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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.

Purpose Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. Methods Between 1 July 2014 and 31 December 2015, we screened 2,360 patients (1,324 males) referred from 169 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. Results Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml–1) and low α-Gal A activity (≤4.0 nmol h–1 ml–1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 15 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 8 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). Conclusion Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Fabry disease is characterized by lyso-Gb3 accumulation, gastrointestinal symptoms and severe nephropathy and myocardiopathy. Clinically relevant lyso-Gb3 concentrations disrupt the gut microbiota homeostasis. Biofilm formation by enteric bacteria is modulated, favoring Bacteroides fragilis growth. In complex gut bacterial communities, lyso-Gb3 reduced bacteria associated with a healthy microbiota (Bifidobacterium, Akkermansia, Lactobacillus, and butyrate-producing Blautia coccoides-Eubacterium rectale, and Clostridium leptum) and butyrate production. We now discuss the clinical and pathophysiological implications of these findings as butyrate has anti-inflammatory properties through epigenetic regulation of histone acetylation that may protect against nephropathy and myocardiopathy. Lyso-Gb3 modulation of the gut microbiota may directly contribute to the gastrointestinal symptoms and indirectly to systemic manifestations of Fabry disease through modulation of the epigenetic regulation of inflammation., JJAC is funded by an FPI grant from Spanish Ministry of Economics and Competitiveness (BES-2014-069007). The authors were further supported by FIS PI15/00298, PI16/02057, PI18/01366, PI19/00815, DTS18/00032, ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064), ISCIII-RETIC REDinREN RD016/0009 Fondos FEDER, Sociedad Española de Nefrología, Comunidad de Madrid B2017/BMD-3686 CIFRA2-CM, Miguel Servet MS14/00133 to MDSN and AGL2016-75951-R.

Fabry disease is a rare X-linked lysosomal storage disorder resulting from deficient activity of α-galactosidase A, leading to the accumulation of glycosphingolipids such as globotriaosylsphingosine (lyso-Gb3). The gastrointestinal symptoms of this disease may be disabling, and the life expectancy of affected patients is shortened by kidney and heart disease. Our hypothesis was that lyso-Gb3 may modify the gut microbiota. The impact of a clinically relevant concentration of lyso-Gb3 on mono- or multispecies bacterial biofilms were evaluated. A complex bacterial community from the simulated transverse colon microbiota was studied using quantitative PCR to estimate different bacterial group concentrations and a HPLC was used to estimate short-chain fatty acids concentrations. We found that lyso-Gb3 increased the biofilm-forming capacity of several individual bacteria, including Bacteroides fragilis and significantly increased the growth of B. fragilis in a multispecies biofilm. Lyso-Gb3 also modified the bacterial composition of the human colon microbiota suspension, increasing bacterial counts of B. fragilis, among others. Finally, lyso-Gb3 modified the formation of short-chain fatty acids, leading to a striking decrease in butyrate concentration. Lyso-Gb3 modifies the biology of gut bacteria, favoring the production of biofilms and altering the composition and short-chain fatty-acid profile of the gut microbiota.

Globotriaosylsphingosine (lyso-Gb3) is a well-established biomarker for diagnosis and prognosis of Fabry disease. This biomarker is measured in biological samples by liquid chromatography-tandem mass spectrometry using an internal standard. The ideal internal standard is a variant of lyso-Gb3 substituted with heavy isotopes, but the total synthesis of such a compound is very labor intensive. In this report, we describe a simple, one-step synthesis of lyso-Gb3 labeled with carbon-13 in all of the galactosyl carbons.

Fabry disease (FD), an X-linked lysosomal storage disease, results from an α-galactosidase A deficiency and altered sphingolipid metabolism. An accumulation of globotriaosylsphingosine (lyso-Gb3) likely triggers the pathological cascade leading to disease phenotype. The pathogenic significance of several Fabry mutations including the R118C α-galactosidase (GLA) gene variant has been disputed. We describe three members of the same family with the R118C variant, each having documented clinical signs of FD, low residual enzyme levels, and an elevated lyso-Gb3 in one heterozygote.Determining the clinical significance of each GLA gene variant remains an ongoing challenge, with potential for inadequate treatment if the diagnosis of FD is missed. Elevated lyso-Gb3 has been shown to be the most reliable noninvasive marker of clinically relevant GLA variants. While the R118C variant will likely lead to a milder phenotype, additional genetic, epigenetic, and environmental factors can ameliorate or exacerbate the expression and impact on the resultant phenotype and associated complications. Patients affected with this variant warrant closer review and better management of disease risk factors.

Background— Currently, no method is available to identify α-galactosidase A (agalA) mutations determining clinically relevant Fabry disease. In our largest European Fabry cohort, we investigated whether a biomarker, specific for the defect, could stratify persons at risk. Methods and Results— A total of 124 individuals with agalA mutations were investigated with a comprehensive clinical workup, genetic analysis, and laboratory testing, including measurements of agalA activity and lyso-Gb3 (degradation product of the accumulating Gb3). Additionally, an extensive family screening with a clinical workup of relatives was performed. The patient population was divided into 2 samples: previously described mutations (n=72) and novel mutations (n=52). The patients with previously described mutations were subdivided into 2 groups: classical mutations, which were known to cause the classic type of Fabry disease with specific symptoms and a high risk for major events in all 3 main organs (heart, kidney, and central nervous system), and atypical mutations without the typical presentation. All patients with atypical mutations (n=17) had lower lyso-Gb3 levels than any of the patients with classical Fabry disease (n=55). A cutoff value of 2.7 ng/mL separated the 2 groups. Six out of 52 patients with novel mutations showed a lyso-Gb3 level Conclusions— Our data show that the biomarker lyso-Gb3 may identify the clinically relevant agalA mutations leading to Fabry disease.

