Loading of cell cultures with cholesterol-dextran particles as a new functional test for Niemann-Pick type C disease.

Deuterium-labeled cholesterol-dextran particles (d4-CholDex), prepared by co-precipitation, were internalized by cultured human skin fibroblasts and HEK293 cells. Subcellular particles from d4-CholDex-treated HEK293 cells were fractionated on iodixanol gradients. More than 60% of d4-cholesterol (d4-UC) in the gradient co-fractionated with lysosomal markers and NPC1. This and formation of d4-cholesteryl esters (d4-CE) in the cells suggests that d4-CholDex is lysosomally processed. In accordance with these findings, we observed an increase in lysosomal cholesterol content by fluorescence microscopy in CholDex-loaded cells. Fibroblast cultures including 13 NPC1-deficient, four heterozygous and six control lines were treated with d4-CholDex at final d4-UC concentration of 0.05 mg/ml (127.98 µmol/L) for 3 h and chased for 48 h in medium without d4-CholDex. Concentrations of d4-UC and d4-CE in harvested cells were measured by tandem mass spectrometry (MS/MS). d4-UC/d4-CE ratios were elevated in NP-C lines compared to controls (n = 6, mean = 4.36, range = 1.89-8.91), with the highest ratios in severe NP-C1 phenotypes and the lowest in adolescent/adult type patients. There were overlaps between NP-C1 forms: early infantile (n = 1, mean = 48.6), late infantile (n = 4, mean = 36.3, range = 20.6-54.0), juvenile (n = 5, mean = 24.7, range = 13.4-38.3), adolescent/adult (n = 3, mean = 14.5, range = 11.7-19.8). The ratios in NP-C1 heterozygotes were mildly elevated (n = 4, mean = 16.4, range = 14.9-17.4) and comparable to patients with adolescent/adult NP-C1. The test can be useful in evaluation of suspected NP-C patients with inconclusive results of biomarker or molecular tests. Its advantages include standardized preparation of particles with longer shelf life at 4 °C, quantitative results, and no requirement for radioactive chemicals.

IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing.

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B.

Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs. Herein, we provide a comprehensive overview of the biochemical and structural abnormalities in pancreatic tissue from two male FD patients with blood group B. In both patients, we found major accumulation of a variety of complex B-GSLs carrying predominantly hexa- and hepta-saccharide structures. The subcellular pathology was dominated by deposits containing B-glycoconjugates and autofluorescent ceroid. The contribution of Gb3Cer to the storage was minor. This abnormal storage pattern was specific for the pancreatic acinar epithelial cells. Other pancreatic cell types including those of islets of Langerhans were affected much less or not at all.Altogether, we provide evidence for a key role of B-antigens in the biochemical and morphological pathology of the exocrine pancreas in FD patients with blood group B. We believe that our findings will trigger further studies aimed at assessing the potential pancreatic dysfunction in this disease.

Neural cells generated from human induced pluripotent stem cells as a model of CNS involvement in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPSII) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates. Human induced pluripotent stem cells (iPSC) represent an alternative system that complements the available MPSII murine model. Herein we report on the reprogramming of peripheral white blood cells from male and female MPSII patients into iPSC using a non-integrating protocol based on the Sendai virus vector system. We differentiated the iPSC lines into IDS deficient and GAG accumulating ß-Tubulin III+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes. The lysosomal system in these cells displayed structural abnormalities reminiscent of those previously found in patient tissues and murine IDS deficient neuronal stem cells. Furthermore, quantitative determination of GAGs revealed a moderate increase in GAG levels in IDS deficient neurons and glia. We also tested the effects of recombinant IDS and found that the exogenous enzyme was internalized from the culture media and partially decreased the intracellular GAG levels in iPSC-derived neural cells; however, it failed to completely prevent accumulation of GAGs. In summary, we demonstrate that this human iPSC based model expresses the cellular and biochemical features of MPSII, and thus represents a useful experimental tool for further pathogenesis studies as well as therapy development and testing.

Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging.

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis.

Diagnostic dilemma: a young woman with Fabry disease symptoms, no family history, and a "sequencing cryptic" α-galactosidase a large deletion.

