Diagnostic dilemmas in Fabry disease: a case series study on GLA mutations of unknown clinical significance.

Fabry disease' (FD) phenotype is heterogeneous: alpha-galactosidase A gene mutations (GLA) can lead to classical or non-classical FD, or no FD. The aim of this study is to describe pitfalls in diagnosing non-classical FD and assess the diagnostic value of plasma globotriaosylsphingosine. This is a case series study. Family 1 (p.A143T) presented with hypertrophic cardiomyopathy (HCM), absent classical FD signs, high residual alpha-galactosidase A activity (AGAL-A) and normal plasma globotriaosylsphingosine. Co-segregating sarcomeric mutations were found. Cardiac biopsy excluded FD. In family 2 (p.P60L), FD was suspected after kidney biopsy in a female with chloroquine use. Males had residual AGAL-A, no classical FD signs and minimally increased plasma globotriaosylsphingosine, indicating that p.P60L is most likely non-pathogenic. Non-specific complications and histology can be explained by chloroquine and alternative causes. Males of two unrelated families (p.R112H) show AGAL-A <5%, but slightly elevated plasma globotriaosylsphingosine (1.2-2.0 classical males >50 nmol/l). Histological evidence suggests a variable penetrance of this mutation. Patients with GLA mutations and non-specific findings such as HCM may have non-classical FD or no FD. Other (genetic) causes of FD-like findings should be excluded, including medication inducing FD-like storage. Plasma globotriaosylsphingosine may serve as a diagnostic tool, but histology of an affected organ is often mandatory.

Detection of anti-MAG antibodies in polyneuropathy associated with IgM monoclonal gammopathy.

BACKGROUND: Detection of serum antibodies to myelin-associated glycoprotein (MAG) by Western blot (WB) is a valuable assay to diagnose a distinct type of demyelinating polyneuropathy with immunoglobulin M (IgM) monoclonal gammopathy. In this study, the diagnostic accuracy of a new and more practical ELISA to detect these antibodies was validated. METHODS: Routine WBs from 2 independent laboratories and ELISA were used to detect anti-MAG IgM in serum from 207 patients with neuropathy and controls. The sensitivity and specificity of these assays were compared and related to the patient clinical and electrophysiologic characteristics. RESULTS: In ELISA, anti-MAG antibodies were found in serum from 49 (72%) of 68 patients with demyelinating polyneuropathy and IgM monoclonal gammopathy. However, in this subgroup of patients, only 30 (44%) and 37 (54%) were positive in the 2 WBs. All of the patients positive in the 2 WBs were also positive in ELISA. A high correlation was found for IgM activity in ELISA to MAG and sulfate-3-glucuronyl paragloboside (SGPG) (Spearman rho = 0.72, p < 0.0001), supporting the notion that the shared sulfated glucuronic acid moiety of MAG and SGPG is preserved. Most patients positive in anti-MAG ELISA had a slowly progressive sensory-motor demyelinating polyneuropathy, even if the WB was negative. In control groups, however, 4 WB-negative patients with a nondemyelinating monoclonal gammopathy-related polyneuropathy were positive in anti-MAG ELISA. The remaining samples were negative in ELISA. CONCLUSION: ELISA is more sensitive than Western blot to diagnose anti-myelin-associated glycoprotein related polyneuropathy, although a positive serology may be found in other forms of polyneuropathy as well.

[Diagnostic value of autoantibodies to MAG by ELISA Bühlmann in 117 immune-mediated peripheral neuropathies associated with monoclonal IgM to SGPG/SGLPG]. / Les autoanticorps IgM anti-MAG par Elisa Bühlmann: apport diagnostique dans 117 neuropathies périphériques auto-immunes associées à une IgM monoclonale à activité anti-SGPG/SGLPG.

