Vitamin C Urinary Loss in Fabry Disease: Clinical and Genomic Characteristics of Vitamin C Renal Leak.

BACKGROUND: Reduced plasma vitamin C concentrations in chronic diseases may result from abnormal urinary excretion of vitamin C: a renal leak. We hypothesized that vitamin C renal leak may be associated with disease-mediated renal dysregulation, resulting in aberrant vitamin C renal reabsorption and increased urinary loss. OBJECTIVES: We investigated the prevalence, clinical characteristics, and genomic associations of vitamin C renal leak in Fabry disease, an X-linked lysosomal disease associated with renal tubular dysfunction and low plasma vitamin C concentrations. METHODS: We conducted a non-randomized cross-sectional cohort study of men aged 24-42 y, with Fabry disease (n = 34) and controls without acute or chronic disease (n = 33). To match anticipated plasma vitamin C concentrations, controls were placed on a low-vitamin C diet 3 wk before inpatient admission. To determine the primary outcome of vitamin C renal leak prevalence, subjects were fasted overnight, and matched urine and fasting plasma vitamin C measurements were obtained the following morning. Vitamin C renal leak was defined as presence of urinary vitamin C at plasma concentrations below 38 µM. Exploratory outcomes assessed the association between renal leak and clinical parameters, and genomic associations with renal leak using single nucleotide polymorphisms (SNPs) in the vitamin C transporter SLC23A1. RESULTS: Compared with controls, the Fabry cohort had 16-fold higher odds of renal leak (6% vs. 52%; OR: 16; 95% CI: 3.30, 162; P < 0.001). Renal leak was associated with higher protein creatinine ratio (P < 0.01) and lower hemoglobin (P = 0.002), but not estimated glomerular filtration rate (P = 0.54). Renal leak, but not plasma vitamin C, was associated with a nonsynonymous single nucleotide polymorphism in vitamin C transporter SLC23A1 (OR: 15; 95% CI: 1.6, 777; P = 0.01). CONCLUSIONS: Increased prevalence of renal leak in adult men with Fabry disease may result from dysregulated vitamin C renal physiology and is associated with abnormal clinical outcomes and genomic variation.

Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy.

Current biomarkers of Fabry nephropathy lack sensitivity in detecting early kidney damage and do not predict progression of nephropathy. Urinary extracellular vesicles (uEVs) and their molecular cargo could reflect early changes in renal impairment as they are secreted by the cells lining the urinary tract. We aimed to conduct a proof-of-concept study to investigate whether analysis of uEV characteristics and expression of uEV-derived microRNAs (miRNAs) could be applicable in studies to predict the development and progression of nephropathy in Fabry disease. A total of 20 Fabry patients were divided into two groups, depending on the presence of nephropathy. Chronological urine samples collected during 10-year follow-up were used for uEVs isolation with size exclusion chromatography. Nanoparticle tracking analysis was used to determine concentration and size of uEVs. We evaluated the expression of five uEV-derived miRNAs by qPCR (miR-23a-3p, miR-29a-3p, miR-30b-5p, miR-34a-5p, miR-200a-3p). There was no difference in the concentration and size of uEVs between patients with and without nephropathy at last follow-up or longitudinally. However, we found increased expression of miR-29a-3p and miR-200a-3p in uEVs isolated from chronological samples of patients with Fabry nephropathy. This may indicate an attempt by the organism to prevent the progression of renal damage leading to end-stage renal disease as previously reported in type 1 diabetes. In addition, we found an increased expression of miR-30b-5p in the 10-year period in uEVs of patients without renal dysfunction. miR-30b-5 was reported to have a protective role in podocyte injury and may possibly be important in Fabry nephropathy. These findings indicate that uEVs and their molecular cargo could be a promising target of studies focusing on elucidation of Fabry nephropathy. Nevertheless, total concentration and size of uEVs were neither indicative of the presence nor progression of Fabry nephropathy, while the role of the analyzed miRNAs in Fabry nephropathy progression was merely indicated and needs further in-depth studies.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

BACKGROUND: Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. METHODS: We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. RESULTS: Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). CONCLUSION: We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.

"Protenuria in SLE: Is it always lupus?"

