies were indicated by (partial) deficiency of [ 14 C]citrulline incorporation in chorionic villi and/or increased levels of argininosuccinate in amniotic fluid. Analysis of the ASL gene in the five patients revealed a considerable allelic heterogeneity. Three novel mutations—R385C (2 patients), V178M and R379C—were detected in homozygous states, whereas one patient was compound heterozygous for the known mutations R193Q and Q286R. In conclusion, there are patients of different ethnic backgrounds who are characterized by residual activity of argininosuccinate lyase and who present with less severe clinical courses. In addition, we present an improved biochemical assay for accurate prenatal and postnatal diagnosis.

In recent years two types of primary hydroxyglutaric acidaemia have been described, characterized by the accumulation of L-2-hydroxyglutaric acid or its D-isomer, respectively. Most patients described suffer from L-2-hydroxyglutaric acidaemia and at least 20 cases have been described. In this disorder typical abnormalities include cerebellar dysfunction, extrapyramidal signs, mental regression and eventual development of seizure disorders (Barth et al 1993).Chalmers et al (1980) were the first to report a patient with accumulation of the D-2-hydroxy isomer only. This patient showed protein-losing enteropathy and normal development. D-2-Hydroxyglutaric acid was not increased in blood. Subsequently described patients have shown a much more severe clinical presentation. Indeed, the patient described by Gibson et al (1993), for instance, presented very early in life with generalized tonic-clonic seizures refractory to anticonvulsant therapy, infantile spasms with hypsarrhythmia, hypotonia, movement disorder, cortical blindness and developmental delay. Furthermore, D-2-hydroxyglutaric acid was elevated in urine, blood and CSE which contrasts with the patient described by Chalmers et al (1980). In an ongoing effort to shed more light on the metabolism of L- and D-2-hydroxyglutaric acid, we concentrated on the degradation of D-2-hydroxyglutaric acid. Since D-2-hydroxyglutaric acid is also known to accumulate in glutaric aciduria type II (Sweetman 1991), in which there is a defect at the level of electron-transfer flavoprotein (ETF) or ETF-dehydrogenase, we first looked for the existence of a mitochondrial flavoprotein reactive with D-2-hydroxyglutaric acid. The results are described below..