Molecular screening of the major mutations in the ARSA gene in patients with metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is an inherited storage disease caused by deficiency of arylsulfatase A (ARSA). Molecular analysis of the major mutations in the ARSA gene was performed in 10 Ukrainian patients (from 9 families) with MLD. According to the age of onset, late infantile MLD was identified in 3 patients, juvenile MLD in 5 patients, and adult MLD in 2 patients (sibs), respectively. The ARSA activity in the patients was 2-26 nmol/h/mg protein (the normal activity has been established in our laboratory as 111.9 +/- 7.1 nmol/h/mg protein). No correlation between enzyme activity and a clinical course of disease was revealed. The IVS2 + 1 mutation was found at 2 of 20 alleles (in a patient with late infantile form) and the P426L mutation was found at 2 of 20 alleles (in two patients with juvenile form). Thus, the total frequency of these two major mutations in the ARSA gene is 20% in Ukrainian MLD patients.     

Metachromatic leukodystrophy in the habbanite Jews: high frequency in a genetic isolate and screening for heterozygotes.

A very high incidence of late infantile metachromatic leukodystrophy (MLD) (1/75 live births) was found in the Jewish Habbanite community which constitutes a genetic isolate of about 1,000-1,200 individuals. Screening in this population for aryl sulfatase A (ASA) levels in married adults revealed a carrier frequency for MLD of 17% and identified six couples of whom both partners were heterozygotes (6% of screened couples). In three pregnancies of these couples, prenatal diagnosis for the detection of ASA in the fetus was performed.     

综述与专论: 异染性脑白质营养不良的研究进展

异染性脑白质营养不良（metachromatic leukodystrophy,MLD）是一种由芳基硫酸酯酶A（arylsulfatase A,ARSA）基因突变导致的罕见遗传性白质脑病。MLD的临床表现及疾病进展速度存在个体差异,但几乎所有的患者最终均会出现运动及认知功能完全丧失。临床上根据患者的发病年龄及病情严重程度分为晚婴型、青少年型及成人型。MLD的临床诊断包括进行性神经系统倒退及典型的头颅核磁共振成像（magnetic resonance imaging,MRI）表现。其临床表现与多种疾病类似,需与其他白质脑病和溶酶体贮积病进行鉴别。MLD暂无有效治疗方法,目前仅能对患者进行对症支持治疗。造血干细胞移植或骨髓移植、酶替代治疗和基因治疗是关于MLD治疗的研究热点。最近研究发现,鞘内注射重组人芳基硫酸酯酶A（rhASA）可延缓疾病的进展。针对MLD家系进行有效的产前分子诊断是预防MLD发生的主要方法。     

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.     

In vitro effect of schistosomicidal drugs on hepatic arylsulfatase B from the schistosoma-infected mouse

Arylsulfatase B (ASB) hydrolyzes the desulfation of N-acetylgalactosamine-4-sulfate at the non-reducing terminal of glycosaminoglycans. This enzyme activity was found to be elevated in mice schistosomiasis. In the present study, the catalytic and immunological properties of purified ASB from the liver of Schistosoma-infected mouse was investigated in the presence and absence of the schistosomicidal drugs praziquantel and Commiphora extract. The in vitro effect of praziquantel was found to be inhibitory with a Ki value of 5.5 x 10(-4) M while that of commiphora extract was as an activator. Furthermore, these drugs did not have an observed effect on the immunological properties of ASB with regard to its binding to its polyclonal rabbit antibody. These results indicate that some schistosomicidal drugs may reverse the alteration of the catalytic properties of the enzyme in schistosomiasis.     

Purification of lysosomal arylsulfatase B from rat liver and its reactivity with lectins in affinity immunoelectrophoresis.

1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.     

Biochemical,psychometric, and neuropsychological studies in heterozygotes for various lipidoses

Summary A sample of 38 clinically unaffected carriers for various lipidoses and their noncarrier relatives was studied with biochemical, psychological, and neuropsychological tests under blind conditions. The largest group of carriers was that for metachromatic leucodystrophy (MLD). The mean activity of arylsulphatase A or cerebroside sulphatase in the obligate carriers was 25%–30% of the control values, some heterozygotes showing little more activity than MLD patients. It was found that compared with the controls all heterozygotes (both obligate and facultative) differ unfavourably in some personality traits and in WISA subtests, including capacity for spatial cognition. These differences are especially obvious in a group of seven MLD carriers from the same family.With respect to reaction times, performance was significantly slower in MLD carriers, and particularly in those with enzyme activity lower than 30% of the control values.     

A noninvasive mucinous cystic neoplasm with intermediate-grade dysplasia of the pancreas and extensive squamous metaplasia: a case report with clinicopathological correlation

?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232

Patients and methods

We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent.

Results

In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria.

Conclusion

MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria.     