Proteinuria is one of the most typical manifestations of kidney involvement in Systemic Lupus Erythematosus (SLE). We report the case of a 23-year-old woman with a 6-year-long history of SLE presenting with proteinuria after a three-year remission on hydroxychloroquine. Kidney histological examination showed alterations inconsistent with lupus nephritis and suggestive of hydroxychloroquine toxicity or Fabry disease. The latter was confirmed by genetic assay.

Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (-C2H4, -C2H4+O, -H2, -H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

Heat shock protein 90 and NFkB levels in serum and urine in patients with chronic glomerulonephritis.

Heat shock proteins play an important role in immune inflammation and the formation and restoration of proteins. In recent years, the importance of heat shock protein 90 (Hsp90) in the activation of immune inflammation through nuclear factor kB (NFkB) has been discussed. To assess the activation of the Hsp90-NFkB system by measuring serum and urinary levels in patients with chronic glomerulonephritis (CGN). This study included 32 patients with active forms of CGN and 14 patients with Fabry nephropathy. The control group included 10 healthy individuals. Twenty-one out of 32 CGN patients had nephrotic syndrome (NS). Eleven out of 32 CGN patients had proteinuria levels from 1 to 3 g/day without nephrotic syndrome. A total of 17 patients had renal dysfunction (estimated glomerular filtration rate < 60 ml/min/1.73m2). Fourteen patients with Fabry nephropathy had proteinuria without nephrotic syndrome. Serum and urine HSP-90 and NFkB p65 levels were determined using an enzyme-linked immunosorbent assay. The levels of HSP-90 and NFkB in the serum of patients with CGN were significantly higher than in healthy individuals and patients with Fabry nephropathy. In patients with Fabry nephropathy, the HSP-90 and NFkB levels in the urine and serum did not significantly differ from those in the control subjects. Serum Hsp90 levels were significantly higher in the CGN patients with NS than in patients without NS, as well as in patients with normal renal function compared with patients with an eGFR < 60 ml/min/1.73 m2 and patients with tubulo-interstitial fibrosis. Higher levels of HSP-90 and NFkB in serum were observed in patients with nephrotic forms of CGN, including focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy. There were no correlations between the clinical signs of CGN and urinary HSP90/NFkB levels. Activation of the HSP-90-NFkB system, which is directly involved in the development of immune inflammation in CGN, was found in patients with an active course of CGN, especially in those with nephrotic syndrome.

Rapid, proteomic urine assay for monitoring progressive organ disease in Fabry disease.

BACKGROUND: Fabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker for monitoring treatment is plasma globotriaosylsphingosine (lyso-Gb3), but it is not informative about the underlying and developing disease pathology. METHODS: We have created a urine proteomic assay containing a panel of biomarkers designed to measure disease-related pathology which include the inflammatory system, lysosome, heart, kidney, endothelium and cardiovascular system. Using a targeted proteomic-based approach, a series of 40 proteins for organ systems affected in Fabry disease were multiplexed into a single 10 min multiple reaction monitoring Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) assay and using only 1 mL of urine. RESULTS: Six urinary proteins were elevated in the early-stage/asymptomatic Fabry group compared with controls including albumin, uromodulin, α1-antitrypsin, glycogen phosphorylase brain form, endothelial protein receptor C and intracellular adhesion molecule 1. Albumin demonstrated an increase in urine and could indicate presymptomatic disease. The only protein elevated in the early-stage/asymptomatic patients that continued to increase with progressive multiorgan involvement was glycogen phosphorylase brain form. Podocalyxin, fibroblast growth factor 23, cubulin and Alpha-1-Microglobulin/Bikunin Precursor (AMBP) were elevated only in disease groups involving kidney disease. Nephrin, a podocyte-specific protein, was elevated in all symptomatic groups. Prosaposin was increased in all symptomatic groups and showed greater specificity (p<0.025-0.0002) according to disease severity. CONCLUSION: This work indicates that protein biomarkers could be helpful and used in conjunction with plasma lyso-Gb3 for monitoring of therapy or disease progression in patients with Fabry disease.

The clinical utility of total concentration of urinary globotriaosylsphingosine plus its analogues in the diagnosis of Fabry disease.

