Prevalence of Fabry disease in male dialysis patients: Argentinean screening study.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by enzyme Alpha-Galactosidase A (α-Gal-A) deficiency, due mutations in GLA gene. Progressive glycolipid accumulation leads to damage in kidney and other organs. The aim of this study was to estimate the prevalence of Fabry disease in Argentinean male patients undergoing dialysis. METHODS: A prospective screening study was carried out measuring the α-Gal-A activity in dried blood spot (DBS) samples of male patients undergoing dialysis from Argentina. Those patients in which DBS α-Gal-A level was low (<4.0 µmol/hr/L), underwent GLA genetic testing for diagnosis confirmation. RESULTS: Nine thousand six hundred and four dialysis male patients from 264 centers distributed over 20 of the 23 provinces of Argentina were investigated. Twenty-four patients showed a decreased or absent α-Gal-A activity in DBS and although genetic analysis found a variant in the GLA gene in every one of these patients, we could confirm FD diagnosis in 22 cases. CONCLUSION: The prevalence rate of FD found in Argentinean male dialysis patients was 0.23%. Classic phenotype was observed in 73% of patients, whereas the remaining 27% presented as late-onset variant. This was the largest study carried out in dialysis patients from a same country at a worldwide level and the first study performed in Argentina.

Characterization of biologically active bovine pituitary FSH purified by immunoaffinity chromatography using a monoclonal antibody.

A substantial amount of highly purified, biologically active bovine FSH was isolated from pituitary extracts by immunoaffinity chromatography based on a novel anti-bovine FSH beta-subunit monoclonal antibody. The biological activity was assessed in vitro using a steroidogenic granulosa cell line constitutively expressing the FSH receptor. Amino acid analysis, N-terminal amino acid sequencing, and peptide mass mapping demonstrated that primary structure modifications do not contribute to the heterogeneity of bovine FSH. The monosaccharide composition of the N-linked oligosaccharides was quantified and remarkably two distinct forms of sialic acids, N-acetyl- and N-glycolyl-neuraminic acids were found. In conclusion, we showed that isoform differences in bovine FSH is likely due only to sugar chain heterogeneity, and we give the first evidence that two substituted sialic acids contribute to the diversity of mammalian glycoprotein hormone isoforms.

Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper.

BACKGROUND: Glycogen storage disease II is characterized by a deficiency of the lysosomal enzyme acid alpha-glucosidase. Currently, glycogen storage disease II is diagnosed by demonstrating the virtual absence or a marked reduction of acid alpha-glucosidase activity in muscle biopsies, cultured fibroblasts, or purified lymphocytes. Early diagnosis and treatment of glycogen storage disease II are considered to be critical for maximum efficacy of the enzyme replacement therapies that are in development. However, these existing diagnostic methods are not suited for newborn screening. We developed an assay useful for newborn screening for glycogen storage disease II. METHODS: A series of three enzyme assays to measure the alpha-glucosidase activities in dried blood spots on filter paper was developed. The measurement of acid alpha-glucosidase activity with minimal interference by other alpha-glucosidases was accomplished using maltose as an inhibitor. The method was used on samples from glycogen storage disease II patients, obligate heterozygotes, and healthy controls. RESULTS: Glycogen storage disease II patients were distinguished from carriers and healthy controls using the series of enzyme assays. CONCLUSIONS: We developed a simple and noninvasive screening method for glycogen storage disease II. The method could be incorporated into newborn screening.

Monoclonal antibodies as a probe for the unfolding of porcine growth hormone.

Monoclonal antibodies (mAbs) were generated against pituitary porcine growth hormone (pGH). Ten mAbs were selected for their specificity and affinity for pGH. These mAbs were of the immunoglobulin G (IgG)(1) kappa subclass, with dissociation constants (K(d)) between 7.42 and 0.26 nM, and recognised seven non-overlapping epitopes. We measured whether the mAbs detected alterations of the pGH three-dimensional structure by comparing the antibody reactivity to native pGH and to pGH experimentally unfolded by heating at 50 degrees C, 75 degrees C and 100 degrees C or by reduction and S-carboxymethylation. The antibody-antigen interactions were studied with two enzyme-linked immunosorbent assays (ELISA), based either on a direct binding or inhibition format. The results show that: 1) one mAb, mAb D12, is a conformation-sensitive antibody that recognises an epitope present only in the native pGH. Because the intact three-dimensional structure is essential for the expression of biological activity, mAb D12 could be used to detect altered pGH molecules in biological samples (blood, pituitary extracts or material produced with recombinant technology), and for the one-step purification of biologically active pGH by immunoaffinity chromatography; 2) one mAb, mAb I4, binds to a linear epitope that is not significantly modified in the denatured hormone. This mAb was able to detect the hormone in assays where protein conformation is usually strongly altered, i.e. immunoblotting and immunohistochemistry; 3) the performances of the other eight mAbs differed significantly in the competitive and non-competitive ELISA.

Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards.

BACKGROUND: Tay-Sachs disease (TSD), Sandhoff disease (SD) and variants are caused by deficient activity of the lysosomal enzymes hexosaminidase A (HA) and total hexosaminidase (TH) (hexosaminidase A plus B), respectively. For diagnosis, these enzymes are usually measured in plasma or extracts of leukocytes. We describe methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots (DBSs) on filter paper. MATERIALS AND METHODS: We studied 163 healthy controls, 9 Tay-Sachs patients, 4 Sandhoff patients, 18 obligate carriers and the newborn-screening cards from two patients with Tay-Sachs and one patient with Sandhoff disease. To tubes containing a 3-mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 degrees C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. RESULTS AND CONCLUSIONS: The described methodology is useful to distinguish patients with Tay-Sachs disease or Sandhoff disease from carriers and controls using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases from a newborn-screening card (NSC) was clearly demonstrated, even after storage for up to 38 months at room temperature. The newborn-screening card has been added to the biological materials that allow the identification of patients with Tay-Sachs disease and Sandhoff disease.

Gaucher and Niemann-Pick diseases--enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards.

BACKGROUND: Gaucher disease (GD) and Niemann-Pick (NP) disease are caused by deficient activity of the lysosomal enzymes acid beta-D-glucosidase (ABG) and acid sphingomyelinase (ASM), respectively. For diagnosis, these enzymes are usually measured in the extracts of leukocytes or cultured fibroblasts. Chitotriosidase (CTE), a chitinolytic enzyme, is markedly increased in the plasma of Gaucher patients. We describe methods for the assay of acid beta-D-glucosidase, acid sphingomyelinase, chitotriosidase, and alpha-N-acetyl-galactosaminidase (NAGA) as a control enzyme in blood spots that were dried onto filter paper. METHODS: To tubes containing a 3 mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 degrees C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. We examined 80 healthy controls, 54 Gaucher patients, 8 Niemann-Pick patients, 27 obligate carriers, and the newborn-screening cards (NSC) from a case of Gaucher and a case of Niemann-Pick disease. RESULTS AND CONCLUSION: The described methodology is useful to identify Gaucher and Niemann-Pick patients and controls, using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases on a newborn-screening card was clearly established. The newborn-screening card has been added to the biological materials that allow the identification of patients with Gaucher and Niemann-Pick diseases.