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Objectives: Our aim was to replace cultured skin fibroblasts in the diagnosis of X-linked adrenoleukodystrophy (X-ALD) by peripheral blood cells., Design and Methods: Very long chain fatty acids (VLCFAs) were analyzed in leukocytes from X-ALD patients, heterozygotes, and controls using gas chromatography-mass spectrometry (GC-MS). Immunofluorescence for adrenoleukodystrophy protein (ALDP) was performed in mononuclear blood cell preparations of X-ALD patients known to be ALDP negative in fibroblasts, heterozygote relatives of these patients, and controls., Results: All X-ALD patients were distinguishable from controls by VLCFA analysis in leukocytes. 91.7% of heterozygotes were identified by combined VLCFA analysis in leukocytes and plasma. All patients investigated lacked ALDP immunoreactivity in mononuclear cells, while heterozygotes showed mosaic patterns of positive and negative cells., Conclusion: Determination of VLCFAs by GC-MS in combination with ALDP immunofluorescence in peripheral blood cells provides a fast and minimally invasive diagnostic method for X-ALD, which, in contrast to plasma analysis, is independent of alimentary influences. Notably, joint evaluation of leukocytes and plasma considerably improves the identification of heterozygotes.

In its severe form, X-linked adrenoleukodystrophy (ALD) is a lethal neurologic disease of children, characterized by progressive cerebral demyelination and adrenal insufficiency. Associated with a biochemical defect of peroxisomal beta-oxidation, very long-chain fatty acids (VLCFA) build up in tissues that have a high turnover of lipids, such as central nervous system (CNS) white matter, adrenal cortex, and testis. Whether the abnormal accumulation of VLCFA is the underlying cause of demyelination or merely an associated biochemical marker is unknown. ALD is caused by mutations in the gene for a peroxisomal membrane protein (ALDP) that shares structural features with ATP-binding-cassette (ABC) transporters. To analyze the cellular function of ALDP and to obtain an animal model of this debilitating disease, we have generated transgenic mice with a targeted inactivation of the ald gene. Motor functions in ALDP-deficient mice developed at schedule, and unexpectedly, adult animals appeared unaffected by neurologic symptoms up to at least 6 months of age. Biochemical analyses demonstrated impaired beta-oxidation in mutant fibroblasts and abnormal accumulation of VLCFAs in the CNS and kidney. In 6-month-old mutants, adrenal cortex cells displayed a ballooned morphology and needle-like lipid inclusions, also found in testis and ovaries. However, lipid inclusions and demyelinating lesions in the CNS were not a feature. Thus, complete absence of ALDP expression results in a VLCFA storage disease but does not impair CNS function of young adult mice by pathologic and clinical criteria. This suggests that additional genetic or environmental conditions must be fulfilled to model the early-onset and lethality of cerebral ALD in transgenic mice.

Two 6-year-old patients with clinical signs of leukodystrophy had no nosological diagnoses in vivo. Neuropathological studies revealed scavenger cells to be clustered in perivascular regions of the demyelinated brains. Histochemical and ultrastructural details of the non-metachromatic storage macrophages suggested lipid storage and prompted a biochemical analysis of cerebral tissue. The detection of increased amounts of very long chain fatty acids in the cholesterol ester fraction from formalin tissue in one patient was consistent with a diagnosis of an adrenoleukodystrophy-like condition, while the marked reduction in beta-galactocerebrosidase activity in a frozen brain sample of the second patient indicate Krabbe disease. The diagnostic potential of post-mortem studies in rare leukodystrophies is addressed.