Fabry disease, an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A). In affected males, the clinical diagnosis is confirmed by the markedly decreased α-Gal A activity. However, in female heterozygotes, the α-Gal A activity can range from low to normal due to random X-chromosomal inactivation, and diagnostic confirmation requires identification of the family's α-Gal A gene mutation. In a young female who had occasional acroparesthesias, corneal opacities, and 15 to 50% of the lower limit of normal leukocyte α-Gal A activity, α-Gal A sequencing in two expert laboratories did not identify a confirmatory mutation, presenting a diagnostic dilemma. A renal biopsy proved diagnostic and renewed efforts to detect an α-Gal A mutation. Subsequent gene dosage analyses identified a large α-Gal A deletion confirming her heterozygosity, and she was started on enzyme replacement therapy. Thus, gene dosage analyses can detect large deletions (>50bp) in suspect heterozygotes for X-linked and autosomal dominant diseases that are "sequencing cryptic," resolving molecular diagnostic dilemmas.

Detection of large gene rearrangements in X-linked genes by dosage analysis: identification of novel α-galactosidase A (GLA) deletions causing Fabry disease.

For most Mendelian disorders, targeted genome sequencing is an effective method to detect causative mutations. However, sequencing PCR-amplified exonic regions and their intronic boundaries can miss large deletions or duplications and mutations that lead to PCR failures in autosomal dominant disorders and in heterozygote detection for X-linked diseases. Here, a method is described for detecting large (>50 bp) deletions/duplications in the X-linked α-galactosidase A (GLA) gene, which cause Fabry disease. Briefly, multiplex PCR mixtures were designed to amplify each GLA exon and an unrelated internal control exon to normalize GLA exonic amplicon peak heights. For each normalized GLA amplicon, the normal control female to male peak-height ratios were 1.8 to 2.2 (expected 2.0), whereas the expected ratios for deletions or duplications would be ∼1.0 or 3.0, respectively. Using this method, three novel deletions, c.369+3_547+954del4096insT, c.194+2049_369+773del2619insCG, and c.207_369+651del814ins231, were detected in unrelated women with signs and/or symptoms suggestive of Fabry disease, but no "sequencing-detectable" mutations. The deletions were confirmed by sequencing their respective GLA RT-PCR products. This method can identify gene rearrangements that may be cryptic to genomic DNA sequencing and can be readily adapted to other X-linked or autosomal dominant genes.

Frequency of unrecognized Fabry disease among young European-American and African-American men with first ischemic stroke.

BACKGROUND AND PURPOSE: The cause of initial ischemic stroke in up to 30% of young patients remains unclear. Fabry disease, due to deficient alpha-galactosidase A (alpha-Gal A) activity, is a vascular endothelial glycosphingolipid storage disease typically presenting in childhood. With advancing age, patients develop renal, cardiac, and cerebrovascular disease and die prematurely. A European study suggested an increased prevalence of unrecognized Fabry disease in patients with cryptogenic stroke. We hypothesized that alpha-Gal A deficiency is a rare cause of initial early-onset ischemic stroke in men. METHODS: The Stroke Prevention in Young Men Study enrolled >550 men (15 to 49 years) with first ischemic stroke in the Baltimore-Washington area in 2004 to 2007. Frozen plasma samples were assayed for alpha-Gal A activity, and DNA from patients with consistently low plasma alpha-Gal A activities were sequenced. RESULTS: The study sample consisted of 558 men (42% African-American; median age 44 years). Stroke was cryptogenic in 154 men (40% African-American). In 10 patients with low plasma alpha-Gal A activities, DNA sequencing identified alterations in the alpha-Gal A gene in 2 patients. The polymorphism, D313Y, which results in low plasma enzyme activity, but near normal levels of cellular activity was seen in one European-American male. The Fabry disease-causing A143T mutation was seen in an African-American male with cryptogenic stroke (0.18% of all strokes: upper 95% CI=0.53%; 0.65% of cryptogenic strokes: upper 95% CI=1.92%). CONCLUSIONS: In this biracial population, unrecognized Fabry disease is a rare but treatable cause of initial ischemic stroke in young men.

Newborn screening for Fabry disease in Taiwan reveals a high incidence of the later-onset GLA mutation c.936+919G>A (IVS4+919G>A).

Fabry disease (alpha-galactosidase A (alpha-Gal A, GLA) deficiency) is a panethnic inborn error of glycosphingolipid metabolism. Because optimal therapeutic outcomes depend on early intervention, a pilot program was designed to assess newborn screening for this disease in 171,977 consecutive Taiwanese newborns by measuring their dry blood spot (DBS) alpha-Gal A activities and beta-galactosidase/alpha-Gal A ratios. Of the 90,288 male screenees, 638 (0.7%) had DBS alpha-Gal A activity <30% of normal mean and/or activity ratios >10. A second DBS assay reduced these to 91 (0.1%). Of these, 11 (including twins) had <5% (Group-A), 64 had 5-30% (Group-B), and 11 had >30% (Group-C) of mean normal leukocyte alpha-Gal A activity. All 11 Group-A, 61 Group-B, and 1 Group-C males had GLA gene mutations. Surprisingly, 86% had the later-onset cryptic splice mutation c.936+919G>A (also called IVS4+919G>A). In contrast, screening 81,689 females detected two heterozygotes. The novel mutations were expressed in vitro, predicting their classical or later-onset phenotypes. Newborn screening identified a surprisingly high frequency of Taiwanese males with Fabry disease (approximately 1 in 1,250), 86% having the IVS4+919G>A mutation previously found in later-onset cardiac phenotype patients. Further studies of the IVS4 later-onset phenotype will determine its natural history and optimal timing for therapeutic intervention.

