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Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding alpha-galactosidase A (AGAL). The impact of X-chromosome inactivation (XCI) on the phenotype of female FD patients remains unclear. In this study we aimed to determine pitfalls of XCI testing in a cohort of 35 female FD patients. XCI was assessed by two methylation-based and two allele-specific expression assays. The results correlated, although some variance among the four assays was observed. GLA transcript analyses identified crossing-over in three patients and detected mRNA instability in three out of four analyzed null alleles. AGAL activity correlated with XCI pattern and was not influenced by the mutation type or by reduced mRNA stability. Therefore, AGAL activity may help to detect crossing-over in patients with unstable GLA alleles. Tissue-specific XCI patterns in six patients, and age-related changes in two patients were observed. To avoid misinterpretation of XCI results in female FD patients we show that (i) a combination of several XCI assays generates more reliable results and minimizes possible biases; (ii) correlating XCI to GLA expression and AGAL activity facilitates identification of cross-over events; (iii) age- and tissue-related XCI specificities of XCI patterning should be considered.

Fabry disease (FD) manifests decreased α-galactosidase A (α-Gal A) activity and multiorgan damage. There are some undiagnosed cases of the condition among patients on dialysis. The prevalence of FD may also vary with the region. Among 227 male patients undergoing maintenance hemodialysis in Awaji Island, a remote island in Japan, 201 (88.5%) were included in this study. Patients with α-Gal A activity

Aims Fabry disease (FD) is an X‐linked genetic disease caused by mutations in the GLA gene that leads to deficient activity of lysosomal enzymes, accumulation of globotriaosylceramide in multi‐organ systems, and variant clinical manifestations. We aimed to detail the clinical and genetic spectrum of FD in Chinese families. Methods and results Five male probands with unexplained left ventricular hypertrophy and their family members were investigated. Genetic screening was available in 11 subjects of the 5 families, 10 of whom proved to be carriers of either GLA gene mutation, including 3 previous reported missense mutations (c.128G > A, c.811G > A, c.950T > C), 1 novel missense mutation (c.37G > C), and 1 novel deletion mutation (c.1241delT). A total of 17 patients were definitely or possibly diagnosed of FD, given their clinical manifestations and hereditary nature of FD. Echocardiography demonstrated normal cardiac structure and function in six female patients. Electrocardiographic pre‐excitation occurred in 80% (4/5) of men and 16.7% (1/6) of women. Six patients (6/14, 42.9%) had chronic kidney disease with decreased renal function and all were male (6/7, 85.7%). Six patients presented with acroparesthesia, hypohidrosis, or both. Three female patients and two male patients experienced sudden death, and one male patient with the mutation (c.128G > A) died of progressive heart failure, between 41 and 66 years of age. Conclusions We reported five unrelated families of FD with different GLA mutations. Clinical manifestations were highly heterogeneous between male and female patients even within the same family. Female patients showed relatively low risks of structural heart disease and renal insufficiency. However, the long‐term outcomes might be adverse in both sexes. Our study underlines the importance of molecular screening of the GLA gene for early identification and clinical decision making in patients with FD.

Background Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women. Patients and Methods We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity. Results 43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome. Conclusions X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.

Approved therapies for Fabry disease (FD) include migalastat, an oral pharmacological chaperone, and agalsidase beta and agalsidase alfa, 2 forms of enzyme replacement therapy. Broad tissue distribution may be beneficial for clinical efficacy in FD, which has severe manifestations in multiple organs. Here, migalastat and agalsidase beta biodistribution were assessed in mice and modeled using physiologically based pharmacokinetic (PBPK) analysis, and migalastat biodistribution was subsequently extrapolated to humans. In mice, migalastat concentration was highest in kidneys and the small intestine, 2 FD‐relevant organs. Agalsidase beta was predominantly sequestered in the liver and spleen (organs unaffected in FD). PBPK modeling predicted that migalastat 123 mg every other day resulted in concentrations exceeding the in vitro half‐maximal effective concentration in kidneys, small intestine, skin, heart, and liver in human subjects. However, extrapolation of mouse agalsidase beta concentrations to humans was unsuccessful. In conclusion, migalastat may distribute to tissues that are inaccessible to intravenous agalsidase beta in mice, and extrapolation of mouse migalastat concentrations to humans showed adequate tissue penetration, particularly in FD‐relevant organs.

Introduction The pathophysiological significance of the Fabry-related, non-classical variant p.D313Y still remains to be solved. This study assesses the involvement of the peripheral nervous system with respect to small fiber neuropathy and neuropathic pain in female patients carrying p.D313Y. Methods This study examined nine females carrying the Fabry-related p.D313Y variant by obtaining skin punch biopsies above the right lateral malleolus. Intraepidermal nerve fiber density was determined for each patient and compared to reference values matched for the patient's decade of life and sex. Moreover, each patient was characterized by a detailed neurological examination and by pain assessment via questionnaire. Results Compared to sex-matched lower fifth percentile reference values per decade, intraepidermal nerve fiber density was decreased in seven out of nine patients. Four patients reported acral paresthesias and neuropathic pain with an average visual analogue scale score of 7 out of 10 points. Two patients experienced acute pain crises. Six out of seven patients diagnosed with small fiber neuropathy had a their medical history of hypo- and/or hyperhidrosis. Discussion The diagnosis of small fiber neuropathy was made in seven out of nine females carrying the non-classical variant p.D313Y. Moreover, neuropathic pain and symptoms indicative of autonomic nervous system dysfunction seem to be common findings that may be of clinical significance and may warrant therapeutic intervention.