Fabry disease is a complex, multisystemic and clinically heterogeneous disease, with elevated excretion of globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)) accumulating in biological fluids caused by deficiency of the enzyme, lysosomal α-galactosidase A. Our aims were to propose a tandem mass spectrometry fragmentation mechanism for lyso-Gb(3), to develop and validate a simple, and robust methodology for the measurement of plasma lyso-Gb(3) using LC-MS/MS in large Fabry cohorts and in controls. Response to treatment was also evaluated.A solid-phase extraction procedure was used to process plasma samples. The 1-β-D-glucosylsphingosine (GSG) internal standard was chosen for its commercial availability. A liquid chromatography method was devised to allow the co-elution of the GSG internal standard with lyso-Gb(3), thus compensating for system variability and reducing the matrix effect. A multiple reaction monitoring method was developed, working in positive electrospray ionization.The validation of the method provided good accuracy and precision: intraday and interday biases of less than 8% and 5%, respectively, and intraday and interday CVs of12% and 7%, respectively. Limit of detection was 0.7 nmol/l and limit of quantification was 2.5 nmol/l. Plasma samples were stable for up to 6h at room temperature, 48 h at 4 °C, and 20 weeks at -20 °C. Regarding untreated Fabry patients, the mean lyso-Gb(3) concentrations were 170 nmol/l for males and 9.7 nmol/l for females, and for treated patients, 40.2 nmol/l for males and 7.5 nmol/l for females.A robust LC-MS/MS methodology is presented for plasma lyso-Gb(3) quantification.

Fabry disease is a hereditary, progressive and multisystemic disorder characterized by variable clinical manifestations and a course that can lead to the death of the patient between the fourth and fifth decades of life if not promptly diagnosed. It is a metabolic, lysosomal storage disorder which is due to the functional deficit of the enzyme alpha-galactosidase A (alfa-GAL A). The deficit alters the metabolism of some glycosphingolipids, mainly globotriaosylceramide (Gb3) and lyso-Gb3 (the deacetylated form), causing their storage in lysosomes of various cell types, especially those of the vascular endothelium. The knowledge of this disease has improved in recent years and it is increasingly clear that it could be a more complex disorder than previously thought and that also other factors are likely to be involved in the onset of its symptoms. (aiaf)

Fabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb(3)), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb(3)), is increased in patients with the disease. Until now, lyso-Gb(3) was not detectable in urine, possibly because of the presence of interfering compounds.We undertook to: 1) characterize lyso-Gb(3) in urine; 2) develop a method to quantitate urinary lyso-Gb(3) by mass spectrometry; 3) evaluate urinary lyso-Gb(3) as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb(3) is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb(3) from 83 Fabry patients and 77 healthy age-matched controls.The intraday and interday bias and precision of the method were15%. Increases in lyso-Gb(3)/creatinine correlated with the concentrations of Gb(3) (r(2)=0.43), type of mutations (p=0.0006), gender (p0.0001) and enzyme replacement therapy status (p=0.0012). Urine from healthy controls contained no detectable lyso-Gb(3). Lyso-Gb(3) did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb(3) of Fabry patients correlated well with a number of indicators of disease severity.Lyso-Gb(3) is a reliable independent biomarker for clinically important characteristics of Fabry disease.

The X-chromosomal-linked lysosomal storage disorder Fabry disease can lead to life-threatening manifestations. The pathological significance of the Fabry mutation D313Y is doubted, because, in general, D313Y patients do not present clinical manifestations conformable with Fabry disease. This is in contrast to the analysis of the alpha-galactosidase A activity, which is reduced in D313Y patients. We report a comprehensive clinical, biochemical and molecular genetic analysis of two patients with a D313Y mutation. The alpha-galactosidase A activity was reduced in both patients. No Fabry symptoms or Fabry organ involvement was detected in these patients. The new biomarker lyso-Gb3, severely increased in classical Fabry patients, was determined and in both patients lyso-Gb3 was below the average of a normal population.Our data for the first time not only clinically but also biochemically supports the hypothesis that the D313Y mutation is not a classical one, but a rare variant mutation.

To neutralize the toxicity of verotoxin (VT) produced by Escherichia coli type O-157, a soluble pseudo-receptor (Lyso Gb3) was synthesized with the deacylated form of the natural receptor, globotrisylceramide (Galalpha 1-4Galbeta1-4-glucosylceramide; Gb3). In this study, we evaluated the characteristics and pharmacological effects of Lyso Gb3, using VT2. It was confirmed that Lyso Gb3 specifically recognized VT2. Lyso Gb3 itself had little influence on the in-vitro growth of Vero cells, but markedly augmented VT2-induced cytotoxicity. In addition, the VT2-induced killing of mice was not decreased, but was, rather, increased by Lyso Gb3. These results indicate that the soluble pseudo-receptor Lyso Gb3 recognized VT2. However, it did not reduce, but, rather, enhanced, VT2-induced toxicity in the presence of the natural receptor, although Lyso Gb3 alone had no toxicity.