BACKGROUND: Fabry disease (FD) is a genetic disorder caused by defective α-galactosidase-A enzyme due to mutations in the GLA gene. A reliable diagnosis in classical FD males can be made by measuring the enzyme activity while diagnosing classical FD females and non-classical FD patients requires mutation analysis. Plasma globotriaosylsphingosine (Lyso-Gb3) has progressively gained more importance as a diagnostic biomarker for FD in recent years. Having another biomarker to complement plasma Lyso-Gb3 will increase the diagnostic accuracy in the era of mass screening, and also precision medicine. This study aims to highlight the clinical utility of the total concentration of urinary Lyso-Gb3 plus its analogues in diagnosing FD. METHOD: Random urine samples collected from 42 FD patients and 48 healthy individuals. Lyso-Gb3 and its analogues were enriched by solid phase extraction and analysed by liquid chromatography tandem mass spectrometry. RESULTS: The total concentration of Lyso-Gb3 plus its analogues in classical FD male and female patients were 1124.4 ±â€¯181.2 and 308.6 ±â€¯78.6 pmol/mmol creat., respectively. The levels in non-classical FD male and female patients were 229.2 ±â€¯169.4 and 314.4 ±â€¯156.4 pmol/mmol creat., respectively. Urinary Lyso-Gb3 and its analogues were virtually undetectable in healthy volunteers making it 100% specific for FD diagnosis. For FD male and female patients, total concentration of urinary Lyso-Gb3 plus its analogue levels ≥0.25 pmol/mmol creat. yielded a diagnostic sensitivity and specificity of 100% (AUC = 1, p < 0.00001). CONCLUSIONS: Our study findings support that the total concentration of Lyso-Gb3 plus its analogues in urine is specific to FD and may provide extra diagnostic utility for both classical and non-classical FD patients.

High-risk screening for Fabry disease in a Canadian cohort of chronic kidney disease patients.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder with a highly heterogeneous clinical presentation. This complex disease is caused by a deficient activity of the enzyme α-galactosidase A, which is involved in the catabolism of glycosphingolipids. The prevalence of Fabry disease is underestimated, due to the presence of atypical variants. High-risk screening protocols are particularly relevant for this disease due to the availability of treatments, such as enzyme replacement and chaperone therapies. As kidney manifestations are present in the majority of male and many female patients with Fabry disease, a high-risk screening protocol was performed for patients with chronic kidney disease of unknown etiology. METHODS: Recruitment of 397 participants took place in four centers across Canada from 2011 to 2017. Globotriaosylceramide (Gb3) was analyzed in dried urine spots by liquid chromatography/tandem mass spectrometry followed by globotriaosylsphingosine (lyso-Gb3) on the repeat analysis. RESULTS: The collection and shipment of urine specimens on filter paper resulted in easier handling/shipment and significant cost-saving. No Fabry patients were detected in this study. CONCLUSIONS: Increased concentrations of urinary Gb3 were observed in 13.6% of patients with chronic kidney disease suggesting that chronic kidney disease or other comorbidities might be associated with increased urinary Gb3 concentrations.

Global glycosphingolipid analysis in urine and plasma of female Fabry disease patients.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase-A, which results in accumulation of the glycosphingolipid (GSL) globotriaosylceramide (Gb3). Gb3 and globotriaosylsphingosine (lyso-Gb3) levels in plasma and urine are used routinely for diagnosis and treatment monitoring. FD female patients are problematic to diagnose and to predict when to begin treatment. Further biomarkers are needed to detect pre-symptomatic females that will develop the chronic symptoms associated with FD. A LC-MS/MS glycosphingolipidomic assay was developed to measure lyso-Gb3 and GSLs from the lysosomal GSL degradation pathway, including globoside (Gb4), Gb3, ceramide dihexosides (CDH) and ceramide monohexosides (CMH). We analysed plasma and urine from a cohort of Fabry patients, grouped according to clinical symptoms and independent of treatment status (asymptomatic females n = 18, symptomatic females n = 18, males n = 27 and control urines n = 16 and control plasmas n = 58). Multivariate and subsequent univariate analysis showed urine GSLs which had highest significance in identifying asymptomatic females were total levels of CDH, in particular the long chain isoforms C22:1,C22:0,C22:1-OH,C22:0-OH,C24:2,C24:0,C24:2-OH,C24:1-OH,C24:0-OH,C26:0 which likely represent Galabiosylceramide (Ga2) and not lactosylceramide. These long chain Ga2 isoforms were found to be 5-fold elevated and more statistically significant (p < 0.0001) than plasma lyso-Gb3 (p < 0.01) in identifying asymptomatic Fabry female patients. Receiver operating characteristic curve analysis gave an area under the curve of 0.82 (p = 0.001) for lyso-Gb3 and 0.88 (p = 0.0006) for long-chain CDH isoforms indicating the long chain CDH isoforms were as, if not more, a better biomarker for the identification of female FD patients.