The purpose of this study was to examine the applicability of the use of samples in dried blood spot (DBS) for the definitive diagnosis of Fabry disease (FD) in males and females and to compare the diagnostic role of α-galactosidase A activity (α-Gal A), levels of lyso-Gb3 and sequencing of the GLA gene in screening patients with suspected FD. Measurement of α-Gal A activity in suspected FD patients in DBS was made followed by lyso-Gb3 determination and GLA gene sequencing. Of the 2381 subjects analyzed, FD was confirmed in 24 patients. Thirteen different variants were considered like pathogenic, five of which had not been previously described (c.143A > G; c.455A > C; c.487G > T; c.554delA; c.1045_1046insA). None of the patients with normal enzyme activity had FD confirmation. The DBS measurement of α-Gal A was more sensitive than lyso-Gb3 levels in both men and women. Definitive diagnosis of FD from a single DBS is possible, allowing samples to be easily sent from anywhere to the reference laboratory.

Enzyme replacement therapy (ERT) with recombinant α‐galactosidase A (r‐αGAL A) for the treatment of Fabry disease has been available for over 15 years. Long‐term treatment may slow down disease progression, but cardiac, renal, and cerebral complications still develop in most patients. In addition, lifelong intravenous treatment is burdensome. Therefore, several new treatment approaches have been explored over the past decade. Chaperone therapy (Migalastat; 1‐deoxygalactonojirimycin) is the only other currently approved therapy for Fabry disease. This oral small molecule aims to improve enzyme activity of mutated α‐galactosidase A and can only be used in patients with specific mutations. Treatments currently under evaluation in (pre)clinical trials are second generation enzyme replacement therapies (Pegunigalsidase‐alfa, Moss‐aGal), substrate reduction therapies (Venglustat and Lucerastat), mRNA‐ and gene‐based therapy. This review summarises the knowledge on currently available and potential future options for the treatment of Fabry disease.

Fabry disease (FD) is caused by a defect in α-galactosidase A gene (GLA) which leads to a progressive accumulation of neutral shingolipids, mainly globotriaosylceramide and its metabolites in several organs. Pulmonary manifestations of FD mimic chronic obstructive pulmonary disease and are disproportionate to smoking status. The effect of enzyme replacement therapy (ERT) on pulmonary function is inconclusive. We studied the effect of ERT on pulmonary function in FD with a mutation p. Arg227Ter (p.R227*) which is one of the most common mutations causing classical FD in Finland and worldwide.Patients were annually examined by multidisciplinary team. Based on the maximal pulmonary oxygen consumption at the baseline, either cardiopulmonary exercise test or combination of spirometry and 6-minute walking test were performed annually during 5-year follow-up.Four males and eight females met the criteria for ERT and were included in this study. Three of 12 patients had obstruction by GOLD criterion before ERT, and one had a borderline obstruction. In 5 years, five patients were classified as obstructive, although the real change in FEV1/FVC was unchanged in the whole cohort. Only one patient was an active smoker.In nonsmokers, pulmonary manifestations in classical FD are mild and might be stabilized by ERT.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Objective Fabry disease (FD) is a rare X‐linked lysosomal storage disorder due to the absent or deficient activity of lysosomal hydrolase a‐galactosidase A (α‐Gal A), which leads to the accumulation of its substrates in various organs and tissues. Classic clinical manifestations include angiokeratomas, proteinuria, renal failure, neuropathic pain, and left ventricular hypertrophy. Fever is one of the rare symptoms that may occur during FD. Methods Three Chinese Han patients with FD referred to Peking Union Medical College Hospital were reported. The complete medical records were established, and detailed data were collected. Whole‐exome sequencing by next‐generation sequencing and α‐Gal A enzyme activity assay were performed to confirm the diagnosis. Results These three patients all presented with recurrent fever of unknown origin initially, accompanied with arthralgia/arthritis and other symptoms. We identified two known variants in the GLA gene, c.1176_1179delGAAG and c.782G>A (p.G261D), and a novel variant c.440G>A (p.G147E) which is likely pathogenic in our patient. Conclusions FD should be considered as a rare cause of recurrent fever of unknown origin. The coexistence of gene variants related to systemic autoinflammatory diseases may make the clinical phenotypes of FD more complex and prone to recurrent fever., This is the first case series report of Chinese Fabry disease (FD) patients in whom recurrent fever of unknown origin was the initial manifestation. Two known variants in the GLA gene, c.1176_1179delGAAG and c.782G>A (p.G261D), and a novel variant c.440G>A (p.G147E) which is likely pathogenic in our patient were detected. We conclude that FD should be considered as a rare cause of recurrent fever of unknown origin.

A novel acidic α-galactosidase (EC 3.2.1.22) designated as Leucopaxillus tricolor α-galactosidase (LTG) has been purified to homogeneity from the fruiting bodies of L. tricolor to 855-fold with a specific activity of 956 U mg-1 by the application of chromatography and gel filtration. The molecular mass of LTG was estimated to be 60 kDa as determined by both SDS-PAGE and by gel filtration. The purified enzyme was identified by LC-MS/MS and four inner amino acid sequences were obtained. When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimal pH and optimal temperature of LTG were pH 5.0 and 50 °C, respectively. The enzyme activity was strongly inhibited by Hg2+ , Fe3 , Cu2+ , Cd2+ , and Mn2+ ions. The chemical modification agent N-bromosuccinimide (NBS) completely inhibited the enzyme activity of LTG, indicating the paramount importance of tryptophan residue(s) to its enzymatic activity. Besides, LTG displayed wide substrate diversity with activity toward a variety of substrates such as stachyose, raffinose, melibiose, locust bean gum, and guar gum. Given the good ability of degrading the non-digestible and flatulence-causing oligosaccharides, this fungus may become a useful source of α-galactosidase for multiple applications.