The neuropathies associated with monoclonal IgM gammopathy reacted with glycoconjugated targets on a very antigenic epitope on the sulfated glucuronic glycolipids corresponding to SGPG and SGLPG (sulfoglucuronyl paragloboside and sulfoglucuronyl lactosaminyl paragloboside), myelin-associated glycoprotein (MAG) and sulfatide. Sometimes monoclonal IgM binds to a broad spectrum of gangliosides. The detection of targets of autoantibodies has considerable importance in the diagnosis and management of patients. It is not known whether the results of antibody tests are equally sensitive and specific for identification of involved auto-antigens. In this study we evaluated the results obtained using IgM reactivity against MAG by enzyme-linked immunosorbent assay (ELISA Bühlmann) with IgM reactivity against SGPG/SGLPG obtained by overlay thin-layer chromatography. We selected 117 patients with anti-SGPG/SGLPG monoclonal gammopathy and peripheral neuropathy and a control group of 102 peripheral neuropathies with 24 having IgM high titres of monoclonal IgM anti-ganglioside antibodies. The anti-MAG sensitivity was 0.97, specificity was 0.86. There is a crossreactivity between 8 (57%) monoclonal IgM antibodies anti-MAG and anti-ganglioside GM1 and 2 (28%) anti-disialylated gangliosides. These results indicate that in clinical practice, anti-MAG ELISA is useful for eliminating anti-MAG neuropathy, as well as for positive diagnosis for titres upper than 10,000 BTU. It is also alpha good test to appreciate clinical improvement after Rituximab treatment.

H-deficient Bombay and para-Bombay red blood cells are most strongly agglutinated by the galactophilic lectins of Aplysia and Pseudomonas aeruginosa that detect I and P1 antigens.

The galactophilic lectins Aplysia gonad lectin (AGL) and Pseudomonas aeruginosa lectin (PA-IL), which detect human I and P1 RBC antigens, were examined for hemagglutination of H+ (group O and B) and H-deficient (Bombay and para-Bombay phenotype) RBCs. The results were compared with those obtained using two other galactophilic lectins, Maclura pomifera lectin (MPL) and Arachis hypogaea (peanut) agglutinin (PNA), which share T-antigen affinity, and two fucose-binding H-specific lectins, Ulex europaeus (UEA-I) and Pseudomonas aeruginosa lectin (PA-IIL), as well as with those achieved with anti-I serum. The results revealed that, in contrast to UEA-I and PA-IIL, which preferentially agglutinated H+ RBCs, and to MPL and PNA, which similarly agglutinated all examined RBCs, AGL, PA-IL, and the anti-I serum agglutinated the H-deficient RBCs more strongly than did the H+ RBCs. These findings could be attributed to increased levels of I and P1 antigens on those RBCs resulting from the use of the free common H-type 2 precursor for their synthesis. Since both PA-IL and PA-IIL are regarded as potential pathogen adhesins, it would be interesting to statistically compare the sensitivities of individuals of H+ and H-deficient RBC populations to P. aeruginosa infections.

Globo-A--a new receptor specificity for attaching Escherichia coli.

Uropathogenic Escherichia coli strains designated as ONAP, based on their O negative A positive agglutination of human P1 erythrocytes, were shown to prefer the globo-A glycolipid as a receptor structure. The dependence on both the A terminal and the globoseries chain was confirmed by agglutination of human AP1, but not Ap or OP1 erythrocytes and by binding to the globo-A glycolipid on TLC plates. Neither Gal alpha 1----4Gal beta nor the A trisaccharide GalNAc alpha 1----3(Fuc alpha 1----2)Gal beta alone functioned as receptors. The bacteria thus appeared to recognize an epitope resulting from the combination of the terminal and internal structures.

Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase.

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.

Exposure of the major human red-cell glycolipid, globoside, to galactose oxidase.

Plasma membrane glycolipids are localized at the outer leaflet of the lipid bilayer, and their carbohydrate portions are exposed to the environment. The efficiency of exposure has, however, not been known. We have been able to determine the availability of the major red cell glycolipid, globoside, to externally added galactose oxidase. Red cells were extensively treated with the enzyme and the oxidized cells reduced with NaBD4. After isolation the extent of exposed globoside was estimated by mass spectrometry. The results show that the exposure of globoside varies in red cells of different individuals from 37-66%. The fatty acid composition of externally available globoside was the same as that of non-oxidized globoside. The exposure was not influenced by protease treatment of intact cells and no correlation was found with different ABO blood groups.

Immunohistochemical localization of glycosphingolipid in urinary renal tubular cells in Fabry's disease.