Podocituria en pacientes pediátricos con enfermedad de Fabry / Podocyturia in paediatric patients with Fabry disease

Introducción: La enfermedad de Fabry (EF) es un trastorno hereditario causado por una deficiencia de la actividad de la enzima alfa-galactosidasa A, cuya transmisión está relacionada con el cromosoma X. Objetivos: Los objetivos del estudio fueron: 1. Cuantificar la presencia de podocitos en pacientes pediátricos con EF y compararla con el valor de la podocituria medida en controles sanos. 2. Determinar en pacientes con EF si una mayor podocituria está relacionada con la albuminuria patológica. 3. Determinar los factores de riesgo asociados con la albuminuria patológica. Métodos: Implementamos un estudio analítico observacional de casos y controles, separados en 2 grupos de acuerdo con la ausencia de enfermedad (grupo control) o con la presencia de enfermedad (grupo Fabry). Resultados: Estudiamos a 31 pacientes, 11 con EF y 20 controles, con una media de edad de 11,6 años. La diferencia entre el tiempo medio transcurrido desde el diagnóstico de EF hasta la medición de la podocituria (40 meses) y la aparición de la albuminuria patológica (34 meses) no fue significativa (p: 0,09). Los podocitos se identificaron mediante tinción para sinaptopodina y las diferencias medias cuantitativas entre ambas podociturias fueron estadísticamente significativas (p: 0,001). La albuminuria fue fisiológica en 4 de las pacientes Fabry y el riesgo relativo para desarrollar albuminuria patológica de acuerdo con la podocituria fue en el grupo control 1,1 y en el grupo Fabry 3,9, con un coeficiente de correlación entre la podocituria y la albuminuria en el grupo Fabry de 0,8354. Finalmente los 2 factores de riesgo asociados al desarrollo de albuminuria patológica fueron la podocituria (OR 14) y la edad mayor a 10 años (OR 18). No encontramos riesgo significativo ni en el filtrado glomerular (FG) (OR 0,5), ni en el género (OR 1,3). El FG medio se mantuvo dentro de valores normales. Conclusión: La detección de podocituria en pacientes pediátricos con EF podría utilizarse como un marcador temprano de daño renal previo y relacionado con la albuminuria patológica

Introduction: Fabry disease (FD) is a hereditary disorder caused by a deficiency of α-galactosidase A enzyme activity. The transmission of the disorder is linked to the X chromosome. Objectives: The objectives of the study were: 1. To quantify the presence of podocytes in paediatric patients with FD and compare them with the value of the measured podocyturia in healthy controls. 2. To determine whether a greater podocyturia is related to the onset of pathological albuminuria in patients with FD. 3. To determine the risk factors associated with pathological albuminuria. Methods: We performed an analytical, observational study of Fabry and control subjects, which were separated into 2groups in accordance with the absence of the disease (control group) or the presence of the disease (Fabry group). Results: We studied 31 patients, 11 with FD and 20 controls, with a mean age of 11.6 years. The difference between the mean time elapsed from the diagnosis of FD to the measurement of podocyturia (40 months) and the onset of pathological albuminuria (34 months) was not significant (p = 0.09). Podocytes were identified by staining for the presence of synaptopodin and the mean quantitative differences between both podocyturias were statistically significant (p = 0.001). Albuminuria was physiological in 4 of the patients with FD and the relative risk to develop pathological albuminuria according to podocyturia was 1.1 in the control group and 3.9 in the Fabry group, with a coefficient of correlation between podocyturia and albuminuria in the Fabry group of 0.8354. Finally, the 2 risk factors associated with the development of pathological albuminuria were podocyturia (OR: 14) and being aged over 10 years (OR: 18). We found no significant risk with regard to glomerular filtrate renal (GFR) (OR: 0.5) or gender (OR: 1.3). The mean GFR remained within normal values. Conclusion: The detection of podocyturia in paediatric patients with FD could be used as an early marker of renal damage, preceding and proportional to the occurrence of pathological albuminuria

Tandem mass spectrometry analysis of urinary podocalyxin and podocin in the investigation of podocyturia in women with preeclampsia and Fabry disease patients.