Fabry disease is an X-linked lysosomal storage disease caused by α-galactosidase A (α-Gal A) deficiency. Kidney and heart failure are frequent complications in adulthood and greatly contribute to patient morbidity and mortality. Because α-Gal A-deficient mouse models do not recapitulate cardiorenal findings observed in patients, a nonmouse model may be beneficial to our understanding of disease pathogenesis. In this study, we evaluated disease processes in a recently generated Fabry rat model. We found that male Fabry rats weighed significantly less than wild-type (WT) males, whereas female Fabry rats weighed significantly more than WT females. Whereas no difference in female survival was detected, we observed that male Fabry rats had a decreased lifespan. Skin histology revealed that inflammation and lipoatrophy may be chief disease mediators in patients. With respect to the kidney and heart, we found that both organs accumulate α-Gal A substrates, including the established biomarkers, globotriaosylceramide and globotriaosylsphingosine. Longitudinal serum and urine chemistry panels demonstrated pronounced renal tubule dysfunction, which was confirmed histologically. Mitral valve thickening was observed in Fabry rats using echocardiography. We conclude that Fabry rats recapitulate important kidney and heart phenotypes experienced by patients and can be further used to study disease mechanisms and test therapies.-Miller, J. J., Aoki, K., Mascari, C. A., Beltrame, A. K., Sokumbi, O., North, P. E., Tiemeyer, M., Kriegel, A. J., Dahms, N. M., α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Self-assembly of nanoparticles provides unique opportunities as nanoplatforms for controlled delivery. By exploiting the important role of noncovalent hydrophobic interactions in the engineering of stable assemblies, nanoassemblies were formed by the self-assembly of fluorinated quantum dots in aqueous medium through fluorine-fluorine interactions. These nanoassemblies encapsulated different enzymes (laccase and α-galactosidase) with encapsulation efficiencies of ≥74 %. Importantly, the encapsulated enzymes maintained their catalytic activity, following Michaelis-Menten kinetics. Under an acidic environment the nanoassemblies were slowly disassembled, thus allowing the release of encapsulated enzymes. The effective release of the assayed enzymes demonstrated the feasibility of this nanoplatform to be used in pH-mediated enzyme delivery. In addition, the as-synthesized nanoassemblies, having a diameter of about 50 nm, presented high colloidal stability and fluorescence emission, which make them a promising multifunctional nanoplatform.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

The clinical significance of the c.427G>A (p.A143T) variant in GLA is a topic of debate within the lysosomal storage disease community. A review of the literature and published case reports found the clinical impact of the variant to range from classic Fabry symptoms to healthy unaffected males with normal alpha- galactosidase enzyme levels, leaving clinicians unsure of how to manage these individuals. As the number of states testing for Fabry disease on their newborn screening panel has increased, more people with this variant are being identified. The goal of this project was to learn how the uncertainty surrounding the clinical significance of the p.A143T variant affects those with this change. A self-response questionnaire was developed to explore this topic. In addition to evaluating participant feelings, the questionnaire explored individuals' beliefs regarding the pathogenicity of the variant. Results suggest that people have diverse feelings regarding reclassification of the p.A143T variant. Around half of those surveyed reported feeling frustrated by the lack of clear information. Despite the ambiguity regarding the health consequences of this variant, many participants felt that knowing this result helps guide medical management.

Background Fabry disease is caused by a deficient or an absent alfa‐galactosidase A activity and is an X‐linked disorder that results in organ damage and a shortened life span, especially in males. The severity of the disease depends on the type of mutation, gender, skewed X‐chromosome inactivation, and other still unknown factors. Methods In this article, we describe the natural course of a common classic Fabry disease mutation, p.Arg227Ter or p.R227*, in Finland. Results Four males and ten females belonged to two extended families. The mean age was 46 years (SD 18.4). Six patients (43%) had cardiac hypertrophy, three patients (21%) had ischemic stroke, and none had severe kidney dysfunction. Three patients had atrial fibrillation; two patients who had atrial fibrillation also had pacemakers. All males over 30 years of age had at least one of the following manifestations: cardiac hypertrophy, stroke, or proteinuria. In females, the severity of Fabry disease varied from classic multiorgan disease to a condition that mimicked the attenuated cardiac variant. No one was totally asymptomatic without any signs of Fabry disease. Cardiac magnetic resonance imaging was performed on nine of 14 patients was the most sensitive for detecting early cardiac manifestations. Five patients (55%) had late gadolinium enhancement‐positive segments. Conclusion Cardiac involvement should be effectively detected in females before considering them asymptomatic mutation carriers.

International audience; BACKGROUND: Fabry disease (OMIM 301500) is an X-linked disorder caused by alpha-galactosidase A (α-Gal A) deficiency. The administration of a pharmacologic chaperone (migalastat) in Fabry patients with amenable mutations has been reported to improve or stabilize organ damages and reduce lyso-Gb3 plasma level. An increase of α-Gal A activity has been observed in vitro in cells expressing amenable GLA mutations when incubated with migalastat. The impact of the drug on α-Gal A in vivo activity has been poorly studied.METHODS: We conducted a retrospective analysis of two unrelated male Fabry patients with p.Asn215Ser (p.N215S) variant.RESULTS: We report the important increase of α-Gal A activity in blood leukocytes reaching normal ranges of activity after about 1 year of treatment with migalastat. Cardiac parameters improved or stabilized with the treatment.CONCLUSION: We confirm in vivo the effects of migalastat that have been observed in N215S carriers in vitro. The increase of α-Gal A activity may be the strongest marker for biochemical efficacy. The normalization of enzyme activity could become the new therapeutic target to achieve.