Reduced brain cholesterol content in arylsulfatase A-deficient mice

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by deficiency in arylsulfatase A (ASA). Concentrations of cholesterol and its metabolites were determined in ASA deficient [ASA(-/-)] mice which serve as an animal model of MLD. We observed a significant reduction in cholesterol content in the brain of adult ASA(-/-) mice when compared to wild-type controls. This was not due to loss of myelin, because ASA(-/-) mice do not demyelinate. Other cholesterol metabolites were not changed significantly in ASA(-/-) mice, except for an increase in lathosterol. Moreover, reduced cholesterol levels were also found in tissue samples from two juvenile MLD cases. Since high cholesterol levels are important for myelination, and various cellular processes, like vesicular trafficking and signal transduction, reduced cholesterol levels might be an important factor in the molecular pathology of MLD.     

Oligomerization capacity of two arylsulfatase A mutants: C300F and P425T

Arylsulfatase A (ARSA) is a lysosomal enzyme implicated in most cases of metachromatic leukodystrophy (MLD). The quaternary structure of ARSA is pH-dependent: at neutral pH, ARSA is a homodimeric protein; at lysosomal (acidic) pH, ARSA is homo-octameric. This dimer-octamer transition seems to be of major importance for the stability of the enzyme in the lysosomal milieu. Sedimentation analysis was used to study the oligomerization capacity of C300F and P425T-substituted ARSA, two MLD-associated forms of the enzyme displaying reduced lysosomal half-lives. P425T-ARSA displays a modest reduction in its octamerization capacity. In contrast, the C300F mutation strongly interferes with the octamerization process of ARSA but not with its dimerization capacity. Interestingly, a major fraction of dimeric ARSA-C300F is composed of covalently linked ARSA molecules, through a thiol-cleavable bond that probably involves Cys414 residues from each monomer. Our data support the notion that the reduced lysosomal half-life of some mutated forms of ARSA is related to deficient octamerization.     

Molecular genetic characterization of two metachromatic leukodystrophy patients who carry the T799G mutation and show different phenotypes; description of a novel null-type mutation

Metachromatic leukodystrophy (MLD) is an autosomal recessive storage disease caused by deficiency of the lysosomal enzyme, arylsulfatase A. Two common mutations causing MLD have been characterized and correlations between phenotype and genotype have been established. A third common mutation, T799G, has also been identified in European MLD patients, and is associated with the late-onset forms of the disease. We report the molecular analysis of two Italian MLD patients, with juvenile and adult onset of the disease, respectively, who carried the T799G mutation in compound heterozygosity with different null mutations. A novel rapid mutation detection method is demonstrated for patient screening. One patient has a novel mutation, a T556C transition that results in the substitution of Pro for Leu at codon 135, and produces no enzymatic activity in transfection experiments. Received: 5 November 1997 / Accepted: 12 January 1998     

Molecular characteristics in Japanese patients with lipidosis: Novel mutations in metachromatic leukodystrophy and Gaucher disease

The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease. Most frequent mutations among them was the 6433C mutation, 40% of 30 chromosomes, whereas the novel mutation of the 3548A found in Japanese patients with neuronopathic Gaucher disease was found in 20% (6 out of 30 chromosomes). The characteristics of these mutations in Japanese patients with Gaucher disease is different from those of Caucasian populations reported previously.     

Juvenile Sandhoff disease: some properties of the residual hexosaminidase in cultured fibroblasts.

The residual hexosaminidase isoenzymes in juvenile Sandhoff and infantile Sandhoff disease fibroblasts, have been determined by starch gel electrophoresis and column isoelectric focusing. Hex A and hex S are the major residual isozymes in fibroblasts from the juvenile patient, while hex B is barely detectable. Only hex S could be detected in fibroblasts from infantile Sandhoff patients. These results suggest that the defects in juvenile and infantile Sandhoff disease may be different allelic modifications of the beta subunit common to hex A and hex B.     

Lysosulfatide (Galactosylsphingosine-3-O-Sulfate) from Metachromatic Leukodystrophy and Normal Human Brain

The glycosphingolipid pattern was examined in three cases of late infantile metachromatic leukodystrophy (MLD): one with a relatively short (2.5 years), one with a long (7.8 years), and one with a very long (13.2 years) survival time. All values were compared with those of age-matched normal controls. The cerebroside concentration was reduced to 25, 12, and 4%, respectively, in the MLD white matter, whereas the sulfatide concentration was increased up to 200% of the control value. The yield of myelin was reduced to less than 15% in the early case and to less than 3 and 1%, respectively, in the two later cases. There was no sign of increased sulfatide proportion in the myelin. The ganglioside pattern was normal in cerebral gray matter, but in the white matter, contents of gangliosides of the lacto series were significantly increased, in particular, the ganglioside suggested by us as being characteristic of reactive astrocytosis. For the first time, lysosulfatide was identified in MLD and normal human brains by mass spectrometry and radioimmunoaffinity TLC using specific monoclonal antibody. Its quantity was found to be similar in normal and MLD brains. These findings support our postulation that the lysoglycosphingolipids are synthesized de novo from sphingosine and that they do not play a key role in pathogenetic mechanisms.     

Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency

Multiple-sulfatase deficiency (MSD) is now considered to be heterogeneous and could be classified into three or four clinical phenotypes according to the onset of the disease: neonatal, late infantile, juvenile and possibly adult type. Neonatal-type MSD shows severe clinical involvement and practically no arylsulfatase A, B and C activities in cultured skin fibroblasts. Furthermore, arylsulfatase A activity in neonatal-type MSD was not enhanced by the addition of thiosulfate. Therefore, it is distinct from late infantile-type MSD. The degradation of 14C-sulfatide can occur in MSD-cultured skin fibroblasts and was much higher than in late infantile-type MLD. The addition of thiol protease such as leupeptin to cultured MSD skin fibroblasts enhanced arylsulfatase A activity as well as the degradation of 14C-sulfatide. This suggests that the decreased activities of arylsulfatase A is due to an acceleration of the enzyme degradation. Enzyme replacement by the addition of arylsulfatases of different sources (human liver, brain, fungus) was carried out in cultured MSD skin fibroblasts. Human enzymes of arylsulfatase A and B were mostly taken up by MSD cells rather than those of fungus origin. By the exposure to leukocytes to cultured skin fibroblasts, MSD cells corrected arylsulfatase A and B activities.     

Mutation analyses in 17 patients with deficiency in acid beta-galactosidase: three novel point mutations and high correlation of mutation W273L with Morquio disease type B

An inherited deficiency in beta-galactosidase can result in GM1 gangliosidosis, with several phenotypes of generalized or chronic psychomotor deterioration, as well as in Morquio disease type B, a characteristic mucopolysaccharidosis free of neurological symptoms. We performed mutation analyses in 17 juvenile and adult patients from various European regions with a deficiency in beta-galactosidase and skeletal abnormalities. Fifteen of these had the Morquio B phenotype and have remained neurologically healthy until now while the two others exhibited psychomotor retardation of juvenile onset. A two-base substitution (851-852TG-->CT; W273L) was present in 14 of the 15 Morquio B cases. Even if one excludes alleles from patients with possible common descent, there was a much higher frequency (79%) among those with Morquio B phenotype for the W273L mutation than previously reported in the literature (37%). That the Morquio phenotype is also expressed in heterozygotes for W273L and alleles typically found in GM1 gangliosidosis makes it possible to predict the phenotype and reliably detect heterozygotes. A single French patient had a novel missense point mutation (Q408P) together with a known mutation (T500A) while the mentally retarded patients were both heterozygous for two mutations known in chronic GM1 gangliosidosis together with two novel missense point mutations (Y270D and H281Y) in the vicinity of W273L. Our results confirm the high impact of Trp 273 for the function of beta-galactosidase and the expression of the Morquio B phenotype. In addition, a second domain around the amino acids 400-500 may also be of significance.     

A new polymorphism of arylsulfatase A within the coding region

A 10-year-old boy with juvenile metachromatic leukodystrophy (MLD) presented with the 459 + 1G→A arylsulfatase A (ASA) mutation on one allele. To detect his complete genotype, the other ASA allele was sequenced and a T-to-C transition at nucleotide 376 in exon 2 was identified. This missense mutation results in a substitution of leucine 76 by proline. Of 20 MLD unrelated controls, 18 carried the L/P76 mutation either in the homozygous (n = 6) or heterozygous (n = 12) state. The presence or absence of L/P76 did not influence leukocyte ASA activity or urinary sulfatide excretion. Apparently, the substitution of leucine 76 by proline is a common ASA polymorphism, neither being related to MLD nor creating ASA pseudodeficiency. However, because of its frequency and location, L/P76 may be of particular importance in genetic studies requiring the differentiation of the ASA alleles within a kindred. Further studies are directed to the as yet unresolved genotype of the index case with juvenile MLD. Received: 5 March 1996 / Revised: 16 April 1996     

Fungal species diversity in juvenile and adult leaves of Eucalyptus globulus from plantations affected by Mycosphaerella leaf disease

In recent years, Mycosphaerella leaf disease (MLD) has become very common in Eucalyptus globulus plantations in Galicia, northwest Spain. The aetiology of MLD is complex and is associated with several species of Mycosphaerella and Teratosphaeria. A survey of the fungal mycobiota associated with juvenile and adult leaves and with leaf litter of the same trees in MLD‐affected plantations was made. The goal was to identify pathogens and endophytes, to determine whether the mycobiota of each leaf type differed and whether leaf litter might be a reservoir of MLD inoculum. Fungi belonging to 113 different species were isolated from the leaves of juvenile and adult trees sampled at 10 locations; 81 species occurred in juvenile and 65 in adult leaves. The average number of species obtained from juvenile leaves was significantly greater (P > 0.01) compared to adult leaves. This difference suggested that juvenile leaves are not only more susceptible to a group of pathogens, but to a wide range of fungi. Therefore, a general resistance mechanism might be lacking or be less effective in juvenile than in adult leaves. Several pathogenic species were identified in both leaf types. Leaf litter and living leaf mycobiotas were very different. However, some of the species they shared were MLD pathogens, suggesting that leaf litter could contribute to the inoculum of MLD.     

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.