Helena Poupětová

Subclinical course of cholesteryl ester storage disease in an adult with hypercholesterolemia, accelerated atherosclerosis, and liver cancer

Few cases of asymptomatic cholesteryl ester storage disease (CESD) due to low enzymatic activity of human lysosomal acid lipase/cholesteryl ester hydrolase (hLAL) have been reported thus far in adults Here, we describe a 51-year-old man with a long clinical history of mixed hyperlipoproteinemia and severe premature atherosclerosis, but with no signs of hepatomegaly, liver dysfunction, or splenomegaly. The disease was discovered by chance in a biopsy performed because of suspected liver cancer (proven to be a cholangiocarcinoma). Residual hLAL activity in peripheral leukocytes was determined to be 6% of control values. DNA sequence and restriction fragment length polymorphism analysis demonstrated that the patient was a compound heterozygote for the prevalent CESD exon 8 splice site mutation (G934A) and the deletion of a C (nucleotide 673, 674, or 675) in exon 6 of the hLAL gene, resulting in premature termination of protein translation at residue 195. The patient died of liver failure as a consequence of extensive tumor infiltration at age 52. Lipid analysis revealed moderate cholesteryl ester storage in the liver and in the suprarenal cortex, and massive accumulation in the testicular histiocytes and Leydig cells, resulting in a pronounced secondary atrophy of the seminiferous tubules. Our case study demonstrates that hepatomegaly is an inconstant feature, even in CESD patients compound heterozygous for a Wolman mutation which results in complete loss of hLAL enzymic activity. It also highlights the need to be aware of this condition as it may be underdiagnosed.

Mutations in TMEM76* Cause Mucopolysaccharidosis IIIC (Sanfilippo C Syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage.

Blood group B glycosphingolipids in α-galactosidase deficiency (Fabry disease): influence of secretor status

Defect in degradation of blood group B-immunoactive glycosphingolipids in Fabry disease (deficiency of lysosomal alpha-galactosidase EC 3.2.1.22) has been studied using highly sensitive and specific TLC-immunostaining analysis of urinary sediments and tonsillar tissues of blood group B patients and healthy controls, secretors and nonsecretors. The B glycolipid antigens with hexasaccharide chains were consistently found increased (25- to 100-fold) in the urinary sediments of three Fabry patients, blood group B or AB secretors. Conversely, they were absent in the urinary sediment of one blood group B nonsecretor patient. In normal secretors, B glycosphingolipids were present only in traces. Moreover, significant increase in B glycolipid antigens (8-fold) was found in the tonsillar tissue of a Fabry patient blood group B secretor. We conclude that the secretor status is responsible for increased concentration of blood group B glycosphingolipids in both urinary cells and tonsils in alpha-galactosidase deficiency. The quantity of stored B-immunoactive glycosphingolipids, however, is much lower than that of the mainly accumulated glycosphingolipid Gb(3)Cer. The results clearly indicate that active or silent Se gene, which controls synthesis of B-antigen precursors, is responsible for notable difference in B-glycosphingolipids expression in Fabry patients - secretors and nonsecretors. Whether this novel aspect may be of prognostic significance, remains to be established.

Pulmonary storage with emphysema as a sign of Niemann–Pick type C2 disease (second complementation group). Report of a case

Abstract. A case is described of Niemann–Pick type C2 disease presenting an infantile pneumopathic phenotype known to occur in this recently established, second, minor complementation group of Niemann–Pick type C (NPC) disease. However, the pulmonary involvement was unique, being dominated, in addition to the usual storage macrophage infiltration of the alveolar and septal compartments, by irregular emphysema attributed to storage cell migration into the bronchiolar lumen. The latter modified considerably the X-ray findings and hindered the initial clinical diagnosis. Otherwise, the storage phenotype, including the range of stored lipids, storage distribution, and cell and organ pathology, was found to be identical to that in the whole Niemann–Pick type C disease group dominated by NPC1. The biochemical findings (cholesterol esterification level) corresponded to the classical biochemical phenotype. Emphysema should thus be considered as a variant of the pulmonary NPC2 storage process, governed most probably by an epigenetic mechanism responsible for storage macrophage migration into the bronchiolar compartment.

Direct tandem mass spectrometric profiling of sulfatides in dry urinary samples for screening of metachromatic leukodystrophy.

BACKGROUND Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine. METHODS The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement. RESULTS Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group. CONCLUSIONS The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening.