The Bacova_02091 gene in the β‐mannan utilization locus of Bacteroides ovatus encodes a family GH36 α‐galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterization of recombinant BoGal36A reveals unique properties compared to other GH36 α‐galactosidases, which preferentially hydrolyse terminal α‐galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (> 90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose‐substituted polymannose backbone.

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.

Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered. Electronic supplementary material The online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.

Fabry disease is a complex, panethnic lysosomal storage disorder. It is characterized by the accumulation of glycosphingolipids in tissues, organs, the vascular endothelium, and biological fluids. The reported incidence in different populations is quite variable, ranging from 1:1400 to 1:117,000. Its complexity lies in the marked genotypic and phenotypic heterogeneity. Despite the fact that it is an X-linked disease, more than 600 mutations affect both males and females. In fact, some females may be affected as severely as males. The purpose of this protocol is to focus on the high-risk screening of patients who might have Fabry disease using a simple, rapid, non-invasive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for urinary globotriaosylceramide (Gb3 ) analysis. Urine filter paper samples are easily collected at home by patients and sent by regular mail. This method has been successfully used for high-risk screening of patients with ophthalmologic manifestations and in an on-going study for high-risk screening of Fabry disease in patients with chronic kidney diseases. © 2017 by John Wiley & Sons, Inc.

Purpose Enzyme replacement therapy (ERT) was offered from year 2001 to patients with Fabry disease. The ophthalmic experience was analysed, as part of a general 10-year status. Methods A retrospective observational series comprising 39 patients (25 females, 14 males) closely followed by the endocrinologists, and with regular ophthalmic control. Time of inclusion was when the option of ERT was started, at age 11–60 years. Eye data (standard eye examination, including retinal imaging) were incomplete in five, due to death or non-attendance, and five patients had refused treatment. Results Vision was normal throughout, except in two young males with total unilateral central retinal artery occlusion, prior to and during enzyme replacement, respectively. Cornea verticillata and conjunctival vessel ectasies were common. Tortuosity of retinal arterioles and venules was recorded in eight and 18 patients, respectively, and phlebopathy in 22, although generally without evidence of loss of vessel wall integrity. Systemic vascular lesions with or without loss of function were recorded for kidney (n = 23), heart (n = 17) and brain (n = 7), and an association was suggested between nephropathy and abnormal morphology of retinal vessels. Conclusions Thirteen of 32 patients on ERT showed a reduction of corneal deposits over the study period. Abnormal ocular vessel morphology was a frequent finding. In contrast to the function loss related to systemic ischaemic lesions, we found no indication of impairment of visual parameters in 37. Compared to other Fabry series, two of 39 patients with serious unilateral occlusive retinal disease may appear a high number. The presence of retinal tortuosity is discussed, possibly reflecting haemodynamic events related to vessel wall deposits, but could also be ‘constitutional’, as part of the Fabry inheritance.

Loss-of-function mutations of the enzyme alpha-galactosidase A (GLA) causes Fabry disease (FD), that is a rare and potentially fatal disease. Identification of these pathological mutations by sequencing is important because it allows an early treatment of the disease. However, before taking any treatment decision, if the mutation identified is unknown, we first need to establish if it is pathological or not. General bioinformatic tools (PolyPhen-2, SIFT, Condel, etc.) can be used for this purpose, but their performance is still limited. Here we present a new tool, specifically derived for the assessment of GLA mutations. We first compared mutations of this enzyme known to cause FD with neutral sequence variants, using several structure and sequence properties. Then, we used these properties to develop a family of prediction methods adapted to different quality requirements. Trained and tested on a set of known Fabry mutations, our methods have a performance (Matthews correlation: 0.56-0.72) comparable or better than that of the more complex method, Polyphen-2 (Matthews correlation: 0.61), and better than those of SIFT (Matthews correl.: 0.54) and Condel (Matthews correl.: 0.51). This result is validated in an independent set of 65 pathological mutations, for which our method displayed the best success rate (91.0%, 87.7%, and 73.8%, for our method, PolyPhen-2 and SIFT, respectively). These data confirmed that our specific approach can effectively contribute to the identification of pathological mutations in GLA, and therefore enhance the use of sequence information in the identification of undiagnosed Fabry patients.

Several families of iminosugar-based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal β- and α-galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1-C-alkyl imino-L-arabinitols are totally inactive toward the three enzymes, 1-C-alkyl imino-D-galactitols were found to be active only toward α-galactosidase A. Finally, 1-N-iminosugars provided the best results, as 4-epi-isofagomine was found to be a good inhibitor of both lysosomal β-galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.