Inflammatory demyelination in the central nervous system in the childhood cerebral phenotype of X-linked adrenoleukodystrophy (X-ALD) bears resemblance to that of multiple sclerosis. With a view to an association of HLA class II genes, specifically HLA-DRB1 subtype DRB1*15 to multiple sclerosis we investigated the HLA class II DR haplotype in 29 unrelated X-ALD patients including 17 childhood cerebral phenotype patients. Our results did not show an association of DRB1*15 and X-ALD, but disclosed a significant association of HLA DRB1*16 alleles and X-ALD in general. This finding suggests that in addition to the X-chromosomal ALD gene an autosomal gene linked to the HLA class II region is involved in the pathogenesis of X-ALD. This gene should affect a pathomechanism common to all ALD variants, such as defective peroxisomal metabolism of very long chain fatty acids.

Fragments of the adrenoleukodystrophy (ALD) cDNA from a patient with adolescent ALD were amplified by polymerase chain reaction and subcloned. Bidirectional sequencing of the entire coding ALD gene disclosed a cytosine to guanine transversion at nucleotide 1451 in exon five, resulting in substitution of proline 484 by arginine. Five of nine siblings of the patient, comprising two cerebral ALD, one adrenomyeloneuropathy, one Addison only as well as the symptomatic mother (all accumulating very long chain fatty acids) carried this mutation, which was not found in the unaffected persons, in five unrelated ALD patients, and in twenty controls. We propose that this missense mutation generated the disease per se as well as the metabolic defect; the different phenotypes, however, must have originated by means of additional pathogenetic factors.

Very long chain fatty acids (VLCFA) were found to be markedly increased and phytanic acid was borderline above normal in formalin-fixed brain white matter of case with an unusual type of familial leukodystrophy with epitheloid cells as described previously by Gullotta et al. [Neuropädiatrie (1970) 2: 173-186]. Increased VLCFA in brain clearly demonstrate that the patient had suffered from a peroxisomal disease. This diagnosis is corroborated by ultrastructural findings in brain showing typical lamellar inclusions. The particular type of peroxisomal disorder present in case (heterozygote of X-linked adrenoleukodystrophy?) remains speculative.

Peroxisomal disorders are genetic metabolic diseases with generalized, multiple, or single functional disturbances of the peroxisome. According to the extent of the functional disturbances 3 groups of diseases can be differentiated: disorders with generalized loss of peroxisomal functions (Zellweger syndrome, ZS; neonatal adrenoleukodystrophy, NALD; infantile Refsum's disease), disorders with multiple enzymatic defects (e.g. rhizomelic chondrodysplasia punctata), and disorders with a single enzymatic defect in the peroxisome, the most important being adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN). Adult Refsum's disease, a genetic neurological disorder with phytanic acid accumulation, is due to a mitochondrial enzyme deficiency, but is often considered together with peroxisomal diseases because of phytanic acid (PHYT) accumulation in most peroxisomal diseases. The main clinical and pathological criteria of the major disorders and the biochemical parameters of their differentiation are presented. Elevated levels of very long chain fatty acids (VLCFA) and/or PHYT are the primary diagnostic markers for all peroxisomal disorders and adult Refsum's disease, respectively. Our investigations disclosed 30 ALD/AMN hemizygotes, 16 ALD/AMN heterozygotes, 8 cases of ZS/NALD and 7 patients with adult Refsum's disease. In addition, 15 cases of peroxisomal disorders were confirmed by biochemical investigations in autopsy material. With regard to peroxisomal disorders, therapeutic concepts exist only for ALD/AMN: corticosteroid substitution for adrenal insufficiency, dietary treatment, and bone marrow transplantation (BMT). Adult Refsum's disease can be treated successfully by dietary therapy. In case of dietary treatment and BMT, assay of VLCFA and/or PHYT is important for the biochemical evaluation of these therapies.

