Glycogen storage disease type 0 (GSD-0) is a rare form of fasting hypoglycemia presenting in infancy or early childhood and accompanied by high blood ketones and low alanine and lactate concentrations. Although feeding relieves symptoms, it often results in postprandial hyperglycemia and hyperlactatemia. The glycogen synthase (GS) activity has been low or immeasurable in liver biopsies, whereas the liver glycogen content has been only moderately decreased. To investigate whether mutations in the liver GS gene ( GYS2 ) on chromosome 12p12.2 were involved in GSD-0, we determined the exon-intron structure of the GYS2 gene and examined nine affected children from five families for linkage of GSD-0 to the GYS2 gene. Mutation screening of the 16 GYS2 exons was done by single-strand conformational polymorphism (SSCP) and direct sequencing. Liver GS deficiency was diagnosed from liver biopsies (GS activity and glycogen content). GS activity in the liver of the affected children was extremely low or nil, resulting in subnormal glycogen content. After suggestive linkage to the GYS2 gene had been established (LOD score ϭ 2.9; P Ͻ 0.01), mutation screening revealed several different mutations in these families, including a premature stop codon in exon 5 (Arg246X), a 5 Ј -donor splice site mutation in intron 6 (G

Patients with glycogen storage disease type II (GSDII, Pompe disease) suffer from progressive muscle weakness due to acid alpha-glucosidase deficiency. The disease is inherited as an autosomal recessive trait with a spectrum of clinical phenotypes. We have investigated 29 cases of GSDII and thereby identified 55 pathogenic mutations of the acid alpha-glucosidase gene (GAA) encoding acid maltase. There were 34 different mutations identified, 22 of which were novel. All of the missense mutations and two other mutations with an unpredictable effect on acid alpha-glucosidase synthesis and function were transiently expressed in COS cells. The effect of a novel splice-site mutation was investigated by real-time PCR analysis. The outcome of our analysis underscores the notion that the clinical phenotype of GSDII is largely dictated by the nature of the mutations in the GAA alleles. This genotype-phenotype correlation makes DNA analysis a valuable tool to help predict the clinical course of the disease.

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (alpha-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded alpha-Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of alpha-Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant alpha-Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant alpha-Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy.

A summary review of leukocyte function in 42 published cases of glycogen storage disease Ib is presented. Polymorphonuclear and monocyte dysfunctions were evidenced in the majority of cases, whereas lymphocytes appeared to be unaffected. Phagocyte dysfunctions comprised in vivo mobilization and motility, in vitro random and directed migration, and one or several component functions of the "metabolic" ("respiratory") burst. On the basis of the available data it is impossible to know whether a primary functional deficit of the glucose 6-phosphate transport protein of the microsomal glucose 6-phosphatase system, as demonstrated in liver, also exists in these phagocytic cells and is responsible for this dysfunction.

Galactokinase deficiency is an inborn error in the first step of galactose metabolism. Its major clinical manifestation is the development of cataracts in the first weeks of life. It has also been suggested that carriers of the deficiency are predisposed to presenile cataracts developing at age 20-50 years. Newborn screening data suggest that the gene frequency is very low worldwide but is higher among the Roma in Europe. Since the cloning of the galactokinase gene (GK1) in 1995, only two disease-causing mutations, both confined to single families, have been identified. Here we present the results of a study of six affected Romani families from Bulgaria, where index patients with galactokinase deficiency have been detected by the mass screening. Genetic linkage mapping placed the disease locus on 17q, and haplotype analysis revealed a small conserved region of homozygosity. Using radiation hybrid mapping, we have shown that GK1 is located in this region. The founder Romani mutation identified in this study is a single nucleotide substitution in GK1 resulting in the replacement of the conserved proline residue at amino acid position 28 with threonine (P28T). The P28T carrier rate in this endogamous population is approximately 5%, suggesting that the mutation may be an important cause of early childhood blindness in countries with a sizeable Roma minority.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.

Early diagnosis of Danon disease is important for genetic counselling and timely cardiac transplantation, the only effective therapeutic option.

Abstract.A male cat 12-1 4 weeks old had walking difficulties and an enlarged abdomen. Facial dysmorphism, plump paws, corneal clouding, granulation of neutrophils, vacuolated lymphocytes, and a positive urine test for sulfated glycosaminoglycans suggested mucopolysaccharidosis. Cultured fibroblasts incorporated %O, into mucopolysaccharides more actively than did fibroblasts of a feline control, and degradation was far inferior. Activity of P-glucuronidase was absent in leukocytes and markedly reduced in fibroblasts, thus establishing the diagnosis of mucopolysaccharidosis VII, a disorder previously described in humans, dogs, and mice. Light microscopic examination revealed foam cells in virtually all organs examined, and electron microscopic examination showed pancytic storage of floccular material characteristic of mucopolysaccharides. Stored sphingolipids in the form of zebra bodies were seen in ganglion cells of the central nervous system and in smooth muscle cells of blood vessels. This case represents another animal model of mucopolysaccharidosis VII with the full disease characteristics known in human patients.Kqv words: P-glucuronidase deficiency; cats; lysosomal storage disease; mucopolysaccharidosis type VII.Mucopolysaccharidoses (MPS) are lysosomal storage disorders, each caused by the deficiency of a lysosomal hydrolase needed for the degradation of mucopolysaccharide (glycosaminoglycan). In humans, different mucopolysaccharidoses (MPS I-VII) have been attributed to specific enzyme defects and hence to the type of undegraded mucopolysaccharides,20 i.e., chondroitin sulfate, dermatan sulfate, heparan sulfate, or keratan sulfate. Animal models for these human disorders are k n~w n . '~.~~ In cats, MPS I (a-iduronidase deficiency) and MPS VI (arylsulfatase B deficiency) have been described,I4 but until now MPS VII (p-glucuronidase deficiency) has only been seen in dogslo.' * , I 3 and m i~e .~.~~ Here, we present the first instance of MPS VII in a cat. Case ReportA black domestic short-haired male cat was examined at age 12-14 weeks for walking difficulty and an enlarged abdomen. He was small for his age, active, attentive, and showed intense licking but otherwise behaved normally. His face was broad, his cheeks were high, and his nose was short. Frontal bossing, corneal clouding, plump front paws with inside rotation, and mild thickening of the skin especially over the paws were also noticed. His ears were small and their tips were distorted. His tongue was lengthened, his abdomen was extended, and his lower thoracic opening was funnel shaped. He walked with his weight shifted to his front paws. Mucopolysaccharidosis was suspected, and a toluidine test for sulfated glycosaminoglycans in urine was positive. When X-ray findings and the excessive granulation of neutrophil granulocytes and intense vacuolation of lymphocytes were seen, biochemical analysis was initiated and P-glucuronidase deficiency was discovered.The clinical course was progressive. The rear legs exhibited reduced proprioceptivity and no ...