Fabry disease, a rare X-linked α-galactosidase A deficiency, causes progressive lysosomal accumulation of globotriaosylceramide (GL-3) in a variety of cell types. As the disease progresses, renal failure, left ventricular hypertrophy, and strokes may occur. Enzyme replacement therapy (ERT), with recombinant α-galactosidase A, is currently available for use to reduce GL-3 deposits. However, although it improves cardiac function and decreases left ventricular mass, GL-3 clearance upon ERT has been demonstrated in cardiac capillary endothelium but not in cardiomyocytes of patients. Relevant models are needed to understand the pathogenesis of cardiac disease and explore new therapeutic approaches. We generated induced pluripotent stem cells (iPSC) from Fabry patients and differentiated them into cardiomyocytes. In these cells, GL-3 accumulates in the lysosomes over time, resulting in phenotypic changes similar to those found in cardiac tissue from Fabry patients. Using this human in vitro model, we demonstrated that substrate reduction therapy via glucosylceramide synthase inhibition was able to prevent accumulation and to clear lysosomal GL-3 in cardiomyocytes. This new in vitro model recapitulates essential features of cardiomyocytes from patients with Fabry disease and therefore provides a useful and relevant tool for further investigations of new therapy.

To determine the effectiveness of enzyme replacement therapy (ERT) for adults and children with Fabry disease. Cohort study including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment and untreated patients contributed natural history data. Consenting adults (N = 289) and children (N = 22) with a confirmed diagnosis of Fabry disease attending a specialist Lysosomal Storage Disorder treatment centre in England. At recruitment 211 adults and seven children were on ERT (range of treatment duration, 0 to 9.7 and 0 to 4.2 years respectively). Clinical outcomes chosen to reflect disease progression included left ventricular mass index (LVMI); proteinuria; estimated glomerular filtration rate (eGFR); pain; hearing and transient ischaemic attacks (TIA)/stroke. We found evidence of a statistically significant association between time on ERT and a small linear decrease in LVMI (p = 0.01); a reduction in the risk of proteinuria after adjusting for angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (p

A number of studies have already investigated the prevalence of Fabry disease (FD) in adult patients with unexplained left ventricular hypertrophy (LVH) with rates varying from 0 % up to 12 % reflecting referral and gender bias as well as differences in diagnostic methodology. We aimed to perform a prospective screening study evaluating the prevalence of FD in male patients older than 30 years with strictly defined unexplained LVH followed by general cardiologists. A predefined number of 100 men with unexplained LVH, defined as maximal wall thickness ≥ 13 mm, were identified during an echocardiographic examination in primary cardiology practice and screened by assessing α-galactosidase A activity in dried blood spots (DBS) or in plasma. Four men (52 ± 4 years, maximal LV wall thickness 18 ± 3 mm) were diagnosed with FD confirmed by enzyme analysis in leukocytes as well as by genetic analysis. Mild extracardiac manifestations of FD were present in two of them. The prevalence of FD in our cohort of male patients followed in primary cardiology practice with strictly defined otherwise unexplained LVH was 4 %. We recommend systematic screening for FD in all men older than 30 years with LVH of unknown etiology even in the absence of obvious extracardiac manifestations of FD.

The advent of effective therapy for Fabry disease (FD) creates two challenges: (1) how do we identify individuals with the condition early in its natural history, and (2) how many people have the condition and require expensive therapy? Between 1981 and 2011, 17 new FD families were referred to the Northern Genetics Service in the North of England, the majority between 2005 and 2001. Since 2009 we have employed biochemical screening in dried blood spots from individuals with high risk phenotypes, such as left ventricular hypertrophy and unexplained renal failure, to identify four of the new index cases; and we have offered comprehensive cascade genetic testing to identify pre-symptomatic and symptomatic cases of FD in their 233 ‘at risk’ relatives. Overall, this combined approach identified a further 23 symptomatic cases of FD and 38 asymptomatic cases. The uptake of cascade genetic testing in this cohort was 48 %, which is similar to other inherited disorders for which there is an effective intervention. The minimum prevalence of GLA mutation in the North of England population at the end of 2011 was 1 in 40,800 and the prevalence of symptomatic FD was 1 in 64,600. Improved uptake of cascade genetic testing would increase these estimates to a maximum of 1 in 21,800 and 1 in 49,000 respectively. We suggest that any protocol for case identification should consist of targeted biochemical screening and coordinated cascade genetic testing.

Background and purpose: GLA is the causative gene of Fabry disease, an X-linked lysosomal storage disorder resulting from a-galactosidase A (a-GAL) deficiency. Stroke is an important manifestation of Fabry disease, and recent epidemiological studies have indicated that up to 4.9% of young male cryptogenic stroke patients have GLA mutations. To determine the importance of GLA mutations in the general stroke population, the frequency of GLA mutations in Japanese male ischaemic stroke (IS) patients with various risk factors and ages was measured. Methods: A total of 475 male IS patients (mean age 69.7 12.5 years), were enrolled in this study. A blood sample was obtained to produce blood spots for measurement of a-GAL activity. Blood samples with decreased enzymatic activity were reassayed and the entire GLA gene was analyzed by direct DNA sequencing if a-Gal A activity was consistently low. Results: a-Gal A activity was decreased in 10 men, five of whom (1.1%) had the GLA gene mutation, p.E66Q. All IS patients with p.E66Q mutation had substantial residual a-Gal A activity, in contrast to patients with classic-type Fabry disease. Clinically, all patients with p.E66Q mutation were > 50 years old and had multiple small-vessel occlusions (lacunar infarctions). Statistical analysis using Fisher’s exact test showed the allele frequency of GLA p.E66Q in patients with small-vessel occlusion to be significantly higher than that in the general Japanese population [odds ratio (OR) = 3.34, P = 0.025). Conclusions: GLA p.E66Q mutation is a genetic risk factor for cerebral small-vessel occlusion in elderly Japanese males.

