Eduard Paschke

[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Publisher Summary This chapter presents procedure for enzymic diagnosis of genetic mucopolysaccharide storage disorders particularly two enzyme defects underlying the Morquio syndrome. For the diagnosis of the rare disorders, fibroblasts are the most convenient enzyme source because of the possibility of performing extensive investigations and because of the relatively high activity found in normal cells. However, enzymic diagnosis can be performed also on leukocytes. α-glucosaminide N-acetyltransferase can be measured in cultured fibroblasts or amniotic fluid cells, leukocytes, and tissues, which is based on the principle that states the substrate, a trisaccharide with the structure O- (α-D-2-amino-2-deoxyglucopyranosyl)-(1 →4)-O-(β-D-glucopyranosyluronicacid)-(1→4)-2,5-anhydro-D-[ 3 H]mannitol, prepared from heparin, can be N-acetylated by acetyl-CoA:α-glucosaminide N-acetyltransferase in the presence of acetyl-CoA. The product bearing an N-acetylated glucosamine residue at the nonreducing terminal, can be hydrolyzed by α-N acetylglucosaminidase to N-acetylglucosamine and a radioactive disaccharide, which can be further split by β-glucuronidase. The positively charged substrate is separated from the neutral or negatively charged products by passage over a cation-exchange resin.

Results of a Nationwide Screening for Anderson-Fabry Disease among Dialysis Patients

Anderson-Fabry disease is possibly underdiagnosed in patients with end-stage renal disease. Nationwide screening was therefore undertaken for Anderson-Fabry disease among dialysis patients in Austria. Screening for alpha-galactosidase A (AGAL) deficiency was performed by a blood spot test. In patients with a positive screening test, AGAL activity in leukocytes was determined. Individuals with decreased leukocyte AGAL activity were subjected to mutation testing in the GLA gene. Fifty (90.9%) of 55 Austrian hemodialysis centers participated in this study; 2480 dialysis patients (80.1% of the Austrian dialysis population) were screened. In 85 patients, the screening test was positive (85 of 2480, 3.42%; women, 3.32%; men, 3.50%). Among these 85 patients, 4 men (in 3 of whom Anderson-Fabry disease was already known before screening) had a severely decreased and 11 subjects had a borderline low AGAL activity. Genetic testing revealed mutations associated with Fabry disease in all four men with severely decreased AGAL activity resulting in a prevalence of 0.161% for the entire study population. A nationwide screening of dialysis patients permitted detection of a hitherto unknown man with Anderson-Fabry disease. The overall prevalence among dialysis patients was at least ten times higher as compared with recent registry data. Screening programs among patients with end-stage renal disease, especially men, should be put in place to identify families with Anderson-Fabry disease who probably may benefit from specific clinical care, and perhaps from enzyme replacement therapy. In dialysis patients, however, there is no evidence to support enzyme replacement therapy at present.

Heparan Sulfate Levels in Mucopolysaccharidoses and Mucolipidoses

SummaryGlycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Genetic and phenotypic heterogeneity in disorders of peroxisome biogenesis--a complementation study involving cell lines from 19 patients.

ABSTRACT: Disorders of peroxisomal biogenesis include the Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum syndrome, and hyperpipecolic acidemia. These names were assigned before the recognition of the peroxisomal defect and the distinction between phenotypes is uncertain. Recent studies have identified at least four complementation groups, and indicate the presence of at least that number of distinct genotypes. The purpose of the present study was to examine the relationship between genotype and phenotype. We studied cultured skin fibroblasts from 19 patients in whom deficiency of peroxisomes had been established. Complementation analysis was performed with the criterion of complementation being the restoration of the capacity to synthesize plasmalogens when fibroblasts from two patients were fused. Six complementation groups were identified, and consisted of one 13 member group, one two member group, and four groups comprising single cases. The phenotype of each group was examined with respect to age of survival, clinical manifestations, and biochemical alterations. The 13 member group included patients with all of the four currently designated phenotypic entities, while the most common phenotype (Zellweger syndrome) was distributed among five of the six groups. We conclude that the currently used clinical categories do not represent distinct genotypes. Apparently different genes code for a similar phenotype and one defective gene may lead to variant phenotypes. Definitive classification and understanding of these disorders await definition of the specific biochemical defect in each of the genotypes.