Although the accumulation of neutral glycosphingolipid (GSL), principally globotriaosylceramide ( GbOse3Cer ), in the kidney of patients with Fabry's disease is well documented, little is known about the type and quantity of lipid present in the renal tubular cells shed in the urine. Using a variety of cytologic technics, the authors examined exfoliated cells found in the urine specimens of patients hemizygous and heterozygous for alpha-galactosidase A deficiency. Renal tubular cells contained periodic acid- Shiff positive material that could be identified easily by Papanicolaou stain. A fluorescein-labeled antibody specific for GbOse3Cer localized this lipid to the cytoplasm. Electron microscopy showed numerous electron-dense multilamellar membranous inclusions within phagolysosomes and electronlucent material within lysosomes of tubular cells. Based on immunofluorescence, heterozygote individuals had similar distribution but less quantity of cytoplasmic GSL. The authors conclude that in Fabry's disease GSL accumulates probably in lysosomes of renal tubular cells. These cells are exfoliated and can be identified specifically in voided urine specimens. Examination of renal tubular cells in urine using the fluorescein antibody technic described here affords a noninvasive means of diagnosing and following the effect of therapy in patients with Fabry's disease.

Localization of globoside and Forssman glycolipids on erythrocyte membranes.

Using the freeze-etch technique, the membrane localization of globoside, a principal glycolipid in human erythrocytes, and Forssman antigen, the chief glycolipid in sheep erythrocytes was evaluated using ferritin and colloidal gold as morphological markers for rabbit antibodies prepared against these glycolipids. Brief trypsinization of human red cell ghosts markedly aggregated intramembranous particles and permitted labeling of globoside, which appeared in a clustered arrangement. The aggregates of ferritin-anti-globoside differed from those of ferritin-wheat germ agglutinin, a label for glycophorin, which corresponded with the aggregates of intramembranous particles. Double-labeling of human trypsinized ghosts with anti-globoside/ Staphylococcal protein A-colloidal gold and ferritin-wheat germ agglutinin indicated that the patterns of labeling were different and that the aggregates of globoside did not bear a direct relationship to the intramembranous particles, which represent transmembrane proteins. Resealed sheep erythrocyte ghosts labeled with ferritin-conjugated rabbit anti-Forssman showed small clusters of Forssman glycolipid on the erythrocyte surface, which could be markedly aggregated with a second goat anti-rabbit antibody, indicating relative mobility of the small glycolipid domains. The distribution of ferritin-anti-Forssman label in sheep ghosts treated at pH 5.5 to aggregate intramembranous particles also did not show definite correspondence between intramembranous particles and the clusters of ferritin-anti-Forssman.

Establishment of lymphoid cell lines from persons with different P blood group phenotypes and the biosynthesis of the antigenic glycosphingolipids.

Human lymphoid cell lines were established from the peripheral lymphocytes of persons with different P blood group phenotypes by in vitro transformation with EB virus. The glycosphingolipid compositions of these cell lines were examined by TLC. The results show that P1k and P2k phenotype cells lack globoside (P antigen) and that two cell lines established from persons with the p phenotype lack both globoside and CTH (Pk antigen), while both the glycosphingolipids were detected in two cell lines established from persons with usual phenotypes (P1 or P2 phenotype). CTH synthetase activity was detected at decreased levels in two p phenotype cell lines, however, globoside synthetase activities could not be significantly detected in all the P phenotype cells.

Headgroup behaviour of an uncharged complex glycolipid.

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide--which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.

A new glycolipid antigen isolated from human erythrocyte membranes reacting with antibodies directed to globo-N-tetraosylceramide (globoside).