BACKGROUND: Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable. METHOD: A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated. RESULTS: The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia. CONCLUSIONS: This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.

Pharmacokinetics, pharmacodynamics, and safety of moss-aGalactosidase A in patients with Fabry disease.

Moss-aGalactosidase A (moss-aGal) is a moss-derived version of human α-galactosidase developed for enzyme replacement therapy in patients with Fabry disease. It exhibits a homogenous N-glycosylation profile with >90% mannose-terminated glycans. In contrast to mammalian cell produced α-galactosidase, moss-aGal does not rely on mannose-6-phosphate receptor mediated endocytosis but targets the mannose receptor for tissue uptake. We conducted a phase 1 clinical trial with moss-aGal in six patients with confirmed diagnosis of Fabry disease during a 28-day schedule. All patients received a single dose of 0.2 mg/kg moss-aGal by i.v.-infusion. Primary endpoints of the trial were safety and pharmacokinetics; secondary endpoints were pharmacodynamics by analyzing urine and plasma Gb3 and lyso-Gb3 concentrations. In all patients, the administered single dose was well tolerated. No safety issues were observed. Pharmacokinetic data revealed a stable nonlinear profile with a short plasma half-life of moss-aGal of 14 minutes. After one single dose of moss-aGal, urinary Gb3 concentrations decreased up to 23% 7 days and up to 60% 28 days post-dose. Plasma concentrations of lyso-Gb3 decreased by 3.8% and of Gb3 by 11% 28 days post-dose. These data reveal that a single dose of moss-aGal was safe, well tolerated, and led to a prolonged reduction of Gb3 excretion. As previously shown, moss-aGal is taken up via the mannose receptor, which is expressed on macrophages but also on endothelial and kidney cells. Thus, these data indicate that moss-aGal may target kidney cells. After these promising results, phase 2/3 clinical trials are in preparation.

Podocyturia in paediatric patients with Fabry disease. / Podocituria en pacientes pediátricos con enfermedad de Fabry.

INTRODUCTION: Fabry disease (FD) is a hereditary disorder caused by a deficiency of α-galactosidase A enzyme activity. The transmission of the disorder is linked to the X chromosome. OBJECTIVES: The objectives of the study were: 1. To quantify the presence of podocytes in paediatric patients with FD and compare them with the value of the measured podocyturia in healthy controls. 2. To determine whether a greater podocyturia is related to the onset of pathological albuminuria in patients with FD. 3. To determine the risk factors associated with pathological albuminuria. METHODS: We performed an analytical, observational study of Fabry and control subjects, which were separated into 2groups in accordance with the absence of the disease (control group) or the presence of the disease (Fabry group). RESULTS: We studied 31 patients, 11 with FD and 20 controls, with a mean age of 11.6 years. The difference between the mean time elapsed from the diagnosis of FD to the measurement of podocyturia (40 months) and the onset of pathological albuminuria (34 months) was not significant (p=0.09). Podocytes were identified by staining for the presence of synaptopodin and the mean quantitative differences between both podocyturias were statistically significant (p=0.001). Albuminuria was physiological in 4 of the patients with FD and the relative risk to develop pathological albuminuria according to podocyturia was 1.1 in the control group and 3.9 in the Fabry group, with a coefficient of correlation between podocyturia and albuminuria in the Fabry group of 0.8354. Finally, the 2 risk factors associated with the development of pathological albuminuria were podocyturia (OR: 14) and being aged over 10 years (OR: 18). We found no significant risk with regard to glomerular filtrate renal (GFR) (OR: 0.5) or gender (OR: 1.3). The mean GFR remained within normal values. CONCLUSION: The detection of podocyturia in paediatric patients with FD could be used as an early marker of renal damage, preceding and proportional to the occurrence of pathological albuminuria.

Clinical parameters, LysoGb3, podocyturia, and kidney biopsy in children with Fabry disease: is a correlation possible?