Pipecolic acid elevation in plasma and cerebrospinal fluid of two patients with pyridoxine-dependent epilepsy

Diagnosis of pyridoxine‐dependent epilepsy is based on the clinical response to high‐dosage application of pyridoxine. Here, we report on 2 patients with pyridoxine‐dependent epilepsy with significant elevation of pipecolic acid concentrations in plasma and cerebrospinal fluid (CSF) and further increase of pipecolic acid in CSF during a 72‐hour pyridoxine withdrawal in 1 of them. Patients with non–pyridoxine‐dependent epilepsy had normal pipecolic acid concentrations in plasma and significantly lower concentrations in CSF. High plasma and CSF pipecolic acid concentrations might provide a diagnostic marker in pyridoxine‐dependent epilepsy. Ann Neurol 2000;48:121–125

Development and Testing of New Screening Method for Keratan Sulfate in Mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA), a progressive lysosomal storage disease, causes skeletal dysplasia through excessive storage of keratan sulfate (KS). We developed an ELISA-sandwich assay that used a MAb specific to KS. Forty-five blood and 59 urine specimens from MPS IVA patients (ages 1–65 y) were analyzed to determine whether KS concentration is a suitable marker for early diagnosis and longitudinal assessment of disease severity. Blood specimens were obtained from patients categorized as phenotypically severe (n = 36) and milder (n = 9). Urine specimens were also analyzed from patients categorized as severe (n = 56) and milder (n = 12), respectively. Blood KS levels (101–1525 ng/mL) in MPS IVA patients were two to eight times higher than those in age-matched controls (15–323 ng/mL). It was found that blood KS level varied with age and clinical severity. Blood KS levels in both MPS IVA and controls peaked between 5 and 10 y of age (mean, 776 versus 234 ng/mL, respectively). Blood levels in severe MPS IVA were 1.5 times higher than in the milder form. In contrast to blood, urine KS levels in both MPS IVA and controls peaked between 1 and 5 y (15.3 versus 0.26 mg/g creatinine), and thereafter declined with age. Urine KS level also varied with age and clinical severity, and the severe MPS IVA phenotype was associated with 6.7 times greater urine KS excretion than the milder one. These findings indicate that the new assay for blood or urine KS may be suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Dystonia and parkinsonism in GM1 type 3 gangliosidosis.

GM1 gangliosidosis is due to β‐galactosidase deficiency. Only patients with type 3 disease survive into adulthood and develop movement disorders. Clinical descriptions of this form are rare, particularly in non‐Japanese patients. We describe four new patients and systematically analyze all previous reports found by a literature search and contacts with the authors for additional information. Generalized dystonia remained the predominant feature throughout the disease course and was often associated with akinetic–rigid parkinsonism. GM1 gangliosidosis must be considered as a cause of early‐onset generalized dystonia, particularly in patients with short stature and skeletal dysplasia.

Pyridoxine responsiveness in novel mutations of the PNPO gene

Objective: To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations. Methods: We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria. Results: We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound heterozygous for a novel missense mutation p.Arg141Cys in exon 5 and a deletion c.279_290del in exon 3. Pathogenicity of the respective mutations was proven by absence in 100 control alleles and expression studies in CHO-K1 cell lines. The response to pyridoxine was prompt in 4, delayed in 2, on EEG only in 2, and initially absent in another 2 patients. Two unrelated patients homozygous for the p.Arg225His mutation experienced status epilepticus when switched to pyridoxal 5′-phosphate (PLP). Conclusions: This study challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5′-phosphate oxidase deficiency and underlines the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug–resistant seizures. Patients with pyridoxine response but normal biomarkers for antiquitin deficiency should undergo PNPO mutation analysis.

Fluorous iminoalditols: a new family of glycosidase inhibitors and pharmacological chaperones.

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N‐substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D‐galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D‐galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.

DLHex-DGJ, a novel derivative of 1-deoxygalactonojirimycin with pharmacological chaperone activity in human GM1-gangliosidosis fibroblasts

G(M1)-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid beta-galactosidase (beta-Gal) degrades two functionally important molecules, G(M1)-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable beta-Gal precursors, normally transported and processed to mature, intralysosomal beta-Gal. In accordance with the MBD phenotype, elevated residual activity against G(M1)-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor proteins rapidly degraded by endoplasmic reticulum-associated protein degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid beta-galactosidase in vitro, and describes its effects on activity, protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of beta-Gal precursors.