Objective The long-term effects of enzyme-replacement therapy (ERT) in Fabry disease are unknown. Thus, the aim of this study was to determine whether ERT in patients with advanced Fabry disease affects progression towards ‘hard’ clinical end-points in comparison with the natural course of the disease. Methods A total of 40 patients with genetically proven Fabry disease (mean age 40 ± 9 years; n = 9 women) were treated prospectively with ERT for 6 years. In addition, 40 subjects from the Fabry Registry, matched for age, sex, chronic kidney disease stage and previous transient ischaemic attack (TIA), served as a comparison group. The main outcome was a composite of stroke, end-stage renal disease (ESRD) and death. Secondary outcomes included changes in myocardial left ventricular (LV) wall thickness and replacement fibrosis, change in glomerular filtration rate (GFR), new TIA and change in neuropathic pain. Results During a median follow-up of 6.0 years (bottom and top quartiles: 5.1, 7.2), 15 events occurred in 13 patients (n = 7 deaths, n = 4 cases of ESRD and n = 4 strokes). Sudden death occurred (n = 6) only in patients with documented ventricular tachycardia and myocardial replacement fibrosis. The annual progression of myocardial LV fibrosis in the entire cohort was 0.6 ± 0.7%. As a result, posterior end-diastolic wall thinning was observed (baseline, 13.2 ± 2.0 mm; follow-up, 11.4 ± 2.1 mm; P

Background Anderson-Fabry disease is an X-linked defect of glycosphingolipid metabolism. Progressive renal insufficiency is a major source of morbidity, additional complications result from cardio- and cerebro-vascular involvement. Survival is reduced among affected males and symptomatic female carriers. This is an update of a Cochrane review first published in 2010, and previously updated in 2013. Objectives To evaluate the effectiveness and safety of enzyme replacement therapy compared to other interventions, placebo or no interventions, for treating Anderson-Fabry disease. Search methods We searched the Cystic Fibrosis and Genetic Disorders Group's Inborn Errors of Metabolism Trials Register (date of the most recent search: 08 July 2016). We also searched 'Clinical Trials' on The Cochrane Library, MEDLINE, Embase and LILACS (date of the most recent search: 24 September 2015). Selection criteria Randomized controlled trials of agalsidase alfa or beta in participants diagnosed with Anderson-Fabry disease. Data collection and analysis Two authors selected relevant trials, assessed methodological quality and extracted data. Main results Nine trials comparing either agalsidase alfa or beta in 351 participants fulfilled the selection criteria. Both trials comparing agalsidase alfa to placebo reported on globotriaosylceramide concentration in plasma and tissue; aggregate results were non-significant. One trial reported pain scores measured by the Brief Pain Inventory severity, there was a statistically significant improvement for participants receiving treatment at up to three months, mean difference -2.10 (95% confidence interval -3.79 to -0.41; at up to five months, mean difference -1.90 (95% confidence interval -3.65 to -0.15); and at up to six months, mean difference -2.00 (95% confidence interval -3.66 to -0.34). There was a significant difference in the Brief Pain Inventory pain-related quality of life at over five months and up to six months, mean difference -2.10 (95% confidence interval -3.92 to -0.28) but not at other time points. Death was not an outcome in either of the trials. One of the three trials comparing agalsidase beta to placebo reported on globotriaosylceramide concentration in plasma and tissue and showed significant improvement: kidney, mean difference -1.70 (95% confidence interval -2.09 to -1.31); heart, mean difference -0.90 (95% confidence interval -1.18 to -0.62); and composite results (renal, cardiac, and cerebrovascular complications and death), mean difference -4.80 (95% confidence interval -5.45 to -4.15). There was no significant difference between groups for death; no trials reported on pain. Only two trials compared agalsidase alfa to agalsidase beta. One of them showed no significant difference between the groups regarding adverse events, risk ratio 0.36 (95% confidence interval 0.08 to 1.59), or any serious adverse events; risk ratio 0.30; (95% confidence interval 0.03 to 2.57). Two trials compared different dosing schedules of agalsidase alfa. One of them involved three different doses (0.2 mg/kg every two weeks; 0.1 mg/kg weekly and; 0.2 mg/kg weekly), the other trial evaluated two further doses to the dosage schedules: 0.4 mg/kg every week and every other week. Both trials failed to show significant differences with various dosing schedules on globotriaosylceramide levels. No significant differences were found among the schedules for the primary efficacy outcome of self-assessed health state, or for pain scores. One trial comparing agalsidase alfa to agalsidase beta showed no significant difference for any adverse events such as dyspnoea and hypertension. The methodological quality of the included trials was generally unclear for the random sequence generation and allocation concealment. Authors' conclusions Trials comparing enzyme replacement therapy to placebo show significant improvement with enzyme replacement therapy in regard to microvascular endothelial deposits of globotriaosylceramide and in pain-related quality of life. There is, however, no evidence identifying if the alfa or beta form is superior or the optimal dose or frequency of enzyme replacement therapy. With regards to safety, adverse events (i.e., rigors, fever) were more significant in the agalsidase beta as compared to placebo. The long-term influence of enzyme replacement therapy on risk of morbidity and mortality related to Anderson-Fabry disease remains to be established. This review highlights the need for continued research into the use of enzyme replacement therapy for Anderson-Fabry disease.

Background In people, lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). Objective The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Methods Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children's Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 μL blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Results Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of β-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), α-mannosidase, α-galactosidase, α-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; α-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II, activities of lysosomal enzymes were markedly increased. Conclusions Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made.

