Brunhilde Molzer

TARGETED INACTIVATION OF THE X-LINKED ADRENOLEUKODYSTROPHY GENE IN MICE

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 β‐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 β‐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. J. Neurosci. Res. 50:829–843, 1997. © 1997 Wiley‐Liss, Inc.

The Cerebrohepatorenal (Zellweger) Syndrome: An Improved Method for the Biochemical Diagnosis and its Potential Value for Prenatal Detection

ABSTRACT: The sequence of reactions involved in plasmalogen biosynthesis has been evaluated in cultured fibroblasts of patients with the cerebrohepatorenal syndrome. A double-label, double-substrate incubation using [1-14C] hexadecanol and 1-0-[9′, 10′-3H]hexadecylglycerol was performed to monitor the relative rates of peroxisomal and microsomal biosynthesic steps. [14C] radioactivity associated with 1′-alkenyl groups of plasmalogens was found to be drastically reduced in fibroblasts of affected patients whereas [3H] incorporation was apparently normal. This finding is specific for cerebrohepatorenal syndrome fibroblasts since cell lines of patients with childhood adrenoleukodystrophy and neuronal ceroidlipofuscinosis utilized the lipid precursors of plasmalogen biosynthesis at normal rates. The results show that the defect in plasmalogen synthesis in the cerebro-hepato-renal syndrome is restricted to the peroxisomal steps. The finding of normal microsomal biosynthetic steps was exploited to devise a novel diagnostic assay in fibroblasts and amniocytes based on the comparison of [3H/14C] isotope ratios within aldehydes released from plasmalogens by acid hydrolysis. The procedure can be completed with a minimal amount of cells since it renders quantitative analyses unnecessary. Therefore, this technique appears ideally suited for the sensitive and safe prenatal diagnosis of the cerebro-hepato-renal syndrome.

Occurrence, distribution and phenotype of arylsulfatase A mutations in patients with metachromatic leukodystrophy

Occurrence, distribution, and phenotype of arylsulfatase A (ASA) mutations were investigated in 27 patients with metachromatic leukodystrophy (MLD) from Central Europe, mainly from Austria (n = 15) and Poland (n = 9). Genomic DNA from leukocytes, fibroblasts, or paraffin-embedded, formalin-fixed brain or nerve tissue, respectively, was tested by natural or mutated primer-modulated PCR restriction, fragment length polymorphism for the eight most common European mutations: R84Q, S96F, 459+1G > A, I179S, A212V, 1204+1G > A, P426L, and 1401del11bp. The overall identification rate of unrelated MLD alleles was the highest, in adult (90%), medium in juvenile (50%), and lowest in late infantile (36%) MLD patients. The two common alleles, 459+1G > A and P426L, together accounted for 42% of all 50 unrelated MLD alleles investigated; I179S was observed in 6 of 50 MLD alleles (12%). Thus, I179S was far more frequent than hitherto thought and appears to be a third common mutation in Europe. Moreover, a different allelic distribution between Austrian and Polish juvenile patients was disclosed, indicating genetic heterogeneity of MLD even within Central Europe. The genotype-phenotype correlation suggested by Polten et al. [N Engl J Med 324:18-22, 1991] was not followed by all of our MLD patients. Moreover, some MLD patients with identical ASA mutations presented with different phenotypes. This may be due, at least in some cases, to the presence of an additional mutation on individual mutant alleles. Therefore, prediction of the clinical course from single mutation analysis is not possible.

Detection of adrenoleukodystrophy by increased C26:0 fatty acid levels in leukocytes.

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.

Late juvenile metachromatic leukodystrophy (MLD) in three patients with a similar clinical course and identical mutation on one allele

Metachromatic leukodystrophy (MLD) is an autosomal, recessively inherited, lysosomal storage disease caused by arylsulfatase A (ASA) activity deficit. Arylsulfatase A initiates the degradation of sulfatide (cerebroside sulfate), which is an essential component of myelin. The main clinical symptoms are caused by progressive demyelination. At least 37 MLD‐related ASA mutations are known to date. I179S (E3P799) is a disease‐related mutation, described for the first time by Fluharty in 1991. This aberration appears to substantially reduce, but not completely eliminate ASA activity, and was detected in individuals with late‐onset (juvenile or adult) forms of MLD. This paper deals with the peculiar clinical course in three unrelated juveniles with late‐onset MLD carrying the I179S mutation on one allele. In the three described patients with the I179S mutation, psychiatric disturbances and intellectual impairment dominated the clinical picture, while the neurological lesions progressed more slowly. Although the symptoms appeared rather early, making it possible to classify this as the juvenile type of MLD, the clinical picture was more that of the adult type. Although the mutations on the second allele in our patients are unknown, one can speculate, that the mutation I179S plays an important role in the characteristic clinical course (psychiatric impairment, slower neurological deterioration, but relatively early onset). It seems that I179S mutation on one allele with another mutation on the other allele reduces ASA activity, but the enzyme can still cope with a part of the substrate influx, leading to late‐juvenile‐onset MLD with such strikingly similar phenotypes remaining a little bit of the adult (psychiatric) type. This could be one more argument in favour of phenotype‐genotype correlation in patients with MLD.