Two glycolipid fractions were separated by high performance liquid chromatography from human erythrocyte membranes and which were reactive with antibodies to globo-N-tetraosylceramide (globoside). The reactivity to the antibody was abolished by treatment with endo-beta-galactosidase of Escherichia freundii which specifically hydrolyzes lacto-N-glycosyl series glycolipids, but does not hydrolyze globo series glycolipids. One of the fractions was isolated by repeated high performance liquid chromatography, and its structure was determined by direct probe mass spectrometry, methylation analysis, and by degradation with exo- and endoglycosidases as follows: GalNAc beta 1 leads to 3Gal beta 1 leads to 4GLcNAc beta 1 leads to 3 Gal beta 1 leads to 4Glc beta 1 leads to 1ceramide. The carbohydrate structure was identical with the asialo core of "G3-ganglioside" (IV3NeuAc2 leads to 3GalNAcLcnOs4Cer) (Watanabe, K., and Hakomori, S. (1979) Biochemistry 14,5502-5504), but the ceramide moiety of this glycolipid was characterized by having C22 and C24 fatty acids in a striking contrast to that G3-ganglioside was characterized by the predominance of C14 fatty acid. Since globoside was previously identified as blood group P antigen (Marcus, D. M., Kundu, S. K., and Suzuki, A. (1981) Semin. Hematol. 18, 63-71), and both globoside and this glycolipid possess the common terminal structure GalNAc beta 1 leads to 3Gal, this glycolipid may represent the second blood group P antigen belonging to the lacto series.

A prospective double-blind study of plasma exchange therapy for the acroparesthesia of Fabry's disease.

During a study of the effect of plasma exchange on glycosphingolipid metabolism, a patient with Fabry's disease noted a dramatic improvement in his painful acroparesthesia. A controlled study was therefore undertaken. Observations were made of nerve conduction times, graded exercise testing, and psychometric evaluations during and after two planned series of three plasma exchanges: one a true plasma exchange and the other a "sham' control in which the patient received his own plasma. All observers and the patient were blinded and unanimously attributed beneficial results to the sham procedure. This study demonstrates the need for controlled studies in diseases prone to unpredictable exacerbation or spontaneous remission and outlines one possible technique of controlling studies involving plasma exchange.

The glycosphingolipids of human plasma lipoproteins.

Human plasma contains low concentrations of four neutral glycosphingolipids (glucosylceramide, lactosylceramide, globotriaosylceramide, and globotetraosylceramide) and GM3 ganglioside which occur as part of the plasma lipoproteins, particularly low density lipoprotein (LDL, d 1.006-1.063 g. mL-1) and to a lesser extent with high density lipoprotein (HDL, d 1.063-1.21 g.mL-1). Plasma glucosylceramide appears to exchange freely between plasma lipoproteins and erythrocytes, and probably also between different lipoprotein fractions, in the circulation. Free exchange of other major neutral glycosphingolipids (GSLs) between lipoproteins and erythrocytes, or between lipoprotein fractions, does not normally occur. The GSL profile of each lipoprotein fraction is the same as the overall GSL composition of unfractionated plasma. In Fabry disease and Gaucher disease, GSL storage diseases, the excess glycolipid in plasma is distributed among the various lipoprotein fractions in the same relative proportions as in healthy individuals. In familial hypercholesterolemia, in which the levels of all plasma GSLs are elevated, the excess GSL is largely associated with the increased concentrations of LDL. In patients with hereditary hypolipoproteinemias, the levels of GSL in plasma are decreased less than those of other lipids. The relative excess of GSL in these patients is distributed among the remaining lipoprotein fractions. Excess GSL such as occurs in Fabry disease, does not appear to have a biologically significant effect on the physical stability of human LDL.

Blood group type glycosphingolipids of human cord blood erythrocytes.

The non-acid glycosphingolipids of cord blood erythrocytes, pooled from blood groups O and A respectively, were characterized by mass spectrometry in combination with immunologic and thin-layer chromatographic analyses. The glycosphingolipid pattern was compared to that of adult erythrocytes. Just as for adult blood, the major glycosphingolipid species were lactosylceramide and globotetraosylceramide (globoside). The triglycosylceramide fraction was quantitatively smaller in cord red blood cells and was also enriched in a triglycosylceramide with a terminal hexosamine, as conclusively shown by mass spectrometry. The amount of blood group A and H active glycosphingolipids appeared to be less in cord blood cells compared to adult cells was glycolipids with longer, probably branched chain structures were not detected. In contrast, increased amounts of penta- and hexaglycosylceramides lacking fucose and possibly being biosynthetic precursor substances to the ordinary blood group A and H glycosphingolipids were found typical for cord blood cells.