A novel GLA mutation in a Fabry family with glucose-6-phosphate dehydrogenase deficiency.

Fabry disease is an X-linked lysosomal disease caused by mutations of the alpha-galactosidase A (GLA) gene at chromosome subband Xq22.1. To date, more than 600 genetic mutations have been identified to determine the nature and frequency of the molecular lesions causing the classical and milder variant phenotypes and for precise carrier detection. We report here a Fabry family (mother, son and daughter) where the alpha-galactosidase A defect was associated with a glucose-6-phosphate dehydrogenase (G6PD) deficiency. Mutation analysis revealed for the GLA gene the presence of a new mutation, i.e., a small deletion (c.452delA) on exon 3 and for the G6PD gene the presence of 2 mutations, p.V68M (G6PD Asahi, G6PD A+) and p.N126D (G6PD A+) on exon 3 and exon 4, respectively.

Large deletion involving exon 5 of the arylsulfatase B gene caused apparent homozygosity in a mucopolysaccharidosis type VI patient.

Apparent homozygosity for the mutation p.R315X present on exon 5 of the arylsulfatase B (ARSB) gene in a mucopolysaccharidosis type VI patient was solved in this study by further testing for a second mutation. Patient cDNA analysis revealed that the entire exon 5 of the ARSB gene was lacking; this new mutation was identified as c.899-1142del. As the genomic DNA sequencing excluded the presence of splicing mutations, polymerase chain reaction analysis was performed for polymorphisms listed in the NCBI SNP database for the ARSB gene. This allowed the mutation at the genomic DNA level to be identified as g.99367-102002del; this gross deletion, involving the entire exon 5 of the gene and parts of introns 4 and 5 led to a frameshift starting at amino acid 300 and resulting in a protein with 39% amino acids different from the normal enzyme. We stress that extensive DNA analysis needs to be performed in case of apparent homozygosity to avoid potential errors in genetic counseling.

Molecular markers for the follow-up of enzyme-replacement therapy in mucopolysaccharidosis type VI disease.

MPS VI (mucopolysaccharidosis type VI) is a lysosomal storage disease in which deficient activity of the enzyme N-acetylgalactosamine 4-sulfatase [ASB (arylsulfatase B)] impairs the stepwise degradation of the GAG (glycosaminoglycan) dermatan sulfate. Clinical studies of ERT (enzyme replacement therapy) by using rhASB (recombinant human ASB) have been reported with promising results. The release of GAG into the urine is currently used as a biomarker of disease, reflecting in some cases disease severity and in all cases therapeutic responsiveness. Using RNA studies in four Italian patients undergoing ERT, we observed that TNFalpha (tumour necrosis factor alpha) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFalpha expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.

Modulation of chondrocyte adhesion to collagen by echistatin.

Primary chondrocytes from quail embryo epiphysis (quail epiphyseal chondrocytes, QEC) can grow either in suspension or in monolayer. In this study, the adhesion of QEC to collagen II was used as a model to study the regulation of the ligand-binding activity of integrin receptors that allows these cells to undergo a rapid transition from suspension to an adherent state. Preincubation of suspension QEC (QECSP) with the disintegrin echistatin increased by 40% their adhesion to collagen II. An inverse relationship between immobilized collagen density and echistatin-induced increase of chondrocyte adhesion was observed, thus suggesting that the disintegrin acts by increasing the ligand-binding affinity of collagen receptor(s). Further, echistatin activity does not appear to depend upon a direct binding of the disintegrin to collagen receptor(s). In fact, immobilized anti-beta1 antibodies, but not immobilized echistatin, served as effective binding sites for QECSP. Echistatin failed to stimulate chondrocyte adhesion to collagen in the presence of metabolic inhibitors, while an activating anti-beta1 antibody was still effective. Thus, echistatin may promote cell adhesion by interfering with energy-dependent signals that keep the collagen receptor(s) in a low-affinity state. Adhesion experiments performed in the presence of pharmacological inhibitors indicate that phosphatidyl inositol 3-kinase (PI3-K)/protein kinase C (PKC) and protein kinase A (PKA) pathways may transmit opposing signals on chondrocyte adhesion, and that collagen receptors are kept in a low-affinity state by PI3-kinase/PKC signalling. Since echistatin is a high-affinity ligand for alphavbeta3 integrin, the effect of the function-blocking anti-alphavbeta3 antibody LM609 was investigated. Like echistatin, LM609 stimulated chondrocyte adhesion to collagen and failed to support their attachment. Therefore, our data suggest that alphavbeta3-antagonists might regulate the binding activity of the beta1 collagen receptor, which in turn leads to the rapid transition of chondrocytes from suspension to an adherent state.

An adult Sanfilippo type A patient with homozygous mutation R206P in the sulfamidase gene.

The Sanfilippo type A syndrome, one of the most frequent forms of mucopolysaccharidosis III, is characterized by severe mental retardation, progressive neurological degeneration, and mild somatic changes. It is due to a deficiency of heparan-N-sulfatase (sulfamidase) activity and consequent excretion of heparan sulfate in the urine. The disease is transmitted through an autosomal recessive mechanism, and more than 60 gene mutations have been identified. Up to now, only 10 cases of attenuated form of Sanfilippo type A syndrome have been described, and the specific mutation has been identified only in two of them. We report here on a female patient, 20 years old, with Sanfilippo type A syndrome presenting with a mild clinical phenotype characterized essentially by a moderate nonevolving mental retardation. The genetic analysis demonstrated that the patient is homozygous for mutation R206P; presence of polymorphism R456H was also found. This study places R206P as a mild mutation underlying Sanfilippo type A disease.