In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.     

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.     

Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity

The occurrence of different mutations on the same arylsulfatase A allele is not uncommon, due to the high frequency of several variants, among which the pseudodeficiency mutations are particularly important. We identified a late infantile metachromatic leukodystrophy patient carrying on one allele the new E253K mutation and the known T391S polymorphism, and on the other allele the common P426L mutation, usually associated with the adult or juvenile form of the disease, and the N350S and *96A>G pseudodeficiency mutations. To analyze the contribution of mutations located on the same allele to enzyme activity reduction, as well as the possible phenotype implications, we performed transient expression experiments using arylsulfatase A cDNAs carrying the identified mutations separately and in combination. Our results indicate that mutants containing multiple mutations cause a greater reduction of ARSA activity than do the corresponding single mutants, the total deficiency likely corresponding to the sum of the reductions attributed to each mutation. Consequently, each mutation may contribute to ARSA activity reduction, and, therefore, to the degree of disease severity. This is particularly important for the alleles containing a disease-causing mutation and the pseudodeficiency mutations: in these alleles pseudodeficiency could play a role in affecting the clinical phenotype.     

A patient presenting a 22q13 deletion associated with an apparently balanced translocation t(16;22): An illustrative case in the investigation of patients with low ARSA activity

A 10-year-old speechless, mentally deficient male, with low arylsulfatase A (ARSA) activity, and presumably, methachromatic leukodystrophy, underwent genetic evaluation. As the clinical picture was not compatible with this diagnosisan ARSA gene and chromosome analysis were performed, showing the presence of a pseudodeficiency ARSA allele and a de novo apparently balanced t(16;22)(p11.2;q13) translocation. A deletion on the long arm of chromosome 22 encompassing the ARSA gene, as shown by FISH and array-CGH, indicated a 22q13 deletion syndrome. This case illustrates the importance of detailed cytogenetic investigation in patients presenting low arylsulfatase A activity and atypical/unspecific clinical features.     

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。     

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.     

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.     

Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy.

We identified a patient suffering from late infantile metachromatic leukodystrophy who genetically seemed to be homozygous for the mutations signifying the arylsulfatase A pseudodeficiency allele. Homozygosity for the pseudodeficiency allele is associated with low arylsulfatase A activity but does not cause a disease. Analysis of the arylsulfatase A gene in this patient revealed a C----T transition in exon 2, causing a Ser 96----Phe substitution in addition to the sequence alterations causing arylsulfatase A pseudodeficiency. Although this mutation was found only in 1 of 78 metachromatic leukodystrophy patients tested, five more patients were identified who seemed hetero- or homozygous for the pseudodeficiency allele. The existence of nonfunctional arylsulfatase A alleles derived from the pseudodeficiency allele calls for caution when the diagnosis of arylsulfatase A pseudodeficiency is based solely on the identification of the mutations characterizing the pseudodeficiency allele.     

Homozygote for mutation c.1204 + 1G > A of the ARSA gene presents with a late-infantile form of metachromatic leukodystrophy and a rare MRI white matter lesion type

The metachromatic leukodystrophy (MLD)--causing mutation c.1204 + 1G > A damages an intron-exon splice site recognition sequence. This results in a complete loss of enzymatic activity of arylsulfatase A (ARSA) protein molecules. We have found a late-infantile type MLD-patient to be homozygous for this mutation, which was not reported earlier, but is consistent with previous suggestions. Interestingly, the cerebral magnetic resonance imaging (MRI) in this patient displayed linear or punctuate structures radiating in the demyelinated white matter, which resembled the patterns described in Pelizaeus-Merzbacher disease. It should be emphasised that whenever a cerebral MRI demonstrates the "tigroid" or "leopard-skin" demyelination pattern not only Pelizaeus-Merzbacher disease, but also metachromatic leukodystrophy diagnosis should be considered; this suggests the necessity of ARSA activity estimations in patients with such specific MRI patterns.     

Apolipoprotein E genotype and LRP1 polymorphisms in patients with different clinical types of metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is a severe, neurodegenerative, metabolic disease which is caused by deficient activity of arylsulfatase A (ARSA). Sulfatides and other substrates of ARSA are stored in central and peripheral nervous systems, and in some other organs. Accumulated sulfatides are especially toxic to oligodendrocytes and Schwann cells leading to progressive demyelination. The kind of apolipoprotein E (apoE) isoform is of essential significance for the modulation of sulfatide quantity in the brain as apoE4 contains more sulfatides than apoE3. Taking into consideration the fact that apoE4 leads to the loss of sulfatides in CSF of Alzheimer's disease patients, we examined if apoE isoforms display any impact on clinical outcome in patients with different forms of MLD in whom sulfatides accumulate. The significant association of age at the onset of MLD symptoms with APOE ε2/ε3/ε4 and LRP1 c.766C>T polymorphisms was shown in multivariate stepwise regression analysis, in which other factors known to affect age at onset of the disease, i.e. clinical type of MLD, family connection of the patient and sex were also analyzed. As expected, the clinical type of MLD explained about 80% of the variance of the dependent variable. The impact of both polymorphisms on age of onset of the disease was considerably lower: 2.0% in the case of APOE polymorphism and 1.0% in the case of LRP1 polymorphism. Thus, the clinical outcome in MLD patients is related principally to the genotype of the ARSA gene, while the polymorphisms in the APOE and LRP1 genes are only slightly modifying factors.     