Abnormal nonstoring capillary endothelium: a novel feature of Gaucher disease. Ultrastructural study of dermal capillaries

Ultrastructural study of skin biopsies in two cases of Gaucher disease (GD) patients (types II and III) revealed hitherto unknown alteration of the blood capillary endothelial cells (ECs) featured by hypertrophy and numerous subplasmalemmal microvesicles underneath both the apical and basal membranes. There was also prominent apical membrane folding with formation of filiform and large cytoplasmic projections, with occasional transcapillary cytoplasmic bridges. Similar, though less frequently expressed, changes were manifested at the basal membrane by numerous cytoplasmic projections into the subendothelial space. Regressive changes with EC breakdown were rare. Lysosomal storage was always absent. Besides EC hypertrophy, there was also increased EC density in the capillary lumen, leading to pronounced changes in capillary architecture with loose or incomplete EC anchoring. There were also signs of EC sprouting. Some pericytes displayed an increase in size and number of cytoplasmic processes, which often extended into distant pericapillary regions. The spectrum of changes suggests that a significant positive growth effect on EC occurs in GD. The putative mechanisms triggered by GBA1 deficiency leading to EC involvement are discussed. The authors are well aware of the fact the results, based on a nontraditional type of bioptic samples, are preliminary, but they are worth following, as further ultrastructural and functional studies of blood endothelium in GD may open a novel field in molecular cell pathophysiology of the disorder: endothelial dysfunction.

Novel mutations associated with metachromatic leukodystrophy: phenotype and expression studies in nine Czech and Slovak patients.

Metachromatic leukodystrophy (MLD) is an inherited demyelinating disorder caused by the deficiency of arylsulphatase A (ASA). This defect leads to an accumulation of galactosylceramide I3‐sulphates (sulphatides) in lysosomes of different tissues. We report on mutations found in a group of nine patients from the Czech and Slovak Republics (former Czechoslovakia). Their diagnosis was confirmed by determination of the activity of arylsulphatase A in leukocytes and by abnormal urinary excretion of sulphatides. All alleles of the patients were identified and eight different mutations were found. They include four novel missense mutations in one infantile (D29N), one juvenile (C294Y), and three adult (C156R, G293S) patients. Four mutations were previously described sequence alterations (459 + 1G > A, G309S, I179S, and P426L). Polymorphisms characteristic for the ASA pseudodeficiency allele were not found in the patients. Substitutions of D29N, C294Y, and G293S in arylsulphatase A caused a severe reduction of enzyme activity in transient expression studies. In contrast, the C156R substitution reduces arylsulphatase A only to 50% of wild type ASA activity. Since no other mutations were found in this patient, the contribution of this mutation to the development of disease remains unclear.

Characterization of gana-1 , a Caenorhabditis elegans gene encoding a single ortholog of vertebrate α-galactosidase and α-N-acetylgalactosaminidase

BackgroundHuman α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA.ResultsBlastP searches for orthologs of human α-GAL and α-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (α-ga lactosidase and α-N-a cetylgalactosaminidase) – gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken α-NAGA, rice α-GAL and human α-GAL suggest a close evolutionary relationship of GANA-1 to both human α-GAL and α-NAGA.Both α-GAL and α-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, α-GAL activity on an artificial substrate was completely inhibited by the α-NAGA inhibitor, N-acetyl-D-galactosamine.A GANA-1:: GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment.Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.Inhibition of gana-1 by RNA interference resulted in a decrease of both α-GAL and α-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype.ConclusionsGANA-1 is a single C. elegans ortholog of both human α-GAL and α-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment.

The coincidence of IgA nephropathy and Fabry disease

BackgroundIgA nephropathy (IgAN) is the most common glomerulonephritis, which may also coexist with other diseases. We present two patients with an unusual coincidence of IgAN and Fabry disease (FD).Case presentationA 26 year-old man underwent a renal biopsy in February 2001. Histopathology showed very advanced IgAN and vascular changes as a result of hypertension. Because of his progressive renal insufficiency the patient began hemodialysis in August 2001. By means of the blood spot test screening method the diagnosis of FD was suspected. Low activity of alpha-galactosidase A in the patient’s plasma and leukocytes and DNA analysis confirmed the diagnosis of FD. Enzyme replacement therapy started in July 2004. Then the patient underwent kidney transplantation in November 2005. Currently, his actual serum creatinine level is 250 μmol/l. Other organ damages included hypertrophic cardiomyopathy, neuropathic pain and febrile crisis. After enzyme replacement therapy, myocardial hypertrophy has stabilized and other symptoms have disappeared. No further progression of the disease has been noted.The other patient, a 30 year-old woman, suffered from long-term hematuria with a good renal function. Recently, proteinuria (2.6 g/day) appeared and a renal biopsy was performed. Histopathology showed IgAN with remarkably enlarged podocytes. A combination of IgAN and a high suspicion of FD was diagnosed. Electron microscopy revealed dense deposits in paramesangial areas typical for IgAN and podocytes with inclusive zebra bodies and myelin figures characteristic of FD. FD was confirmed by the decreased alpha-galactosidase A activity in plasma and leukocytes and by DNA and RNA analysis. Enzyme replacement therapy and family screening were initiated.ConclusionsOur results emphasize the role of complexity in the process of diagnostic evaluation of kidney biopsy samples. Electron microscopy represents an integral part of histopathology, and genetic analysis plays a more and more important role in the final diagnosis, which is followed by causal treatment.