Leukocyte Sulfatidase for the Reliable Diagnosis of Metachromatic Leukodystrophy

A simple assay technique for the determination of sulfatidase activity in leukocytes has been developed for the reliable diagnosis of metachromatic leukodystrophy (MLD). Sulfatide is tritiated in sphingosine and fatty acid by reduction with [3H]sodium borohydride in alkali in the presence of palladium chloride. This labeled natural substrate for aryl sulfatase A (AsA) is hydrolyzed by normal human leukocytes in 25 mM-acetate buffer, pH 5.0, in the presence of 0.3% sodium taurodeoxycholate. The enzyme activity is greatly improved after dialysis, exhibiting better linearity with protein concentration. It is stimulated maximally by 5 mM-MnCl2 with an apparent Km of 0.17 mM for the substrate. Patients with MLD exhibited virtually no detectable sulfatidase activity although they had residual AsA activity that was measured with the synthetic substrate, p-nitrocatechol sulfate (NCS). Potential heterozygotes could be identified by the sulfatidase assay in instances where the NCS assay for AsA was inconclusive. Several individuals with levels of AsA activity characteristic of MLD, including a few healthy carriers and certain patients with unknown neurological diseases, were shown not to have MLD by the presence of measurable levels of sulfatidase in their leukocytes.     

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.     

Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy

We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep-Pak column, derivatization with o-phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9-35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (approximately 2 pmol/mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223-1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3-45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.     

Exocytosis of storage material in a lysosomal disorder

Lysosomal exocytosis is a ubiquitously occurring process, which has a physiological role in repair of wounds of the plasma membrane. Lysosomal storage disorders are a group of more than 40 different diseases, which are characterized by intralysosomal storage of various substances. Metachromatic leukodystrophy is a lysosomal disease caused by the deficiency of arylsulfatase A, which results in the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in, e.g., oligodendrocytes and distal tubule kidney cells. Here we show that sulfatide storing cultured primary kidney cells of arylsulfatase A deficient mice can undergo calcium induced lysosomal exocytosis and that this results in the delivery of storage material to the culture medium. In metachromatic leukodystrophy extracellular sulfatide has been found in urine and cerebrospinal fluid. Lysosomal exocytosis may explain the presence of sulfatide in these body fluids.     

Synthetic sulfogalactosylceramide (sulfatide) and its use for the mass spectrometric quantitative urinary determination in metachromatic leukodystrophies

3-O-Sulfogalactosylceramides (sulfatides) accumulate in the genetic disease metachromatic leukodystrophy which is due to a defect in the catabolic enzyme, arylsulfatase A. Clinical diagnosis is usually confirmed by in vitro enzymatic deficiency of arylsulfatase A activity. The diagnosis may be complicated because of arylsulfatase A pseudo-deficiencies and another cause of MLD, sphingolipid activator B deficiency. As large quantities of sulfatides can be found in the urine in this disease, sulfatiduria appears as an extremely useful test. As recently enzyme replacement is underway, the quantitative determination, using an internal standard, appears particularly useful as a follow-up. Thus a non-physiological sulfatide was synthesized for this purpose, i.e. 3-O-sulfo-β-d-C17 galactosylceramide (3-O-Sulfo-d-Galactosyl-β1′→1-N-Heptadecanoyl-d-erythro-Sphingosine). It has been prepared through condensation of an azidosphingosine derivative with a protected d-galactopyranosyltrichloroacetimidate. Reduction of the azide was followed by acylation of a C-17 fatty acid. The key step was achieved by selective sulfation of the desired hydroxyl group on the sugar residue of the galactosylceramide using the stannylene methodology to give a 3′-sulfated beta-galactosyl C-17 ceramide. An erratum to this article can be found at     

Three Novel Mutant Arylsulfatase A Alleles Causing Metachromatic Leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. This leads to the accumulation of 3-O-sulfogalactosylceramide, which results in severe demyelination. Here we describe a novel non-sense mutation W124ter and two disease-causing missense mutations E382Q and C500F in arylsulfatase A gene. Another so far unknown allele harbors three sequence alterations: two polymorphisms (N350S, R496H) and a missense mutation (R288H). The R288H substitution and the N350S polymorphism have previously been found on one allele together with a polymorphism in a polyadenylation signal characteristic for the arylsulfatase A pseudodeficiency allele. The R496H has been shown to occur on another allele. The presence of the R288H, N350S, and R496H substitution on one allele in the absence of the polyadenylation site polymorphism shows that this allele has probably arisen by recombination between the nucleotides of codon 350 and 496.     