Immunoglobulin (Ig) concentrations were investigated in white matter samples of two adrenoleukodystrophy (ALD), three multiple sclerosis (MS), two systemic lupus erythematosus (SLE), one rheumatoid arthritis, and three control brains obtained at autopsy. "Free" Igs were extracted at pH 7.4; subsequently, bound Igs were extracted at pH 2.5 and 10.8, respectively. Igs were quantified by radial immunodiffusion. In ALD material there was an increase of free IgG and IgA, in one sample also of IgM, as compared to controls. No significant amounts of Igs were detected in the pH 2.5 and 10.8 extracts of ALD brain. Similarly to ALD, an increase of free IgG and IgA was a characteristic finding in MS brain; in contrast to ALD and control material, significant amounts of bound Igs (IgG) extractable at acid or alkaline pH, respectively, were present in MS tissue. In both SLE brains increase of free IgM was conspicuous. Preliminary studies on binding of Igs extracted at pH 7.4 from brain to frozen sections of normal human and bovine brain tissue revealed different binding properties of Igs from ALD, MS, SLE, and control brains. Immunochemical findings in ALD indicating pathologic accumulation of Igs in brain tissue were paralleled by immunocytochemical observations demonstrating accumulation of lymphoid cells staining for IgG, IgA, and IgM, respectively, mainly in areas of recent demyelination. Participation of Igs in the pathogenesis of ALD lesions may be considered but needs further confirmation.

Very long chain fatty acids of peripheral blood leukocytes were analyzed by gas chromatography in nine members of a family including two hemizygotes and one obligate heterozygote for adrenoleukodystrophy (ALD), as well as in twelve controls. Comparative investigations were done in cultured fibroblasts. Elevated C26:0 levels were observed in leukocytes and fibroblasts of ALD hemizygotes. The obligate heterozygote displayed a clear-cut increase of C26:0 concentration in leukocytes but not in fibroblasts. Determination of C26:0 in leukocytes may serve as diagnostic tool in the detection of ALD gene carriers.

Very long chain fatty acids were investigated by gas chromatography in fibroblasts of patients with genetic peroxisomal diseases (cerebro-hepato-renal (Zellweger) syndrome, childhood adrenoleukodystrophy, adrenomyeloneuropathy, neonatal adrenoleukodystrophy) and of controls. Concentrations of C 26:0 were increased to about the same extent in all disorders investigated. C 26:1 concentrations, on the other hand, were considerably elevated only in the cerebro-hepato-renal syndrome. In all control, adrenoleukodystrophy, and adrenomyeloneuropathy cases the C 22:0 concentration was higher than the respective C 26:0 concentration; the reverse was found in the cerebro-hepato-renal syndrome. These differences seem to reflect different impairment of peroxisomes in the cerebro-hepato-renal syndrome and adrenoleukodystrophy variants, respectively. Additional experiments to characterize C 26:1 by thin layer chromatography, gas chromatography and mass spectrometry revealed the presence of two straight-chain C 26:1 isomers with similar fragmentation patterns.

A 43-year-old female with adrenoleukodystrophy (ALD) is described, who developed spastic tetraparesis, suffered grand mal seizures, and became stuporous and demented during the last 5 years of her life. Computed tomography revealed symmetrical hypodense lesions in the peritrigonal regions. Adrenal insufficiency was not evident except for skin pigmentation. The ultrastructure of a rectal biopsy specimen showed inclusions with lamellae and interspersed clefts in macrophages of the submucosal layer. At autopsy, the adrenals were found to contain large foam cells filled with similar inclusions. The brain cortex and the spinal cord were histologically normal. However, cerebral white matter exhibited widespread demyelination which spared only the arcuate fibres. In regions of less severe demyelination scattered inflammatory cells were seen. On electron microscopy, aggregates of typical paired leaflets with distinct intermediate lines were demonstrated in perivascular macrophages. Histochemical study showed these cells to contain free as well as esterified cholesterol. Gas chromatographic analysis of very long chain fatty acids (VLFA) from the demyelinated cerebral white matter showed a marked increase of C26:0 fatty acid in cholesterol esters and above-normal values for C24:0 and C24:1 in gangliosides. It is suggested that the condition was a heterozygote form of X-linked ALD. Patients with neurodegenerative symptoms with or without adrenal insufficiency can easily be screened for X-linked ALD by VLFA analysis in blood or cultured fibroblasts.