In this article, we describe a hydrophobic interaction chromatography (HIC) method to remodel the carbohydrates on recombinant human β-glucocerebrosidase (GCR) and the use of hydroxyl ethyl starch (HES) an ethylated starch polymer, to improve this process. GCR is a therapeutic protein used in the treatment of Gaucher disease, a life threatening condition in which patients lack sufficient functional levels of this enzyme. Gaucher disease is the most common inherited lysosomal storage disorder resulting in hepatomegaly, splenomegaly, and bone and lung pathology due to the accumulation of glucosylceramide in the lysosomes of macrophages (Beutler and Grabowski, 2001). The oligosaccharide remodeling of GCR, performed on HIC using three enzymes that remove sugars, increases macrophage uptake through the mannose receptor and thereby lowers its therapeutic dose versus unmodified GCR (Furbish et al., 1981; Van Patten et al., 2007). In this article we describe findings that the addition of HES lowered the amounts of three deglycosylating enzymes needed for remodeling GCR. HES also stabilized the activity of α-glucosidase, α-galactosidase, and GCR under conditions in which these three enzymes rapidly lose activity in the absence of this polymer. Circular dichroism (CD) and second derivative UV spectroscopy revealed that the secondary and tertiary structure of α-glucosidase was unchanged while for GCR there was a slight compaction of the secondary structure but no apparent affect on the tertiary structure. The thermal stability of both GCR and α-glucosidase were enhanced by HES as both molecules showed an increased transition midpoint (Tm). Biotechnol. Bioeng. 2012; 109:1217–1227. © 2011 Wiley Periodicals, Inc.

Fabry disease (FD) is an X-linked lysosomal storage disorder which may lead to impaired peripheral nerve function, mostly affecting small nerve fibers, and to neuropathic pain. Characteristics of the neuropathy associated with FD and the covariates for its development and temporal course have not been described in a large cohort. We studied small fiber function and morphology in 120 Fabry patients at baseline and in subgroups of these until 4-year follow-up. Baseline neurological (89/120) and electrophysiological (106/120) examination was mostly normal. Quantitative sensory testing revealed impaired cold detection thresholds in 84% of men and 39% of women. Lower leg intraepidermal nerve fiber density (IENFD) was reduced to 46% in Fabry patients compared to controls and to 12.5% in men with impaired renal function. Patients with abnormal IENFD more often had pain. Group means for IENFD did not improve under enzyme replacement therapy (ERT), but IENFD in the back increased under ERT in 4/15 patients with good renal function and clinical improvement. Cutaneous cytokine gene expression did not differ from controls. We conclude that ERT may improve proximal skin innervation in patients with good renal function, but does not protect small fiber function in men with impaired renal function.

The functional significance of missense mutations in genes encoding acid glycosidases of lysosomal storage disorders (LSDs) is not always clear. Here we describe a method of investigating functional properties of variant enzymes in vitro using a human embryonic kidney epithelial cell line. Site-directed mutagenesis was performed on the parental plasmids containing cDNA encoding for alpha-galactosidase A (α-Gal A) and acid maltase (α-Glu) to prepare plasmids encoding relevant point mutations. Mutant plasmids were transfected into HEK 293 T cells, and transient over-expression of variant enzymes was measured after 3 days. We have illustrated the method by examining enzymatic activities of four unknown α-Gal A and one α-Glu variants identified in our patients with Anderson-Fabry disease and Pompe diseases respectively. Comparison with control variants known to be either pathogenic or non-pathogenic together with over-expression of wild-type enzyme allowed determination of the pathogenicity of the mutation. One leader sequence novel variant of α-Gal A (p.A15T) was shown not to significantly reduce enzyme activity, whereas three other novel α-Gal A variants (p.D93Y, p.L372P and p.T410I) were shown to be pathogenic as they resulted in significant reduction of enzyme activity. A novel α-Glu variant (p.L72R) was shown to be pathogenic as this significantly reduced enzyme activity. Certain acid glycosidase variants that have been described in association with late-onset LSDs and which are known to have variable residual plasma and leukocyte enzyme activity in patients appear to show intermediate to low enzyme activity (p.N215S and p.Q279E α-Gal A respectively) in the over-expression system.

The Fabry Outcome Survey (FOS) was established to extend the knowledge of the natural history of Fabry disease and to assess the effects of enzyme replacement therapy (ERT) with agalsidase alfa. As of March 2009, 64 boys and 34 girls with Fabry disease had enrolled in the FOS and been treated with agalsidase alfa for at least 6 months. The prevalence of symptoms tended to be reduced after 12 and 24 months of ERT in patients who experienced symptoms at baseline. In the entire population, non-significant decreases in the prevalence of gastrointestinal problems in boys and pain crises in girls were observed after 12-24 months. Kidney function and left ventricular mass indexed to height remained stable. Fifty-eight treatment-related adverse events were reported in 23 patients (21 boys and 2 girls), including 55 infusion reactions. Anti-agalsidase alfa IgG antibodies were found in two boys. No IgE antibodies were reported. This study represents the largest observational study of paediatric Fabry disease patients treated with ERT and indicates continued safety of long-term ERT in children. Continued long-term follow-up is recommended to determine early initiation of ERT, which could potentially slow or prevent the progression of serious morbidities of Fabry disease.