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.     

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。     

Critical issues for the proper diagnosis of Metachromatic Leukodystrophy

Metachromatic Leukodystrophy is a lysosomal storage disorder caused by Arylsulfatase A deficiency. Diagnosis is usually performed by measurement of enzymatic activity and/or characterization of the gene mutations. Here we describe a family case in which the determination of enzyme activity alone did not allow diagnosis of the pre-symptomatic sibling of the index case. Only combination of gene sequencing with thorough biochemical analysis allowed the correct diagnosis of the sibling, who was promptly directed to treatment.     

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.     

Alexander Disease: A Leukodystrophy Caused by a Mutation in GFAP

Alexander disease, a rare fatal disorder of the central nervous system, causes progressive loss of motor and mental function. Until recently it was of unknown etiology, almost all cases were sporadic, and there was no effective treatment. It was most common in an infantile form, somewhat less so in a juvenile form, and was rarely seen in an adult-onset form. A number of investigators have now shown that almost all cases of Alexander disease have a dominant mutation in one allele of the gene for glial fibrillary acidic protein (GFAP) that causes replacement of one amino acid for another. Only in very rare cases of the adult-onset form is the mutation present in either parent. Thus, in almost all cases, the mutation arises as a spontaneous event, possibly in the germ cell of one parent.     

Brain Galactolipid Content in a Patient with Pseudo-Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno-, and Other Leukodystrophies

A 4-year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudoarylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachromatic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodystrophy. A general mechanism of this anomaly in severe demyelination is considered.     

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

Prenatal origins of brachymesophalangia-5

As shown in 91 embryos and fetuses in the 41–235 mm crown-rump length, brachymesophalangia-5 may be identified in prenatal time. Although the ratios involving different combinations of length of mid-5 relative to other segments yield different frequencies of BMP-5, the short middle segment of the little finger is already associated with clinodactyly and developmental eccentricity as early as the ninth week of prenatal development.     

Prenatal selection and dermatoglyphic patterns

Although human dermatoglyphics have been extensively studied, little is known of the prenatal origins of dermatoglyphic patterns. Digital patterns, i.e., loops, whorls, and arches, were obtained from 81 human abortuses, ranging in age from 11 to 25 weeks post-fertilization. Patterns were discernible with the earliest indications of epidermal ridge development. Findings indicate that pattern frequencies during early prenatal development differ from those of later fetal and postnatal periods. Furthermore, a high frequency of arches is seen associated with spontaneous abortion, suggesting evidence for prenatal selection in human abortuses.     

黑龙江地区466例孕妇羊水穿刺产前诊断结果分析

目的:探讨羊水细胞染色体异常核型与各产前诊断之间的关系。方法:466例高危孕妇行羊膜腔穿刺术后羊水细胞培养及染色体核型分析。结果:异常核型66例,异常率14.16%,包括染色体数目异常27例,三体综合征22例(21-三体15例、18-三体6例、13-三体1例),占异常染色体核型的33.33%,占染色体数目异常的81.48%;染色体结构异常39例,主要包括染色体多态性、平衡易位、倒位和衍生等,占染色体异常核型的59.10%。异常核型检出率中血清学筛查高危组(14.44%)要高于高龄妊娠组(10.89%)和有不良孕产史组(11.11%)(P0.05);超声提示胎儿发育异常组(23.26%)要高于血清筛查高危组(P0.05)。结论:血清筛查高危和超声提示胎儿发育异常是黑龙江地区最主要的产前诊断指征,异常核型以21-三体综合征检出率最高。通过对高危孕妇羊水细胞染色体的核型分析可发现部分染色体疾病,从而避免此类出生缺陷儿的出生。     

Effects of Prenatal Phenobarbital on Benzodiazepine Receptor Development

Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.     

Prenatal detection of a paracentric inversion 16(q11.2q13)

We report the prenatal detection of an inherited paracentric inversion 16(q11.2q13).