The genetics of the aryl sulfatase a locus

A genetic analysis was performed in an isolate in which metachromatic leukodystrophy (MLD) and aryl sulfatase A (ASA) pseudodeficiency are relatively frequent. The frequency of matings at risk and the frequency of ASA pseudodeficiency among parents of MLD patients are compatible with allelism between the gene determining MLD and the gene determining ASA pseudodeficiency. Two independent pedigrees including MLD patients and ASA-deficient healthy individuals also fit the model of allelism.     

Pseudo arylsulfatase a deficiency: Evidence for a structurally altered enzyme

Analysis of arylsulfatase A from pseudo arylsulfatase A deficiency fibroblasts by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoradiochemical nitrocellulose blot radiography revealed two subunit bands which migrated faster than subunit bands of enzyme from normal fibroblasts. Immunoreactive material was present only at levels comparable to enzyme activity. These findings imply that arylsulfatase A in pseudodeficiency is structurally altered, but it is catalytically equivalent to normal arylsulfatase A. This altered enzyme must be the product of the pseudodeficiency gene since no immunoreactive product of the metachromatic leukodystrophy gene could be detected in metachromatic leukodystrophy cells by the procedure employed. It is not clear from the present data if the attenuated arylsulfatase A activity in pseudodeficiency results from a decreased rate of synthesis or an increased lability of the mutant enzyme.     

An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy

Summary Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). A substantial ASA deficiency has also been described in clinically healthy persons, a condition for which the term pseudodeficiency was introduced. The discrimination of both kinds of deficiencies based on ASA activity determination is difficult and unreliable. This creates a serious problem in the genetic counseling and diagnosis of MLD. The mutations characteristic for the pseudodeficiency (PD) allele have recently been identified. A non-radioactive assay based on the polymerase chain reaction is described, which allows the rapid detection of the ASA pd allele. The assay utilizes pairs of primers that allow either the amplification of the ASA PD allele or of other ASA alleles, since their 3 residues match either the ASA PD allele or other ASA alleles.     

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     

Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease

Several allelic mutations at the arylsulfatase A (ASA) locus cause substantial deficiencies of this lysosomal enzyme. Depending on the genetically determined degree of the deficiency, the clinical outcome may be very different—either metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder affecting the nervous system, or, more frequently, the so-called pseudodeficiency (PD), which has no apparent clinical consequence. Because of compound heterozygosity for MLD and PD, 1/1,000 individuals in the population have low residual enzyme activities, which are intermediate between those of MLD patients and those of PD homozygous normal individuals. In order to assess whether PD/MLD compound heterozygotes bear a health risk, we examined clinically and biochemically 16 individuals with this genotype. Of these subjects, two had neurological symptoms and two showed lesions, without clinical symptoms, in magnetic resonance imaging of the brain. None of these symptoms was progressive, nor did they resemble those of MLD. Nerve conduction velocities were normal in these probands, and they secreted only low amounts of sulfatide in the urine. We conclude that the observed neurological symptoms are unrelated to the ASA genotype and that PD/MLD compound heterozygotes are not at an increased risk for developing progressive nervous system diseases.     

Arylsulfatase and beta-glucuronidase expression in green sunfish,bluegill, and their reciprocal interspecific hybrids

Fish arylsulfatases (arylsulfate sulfohydrolase; EC 3.1.6.1) were resolved into cationic arylsulfatase A-like (ARSA) and anionic arylsulfatase B-like (ARSB) fractions by DEAE-Sephacel chromatography. Green sunfish (GSF) hepatic ARSA was more acidic and more thermostable than bluegill (BG) ARSA. GSF × BG interspecific hybrids preferentially expressed GSF ARSA, while BG × GSF hybrids appeared to produce a dimeric enzyme consisting of both GSF and BG ARSA polypeptides. GSF hepatic betaglucuronidase (GUS) also proved to be more thermostable than BG GUS. Thermostabilities of GUS produced by reciprocal interspecific hybrids were very similar to that of GSF GUS. Either GSF GUS is preferentially expressed in both interspecific hybrids or both the GSF and BG GUS polypeptides are synthesized in comparable amounts, and the GSF GUS polypeptide sufficiently stabilizes the heterotetramers produced by the hybrids to produce denaturation profiles closely approximating that of the GSF enzyme.     

A 9-bp deletion (2320del9) on the background of the arylsulfatase A pseudodeficiency allele in a metachromatic leukodystrophy patient and in a patient with nonprogressive neurological symptoms

A 9-bp deletion (2320del9) was detected in the arylsulfatase A genes of a patient with late infantile metachromatic leukodystrophy and of a patient with nonprogressive neurological symptoms and very low arylsulfatase A activity. Both patients are heterozygous for the deletion, which involves codons 406–408 and causes loss of a Ser-Asp-Thr tract in the predicted protein. In both patients the 9-bp deletion lies in a pseudodeficiency allele. The patient with metachromatic leukodystrophy carries the common 459 + 1G > A mutation in the other allele. The other patient is homozygous for the pseudodeficiency allele, and consequently is a compound heterozygote for a metachromatic leukodystrophy allele and a pseudodeficiency allele. We hypothesize that the compound heterozygosity predisposes to the development of nonprogressive neurological symptoms in the presence of additional, still unknown, genetic or nongenetic factors. Received: 18 April 1997 / Accepted: 16 August 1997     

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.