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase enzyme deficiency. We present clinical, biochemical, and histologic findings in children with classical phenotypic presentation of Fabry disease. METHODS: A retrospective analysis was performed using charts from 14 children with confirmed diagnosis. Clinical parameters were evaluated. Globotriaosylsphingosine -lysoGb3- detection in plasma, podocyturia, and kidney biopsy were carried out in all cases. RESULTS: All patients except one demonstrated at least one symptom of Fabry disease. LysoGb3 levels were above the normal range in all patients. Podocyturia was documented in all patients. Kidney biopsy revealed glomerular, interstitial, vascular, and tubular changes on light microscopy in nearly all patients. Electron microscopy showed podocyte inclusions in all patients. CONCLUSIONS: No difference in symptomatology was discernible between boys and girls. Podocyturia was detectable in children serving as a possible early marker of kidney injury. LysoGb3 was elevated in all cases, emphasizing the importance for diagnosis especially in female patients with normal αGal A activity. A possible association between lysoGb3 and symptom severity and histological involvement in kidney biopsy should be assessed in prospective studies with enough statistical power to determine if lysoGb3 can be used to predict nephropathy in children with Fabry disease.

Urine-derived cells: a promising diagnostic tool in Fabry disease patients.

Fabry disease is a lysosomal storage disorder resulting from impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. Currently, powerful diagnostic tools and in vivo research models to study Fabry disease are missing, which is a major obstacle for further improvements in diagnosis and therapy. Here, we explore the utility of urine-derived primary cells of Fabry disease patients. Viable cells were isolated and cultured from fresh urine void. The obtained cell culture, modeling the renal epithelium, is characterized by patient-specific information. We demonstrate that this non-invasive source of patient cells provides an adequate cellular in vivo model as cells exhibit decreased α-Gal A enzyme activity and concomitant globotriaosylceramide accumulation. Subsequent quantitative proteomic analyses revealed dysregulation of endosomal and lysosomal proteins indicating an involvement of the Coordinated Lysosomal Expression and Regulation (CLEAR) network in the disease pathology. This proteomic pattern resembled data from our previously described human podocyte model of Fabry disease. Taken together, the employment of urine-derived primary cells of Fabry disease patients might have diagnostic and prognostic implications in the future. Our findings pave the way towards a more detailed understanding of pathophysiological mechanisms and may allow the development of future tailored therapeutic strategies.

Mulberries in the urine: a tell-tale sign of Fabry disease.

Fabry disease is a treatable progressive illness of inborn error causing eventual multiple organ dysfunction in advanced untreated cases. We report on a classic Fabry child patient presenting with urinary mulberry cells and bodies without renal involvement. This report emphasizes the usefulness of urinary microscopic findings in the early diagnosis of Fabry disease.

Globotriaosylsphingosine (Lyso-Gb3) as a biomarker for cardiac variant (N215S) Fabry disease.

Fabry disease (FD) is a multi-systemic X-linked lysosomal disorder caused by the deficient activity of α-galactosidase-A enzyme, which leads to accumulation of glycosphingolipids in various body tissues. The N215S mutation is a known variant of FD, with a late onset cardiac phenotype. Consensus guidelines acknowledged the use of globotriaosylsphingosine (Lyso-Gb3) as a diagnostic marker for classical FD but its utility for cardiac variant FD is not clear. We aim to characterize the clinical features and evaluate the diagnostic accuracy of plasma and urinary Lyso-Gb3 levels in N215S cardiac variant FD patients. Thirty-four FD patients with the late-onset N215S cardiac variant mutation were enrolled along with 62 classical FD patients and 109 healthy controls. Plasma and urinary Lyso-Gb3 and its analogues were analyzed by LC-MS/MS. Both FD males and females with N215S mutation showed Lyso-Gb3 levels of (mean ± SEM) 9.7 ± 1.0 and 5.4 ± 0.8 nM, respectively. These levels were significantly higher than healthy control and lower than classical FD patients (p < 0.0001). Plasma Lyso-Gb3 levels equal to or higher than 2.7 nM yielded a diagnostic sensitivity and specificity of 100% (AUC = 1, p < 0.0001). Cardiac involvement was frequent with 16/34 (47%) developing left ventricular hypertrophy. Three patients who underwent renal biopsy had the characteristic sphingolipid deposition in the podocytes while 6/19 (32%) had evidence of white matter changes or infarct on brain MRI. Taken together, cardiac variant N215S mutation is rather an attenuated form of classical FD. Plasma Lyso-Gb3 is a diagnostic hallmark to differentiate N215S variant phenotype from subjects with no FD.