Replacement of alpha-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 alpha-galactosidases A (agalsidases, Fabrazyme, and Replagal) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of ((3)H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with Fabrazyme, 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.

Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation.

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.

A nationwide blood spot screening study for Fabry disease in the Czech Republic haemodialysis patient population.

BACKGROUND: Fabry disease (FD) is a genetic disorder characterized by accumulation of trihexosylceramide in lysosomes of various tissues leading to multiorgan manifestations, including progressive renal disease. Previous screening studies have shown that a non-neglectable proportion of haemodialysis(HD) patients have unsuspected FD. An extensive FD screening study, the largest to date, has been conducted in HD patients in Czech Republic. We aimed to uncover previously undiagnosed FD patients, to enable them to benefit from cause-specific therapeutic intervention with enzyme replacement therapy (ERT). METHODS: Large-scale screening was executed using a convenient automated enzymatic (alpha-galactosidose A, alpha-Gal A) dried blood spot on filter paper fluorescence method. RESULTS: In total, 3370 (45.1% males, 54.9% females) out of 4058 HD patients (83%) in Czech Republic participated in this blood spot screening (BSS) study. Abnormal low fluorescence readings were obtained in 117 patients (3.5%). Subsequent determination of plasma alpha-Gal A activity identified four males and three females with deficient plasma enzyme activity. Determination of alpha-Gal A activity in peripheral blood leucocytes and confirmatory molecular analysis resulted in four newly diagnosed Fabry males and one female. Subsequent family screening identified 10 family members with genotypically proven FD. Based on these screening results, ERT could be offered to five male FD patients. CONCLUSIONS: BSS represents a promising screening tool that has proven to be convenient and effective in uncovering unrecognized FD patients among the chronic HD population in Czech Republic.

Relationship between X-inactivation and clinical involvement in Fabry heterozygotes. Eleven novel mutations in the alpha-galactosidase A gene in the Czech and Slovak population.

We have identified 21 different alpha-galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.

Recurrence of Fabry disease as a result of paternal germline mosaicism for alpha-galactosidase a gene mutation.

We present two sisters with a severe form of Fabry disease, who both carry the same mutation in the alpha-galactosidase A (alpha-gal A) gene (Q330X). Each of the sisters developed renal failure in the third decade of life; the older sibling underwent renal transplantation at 40 years of age. The severe phenotype of the siblings correlates with results of the X-inactivation study: examination of methylation status in human androgene receptor (HUMARA) gene suggests preferential inactivation of the wild-type allele in both patients. Patients' parents had no symptoms of Fabry disease and were tested negative for the mutation Q330X in DNA isolated from peripheral leukocytes, mouth wash cells, and urinary sediment cells. Genotype analysis using DXS7424 marker showed paternal origin of the mutation. The father's sperm was then tested for presence of the mutation to examine the possibility of the germline mosaicism. Both mutant and wild-type alleles were found in DNA isolated from father's sperm. The apparent explanation of these findings is germline mosaicism due to mutation event during the embryonic development of sperm producing cells (spermatogonia). This is the first case of germline mosaicism in Fabry disease reported in the literature.

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

BACKGROUND: Human alpha-galactosidase A (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD)--Fabry (alpha-GAL deficiency) and Schindler (alpha-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 alpha-GAL and alpha-NAGA. RESULTS: BlastP searches for orthologs of human alpha-GAL and alpha-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (alpha-galactosidase and alpha-N-acetylgalactosaminidase)--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 alpha-NAGA, rice alpha-GAL and human alpha-GAL suggest a close evolutionary relationship of GANA-1 to both human alpha-GAL and alpha-NAGA. Both alpha-GAL and alpha-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, alpha-GAL activity on an artificial substrate was completely inhibited by the alpha-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 alpha-GAL and alpha-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype. CONCLUSIONS: GANA-1 is a single C. elegans ortholog of both human alpha-GAL and alpha-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.

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases.

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.