Elevated sulfatide excretion in compound heterozygotes of metachromatic leukodystrophy and ASA-pseudodeficiency allele

OBJECTIVE Use of sulfatide excretion in differentiating MLD/PD-heterozygotes from MLD-patients and PD/PD-homozygotes. DESIGN AND METHODS Sulfatide was extracted from urine sediment with chlorotom/methanol (2:1, v/v). The quantity of sulfatide was measured densitometrically (lambda = 580 nm) after thin-layer chromatography. ASA and beta-galactosidase activities were assayed enzymatically. RESULTS MLD/PD-heterozygotes excreted sulfatide in the range of 4.8-36.3 nmol/mg lipid (mean +/- SD = 17.8 +/- 10.7), whereas sulfatide in MLD-patients ranged from 74.3-411.6 nmol/mg lipid (mean +/- SD = 184.5 +/- 130.8) and in PD/PD-hormozygotes sulfatide excretion remained in normal range of 0.0-5.9 nmol/mg lipid (mean +/- SD = 1.64 +/- 2.12). ASA activities in these groups were very low or lowered. CONCLUSIONS The quantitative measurement of sulfatide in urine allows differentiation between MLD/PD-heterozygotes and MLD-heterozygotes, as well as between MLD/PD-heterozygotes with very low ASA activity and MLD-patients or PD/PD-hormozygotes. The quantitative measurement of sulfatide in urine differs between MLD-carriers and controls.

Multiple Sclerosis-Like Syndrome in a Woman Heterozygous for Adrenoleukodystrophy

A 28-year-old asymptomatic woman was diagnosed to be heterozygous for adrenoleukodystrophy (ALD) by elevated very long-chain fatty acids in serum and fibroblasts after ADL had been diagnosed in her son. A year later she had transient unilateral blurred vision. Evoked potentials and brain magnetic resonance imaging showed further separate cerebral white matter lesions suggesting multiple sclerosis (MS). MS-like syndromes in women heterozygous for ALD may be more frequent than previously recognized.

Molecular and phenotypic characteristics of metachromatic leukodystrophy patients from Poland.

The occurrence and genotype–phenotype correlations of the eight most common mutations in the arylsulfatase A (ARSA) gene were studied in 43 unrelated Polish patients suffering from different types of metachromatic leukodystrophy (MLD). Screening for mutations p.R84Q, p.S96F, c.459+1G>A, p.I179S, p.A212V, c.1204+1G>A, p.P426L, and c.1401–1411del allowed the identification of 53.5% of the mutant alleles. In the whole investigated group of patients, mutations c.459+1G>A and p.P426L were the most frequent, 19 and 17%, respectively. The prevalence of the third most frequent mutation, i.e. p.I179S (13%), seems to be higher than that in other populations. The incidence of c.1204+1G>A was 5%, which is higher than reported earlier (2%). It seems that p.I179S and c.1204+1G>A are more prevalent in MLD patients from Poland than from other countries. In the group examined by us, mutations p.R84Q, p.S96F, p.A212V, and c.1401–1411del were not detected; thus, 46.5% of MLD alleles remained unidentified. This indicates that other, novel or already described, but rare, mutations exist in Polish population. In late infantile homozygotes for c.459+1G>A and one homozygote for c.1204+1G>A, first clinical symptom was motor deterioration. In adult homozygotes for p.P426L, the disease onset manifested as gait disturbances, followed by choreoathetotic movements, difficulties in swallowing, dysarthria, tremor, and nystagmus. In the carriers of the p.I179S mutation, the hallmark of the clinical picture was psychotic disturbances.

Very long chain fatty acids in genetic peroxisomal disease fibroblasts: Differences between the cerebro-hepato-renal (Zellweger) syndrome and adrenoleukodystrophy variants

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.

Simultaneous detection of the two most frequent metachromatic leukodystrophy mutations

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurometabolic disorder caused by deficiency of arylsulfatase A (ASA). To detect ASA mutations E2S609 and E8P2382, the two most frequent MLD mutations, a non-radioactive polymerase chain reaction (PCR)-based assay was developed. This assay is a multiple “mutated primer-modulated PCR restriction fragment length polymorphism”. The primers related to each mutation mismatch to create anXbaI orPstI restriction site in mutation E2S609 or E8P2382, respectively. The assay was designed to give four fragments of 160, 130, 100, and 70 bp, easy to distinguish. An internal control fragment is not necessary since both primer pairs amplify different regions of the ASA gene and fragments will be obtained in all allelic possibilities. This technique produced clear-cut results when genomic DNA, isolated either from leukocytes, cultured human fibroblasts, or paraffin-embedded autopsy material, was used as template. The assay will be of help in comparative studies on the relation between MLD genotype and phenotype, a problem not yet fully understood. Since our method was shown to work also on DNA from paraffin-embedded autopsy material, genotype/phenotype studies would not be restricted to in vivo investigations but could be done also on post mortem material, thus including investigations on a large group of cases and also studies on the relation between genotype and neuropathological features.