Saskia M. Rombach

Elevated globotriaosylsphingosine is a hallmark of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of α-galactosidase A that affects males and shows disease expression in heterozygotes. The characteristic progressive renal insufficiency, cardiac involvement, and neuropathology usually are ascribed to globotriaosylceramide accumulation in the endothelium. However, no direct correlation exists between lipid storage and clinical manifestations, and treatment of patients with recombinant enzymes does not reverse several key signs despite clearance of lipid from the endothelium. We therefore investigated the possibility that globotriaosylceramide metabolites are a missing link in the pathogenesis. We report that deacylated globotriaosylceramide, globotriaosylsphingosine, and a minor additional metabolite are dramatically increased in plasma of classically affected male Fabry patients and plasma and tissues of Fabry mice. Plasma globotriaosylceramide levels are reduced by therapy. We show that globotriaosylsphingosine is an inhibitor of α-galactosidase A activity. Furthermore, exposure of smooth muscle cells, but not fibroblasts, to globotriaosylsphingosine at concentrations observed in plasma of patients promotes proliferation. The increased intima-media thickness in Fabry patients therefore may be related to the presence of this metabolite. Our findings suggest that measurement of circulating globotriaosylsphingosine will be useful to monitor Fabry disease and may contribute to a better understanding of the disorder.

Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-Galactosidase A, causing accumulation of globotriaosylceramide and elevated plasma globotriaosylsphingosine (lysoGb3). The diagnostic value and clinical relevance of plasma lysoGb3 concentration was investigated. All male and adult female patients with classical Fabry disease could be discerned by an elevated plasma lysoGb3. In young pre-symptomatic Fabry heterozygotes, lysoGb3 levels can be normal. Individuals carrying the R112H and P60L mutations, without classical Fabry symptoms, showed no elevated plasma lysoGb3. Multiple regression analysis showed that there is no correlation of plasma lysoGb3 concentration with total disease severity score in Fabry males. However, plasma lysoGb3 concentration did correlate with white matter lesions (odds ratio: 6.1 per 100 nM lysoGb3 increase (95% CI: 1.4-25.9, p=0.015). In females, plasma lysoGb3 concentration correlated with overall disease severity. Furthermore, plasma lysoGb3 level was related to left ventricular mass (19.5+/-5.5 g increase per 10 nM lysoGb3 increase; p=0.001). In addition, it was assessed whether lifetime exposure to lysoGb3 correlates with disease manifestations. Male Fabry patients with a high lysoGb3 exposure (>10,000 U), were moderately or severely affected, only one mildly. Female patients with a low exposure (<1000 U) were asymptomatic or mildly affected. A large proportion of the females with an exposure >1000 U showed disease complications. Plasma lysoGb3 is useful for the diagnosis of Fabry disease. LysoGb3 is an independent risk factor for development of cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in females. Disease severity correlates with exposure to plasma lysoGb3.

Reduction of elevated plasma globotriaosylsphingosine in patients with classic Fabry disease following enzyme replacement therapy

Fabry disease is treated by two-weekly infusions with α-galactosidase A, which is deficient in this X-linked globotriaosylceramide (Gb3) storage disorder. Elevated plasma globotriaosylsphingosine (lysoGb3) is a hallmark of classical Fabry disease. We investigated effects of enzyme replacement therapy (ERT) on plasma levels of lysoGb3 and Gb3 in patients with classical Fabry disease treated with agalsidase alfa at 0.2mg/kg, agalsidase beta at 0.2mg/kg or at 1.0mg/kg bodyweight. Each treatment regimen led to prominent reductions of plasma lysoGb3 in Fabry males within 3 months (P=0.0313), followed by relative stability later on. Many males developed antibodies against α-galactosidase A, particularly those treated with agalsidase beta. Patients with antibodies tended towards smaller correction in plasma lysoGb3 concentration, whereas treatment with high dose agalsidase beta allowed a reduction comparable to patients without antibodies. Pre-treatment plasma lysoGb3 concentrations of Fabry females were relatively low. In all females and with each treatment regimen, ERT gave reduction or stabilisation of plasma lysoGb3. Our investigation revealed that ERT of Fabry patients reduces plasma lysoGb3, regardless of the recombinant enzyme used. This finding shows that ERT can correct a characteristic biochemical abnormality in Fabry patients.