Eur J Clin Invest 2011; 41 (7): 710–718 Abstract Aims This study estimated the resource implications and budget impact of managing adults with Fabry disease in Italy, from the perspective of the Servizio Sanitario Nazionale (SSN). Methods A decision model was constructed using published clinical outcomes and clinician-derived resource utilisation estimates depicting the management of adults with Fabry disease in Italy. Results The expected annual cost of managing 220 existing and 20 new Fabry patients in Italy was estimated to be €28·3 million. In an average year, patients receiving enzyme replacement therapy (ERT) with 0·2 mg kg−1 agalsidase alfa (Replagal®; Shire Human Genetic Therapies, Basingstoke, Hampshire, UK) or 1·0 mg kg−1 agalsidase beta (Fabrazyme®; Genzyme Europe BV, Naarden, The Netherlands) are collectively expected to make 4500 hospital attendances to a day ward for infusions, which equates to 2000 eight-h days on the day ward associated with ERT. If all ERT-treated patients received their infusions at home, there would be a marginal reduction in the annual health care cost to manage these patients, and the total annual number of days on the day ward associated with ERT in the second year could potentially be reduced from a mean 2000 to zero, thereby releasing substantial hospital resources for use by non-Fabry patients. Currently, only agalsidase alfa is licensed for home treatment in Italy; hence, only patients receiving this enzyme could be offered home treatment. Conclusions Use of agalsidase alfa (0·2 mg kg−1) instead of agalsidase beta (1·0 mg kg−1) has the potential to reduce health care costs and release hospital resources in different specialities for alternative use by non-Fabry patients, thereby improving the efficiency of the public health care system in Italy.

Selective monofluorination of the α-glycosidase inhibitor and antidiabetic agent miglitol at positions C(2') or C(6) creates competitive inhibitors of glycosidases. Introducing a fluorine substituent at position C(6) results in a reduced binding to the enzyme whereas fluorination at C(2') produces an inhibitor with an activity four times higher than the parent compound. This compound is selective for the α-galactosidase from green coffee beans. Its screening against a panel of human cell lines showed a low cytotoxicity, therefore, making this compound an interesting candidate for further clinical investigations.

Background: Early occurrence of small-fibre neuropathy (SFN) is a common feature of Fabry disease (FD) – an X-linked storage disorder caused by reduced activity of the α-galactosidase A (α-GAL). Although SFN may result from different disorders, the cause is often unclear. Therefore, we investigated the frequency of FD in patients with SFN of unknown aetiology. Methods: Patients with idiopathic SFN, established by sensory quantitative testing and/or skin biopsy, were examined for mutations in the α-GAL gene. Where mutations in the α-GAL gene were identified, levels of globotriaosylceramide (Gb3) were measured in urine and blood and the α-GAL activity was evaluated. When new mutations were detected, a diagnostic work-up was performed as well as a Gb3 accumulation in the skin, lyso-Gb3 in blood and Gb3_24 in urine were proved. Results: Twenty-four of 29 eligible patients were enrolled in the study. Mutations in the α-GAL gene were observed in five patients. A typical mutation for FD (c.424T>C, [C142R]) was detected in one patient. In four patients, a complex intronic haplotype within the α-GAL gene (IVS0-10C>T [rs2071225], IVS4-16A>G [rs2071397], IVS6-22C>T [rs2071228]) was identified. The relevance of this haplotype in the pathogenesis of FD remains unclear until now. However, these patients showed increased concentrations of Gb3 and/or lyso-Gb3, while no further manifestations for FD could be proved. Conclusions: Fabry disease should be considered in patients with SFN of unknown aetiology, and screening for FD should be included in the diagnostic guidelines for SFN. The significance of the intronic haplotype regarding SFN needs further evaluation.

Newborn screening for Fabry disease in Taiwan Chinese has revealed a high incidence of the late-onset GLA mutation IVS4 + 919G→A (∼1 in 1,500–1,600 males). We studied 94 adults with this mutation [22 men, 72 women; mean age: men 57.8 ± 6.0 (range 42–68), women 39.1 ± 14.1 years (range 19–82)]. Plasma α-galactosidase A activity assay was 10.4 ± 11.2% of normal in the men and 48.6 ± 19.5% of normal in the women. Echocardiography in 90 of the adults revealed left ventricular hypertrophy (LVH) in 19 (21%), including 14 of 21 men (67%) and 5 of 69 women (7%). Microalbuminuria, based on the urine albumin-to-creatinine ratio measured on at least two occasions, was present in 17 of 86 subjects (20%) (men: 5/20, 25%; women 12/66, 18%). At least one ocular manifestation consistent with Fabry disease was present in 41 of 52 subjects (79%) who underwent ophthalmologic examination, including 8 (15%) with conjunctival vessel tortuosity, 15 (29%) with cornea verticillata, 10 (19%) with Fabry cataract, and 34 (65%) with retinal vessel tortuosity. Among subjects over 40 years of age, men were more likely than women to have LVH [14/21 (67%) vs 5/25 (20%), p < 0.001]. Cardiovascular, renal and ocular abnormalities are highly prevalent in adult Taiwan Chinese subjects with the Fabry mutation IVS4 + 919G→A. Our findings contribute to the limited understanding of the course of this late-onset disease variant and underscore the need for close follow up in such patients.

A microassay method for early prenatal diagnosis of inborn error of metabolism using cultured amniotic cells was developed and Fabry's disease was diagnosed prenatally. After several days of culture small freeze-dried cell samples (about 1 μg of dry weight) were prepared and weighed on a quartz fiber fish-pole balance, then incubated in 5μl of assay mixture in the microtube for a-galactosidase. Severe deficiency of the enzyme activity and male karyotype suggested that the fetus was hemizygous. After termination of pregnancy, diagnosis was confirmed by proving the enzyme deficiency in various tissues. Morphological study revealed that abnormal accumulation started early in the prenatal stage.

Objective The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. Materials and methods Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. Results We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. Conclusions However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.