Functional studies of new GLA gene mutations leading to conformational Fabry disease.

Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of alpha-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants in FD patients which lead to alpha-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations, c.155G>A (p.C52Y), c.548G>C (p.G183A), c.647A>G (p.Y216C) in as many individuals (two male; one female) and performed in vitro expression studies and Western blot analysis in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When mutant proteins overexpressed in COS-1 cells and in patients' lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients, but it is not always possible to issue the enzyme's active form in all involved organs. Our study endorses the hypothesis that an active site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.

Aging of silastic peritoneal catheters.

Increasing the survival of patients on CAPD is related to the long-term reliability of the peritoneal access. Six silicone Tenckhoff catheters (with strip or diffuse barium sulphate inclusion) removed after 39-69 months because of the appearance of external segment fissures, were analysed by scanning electron microscopy (SEM) and infra-red spectroscopy with attenuated total refractance (ATR). The extracorporeal portion of the catheters showed (by ATR) a more prominent oxidation peak on the external than the internal surface; SEM showed marks and cracks on the external surface and exfoliation and flattening of the silastic reticle on the intraluminal surface. No evidence of oxidation was found in the intra-abdominal portion of the catheters but biofilm was found. We suggest that barium sulphate may render the silastic brittle and physiological and environmental long-term factors (such as uv-rays, temperature, sweat and disinfectants) could cause oxidation and loss of physico-chemical properties, with critical aging of the silastic and loss of catheter resistance to mechanical injury.

Dextran deposits in tissues of patients undergoing haemodialysis.

Recently the possible storage of dextran-related material in patients undergoing regular haemodialysis has been suggested. We examined biopsy and autopsy specimens of 32 patients treated with regular haemodialysis for 61 +/- 34 months. All patients received dextran-40 as a plasma expander because of hypotension during haemodialysis. The same study was carried out in a control group of 11 haemodialysed patients who were given other plasma expanders. In the 11 patients who received larger doses of dextran-40 (0.38 g/kg body weight per week) we found particles in the cytoplasm of macrophages in various organs, which proved PAS positive and diastase resistant on light microscopy, and birefringent on polarisation. Electron microscopy revealed a fibrillar structure, but ionic analysis by electronic sampler on scanning electron microscopy excluded the presence of silicon. No intracellular inclusions were observed in the control group, nor in the patients given dextran-40 in doses lower than 0.08 g/kg body weight per week. As we also found a linear relationship between the number of particles and the dextran-40 doses given, we hypothesise that the material demonstrated in the macrophages is a structurally modified dextran.

Retinol binding protein in serum and in urine of glomerular and tubular nephropathies.

RBP behavior was studied in different kidney diseases. Serum RBP was increased in chronic renal diseases when the serum creatinine increased. RBP clearance and RBP clearance as a percentage of albumin clearance increased when GFR decreased; this is more evident in tubular kidney diseases. Urinary RBP loss as a percentage of urinary albumin loss increased especially in kidney diseases with persistent tubular lesions.

A new end-to-end anastomosis formed without sutures for haemodialysis arteriovenous fistulas.

Carrying out vascular sutures is often a serious trouble during the surgical preparation of arteriovenous fistulas for haemodialysis. A new technique is suggested to perform an end-to-end anastomosis by inserting the artery 5-6 mm into the vein and then cementing the vessels by means of a cyanoacrilic tissue adhesive. The duration of the surgical procedure is reduced to half and this technique is much more simple than the usual ones. In 6 patients thus treated no troubles could be found either immediately or afterwards.