Long term enzyme replacement therapy for Fabry disease: effectiveness on kidney, heart and brain

BackgroundFabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A deficiency leading to renal, cardiac, cerebrovascular disease and premature death. Treatment with α-galactosidase A (enzyme replacement therapy, ERT) stabilises disease in some patients, but long term effectiveness is unclear.MethodsRenal, cardiac, and cerebral outcomes were prospectively studied in males and females with Fabry disease treated with ERT. Additionally, the occurrence of major cardiac events, stroke, end-stage renal disease and death was compared to a natural history (NH) cohort meeting treatment criteria.ResultsOf 75 patients on ERT (median treatment duration 5.2 years, range 0.05-11.0), prospective follow-up was available for 57 adult patients (30 males) and 6 adolescents. Renal function declined in males (-3.4 ml/min/1.73 m2 per year, SE 0.2; p < 0.001) despite ERT, but followed the normal course in females (-0.8 ml/min/1.73 m2 per year, SE 0.3; p = 0.001). Cardiac mass increased during ERT in males (+ 1.2 gram/m2.7, SE 0.3; p < 0.001), but remained stable in females (-0.3 gram/m2.7 per year, SE 0.4; p = 0.52). ERT did not prevent the occurrence of cerebral white matter lesions. Comparison of ERT treated to untreated patients revealed that the odds to develop a first complication increased with age (OR 1.05 (95% CI: 1.0-1.1) per year, p = 0.012). For development of a first or second complication the odds declined with longer treatment duration (OR 0.81 (95% CI: 0.68-0.96) per year of ERT, p = 0.015;OR 0.52 (0.31-0.88), p = 0.014 respectively).ConclusionsLong term ERT does not prevent disease progression, but the risk of developing a first or second complication declines with increasing treatment duration. ERT in advanced Fabry disease seems of doubtful benefit.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

Vasculopathy in patients with Fabry disease: Current controversies and research directions

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of the enzyme alpha-galactosidase A. The principal clinical manifestations of Fabry disease consist of cardiovascular complications including cerebrovascular, renal and cardiac disease but the pathophysiology of this specific vasculopathy is unclear. With the development of targeted treatment for Fabry disease, i.e. enzyme replacement therapy, it has become apparent that the removal of stored glycosphingolipid from the endothelial cells does not prevent progression of vascular disease in many patients. The aim of this study is to review the current available literature on vascular function tests, imaging and pathology studies and propose a hypothesis on the evolution of arterial complications in Fabry disease. Clearly, although premature atherosclerosis is suggested to occur, most studies describe absence of characteristic plaque formation. Smooth muscle cell hypertrophy, is probably the earliest feature of a complex vasculopathy, as in females and atypical cardiac variants, who have residual enzyme activity, no endothelial storage of significance is found. Subsequently, processes occur as observed in neo intima formation however with formation of more fibrotic structures. In the presence of a hyperdynamic circulation in combination with a less compliant vascular wall, it is hypothesized that upregulation of local renin angiotensine systems may occur. Angiotensin II is known to increase adhesion molecules, cytokines and chemokines and exerts a pro-inflammatory effect on leucocytes, endothelial cells and vascular smooth muscle cells. This enhances release of pro-thrombotic factors and opposes actions mediated through angiotensin 2 (AT2) receptor, including the release of nitric oxide (NO). A combination of reduced vascular compliance and activation of pro-thrombotic factors can lead to vascular complications in Fabry disease.

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. Methods Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. Results Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB− males (p = 0.04). Urinary Gb3 levels decreased markedly in AB− but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. Conclusion In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.

Consequences of a global enzyme shortage of agalsidase beta in adult Dutch Fabry patients

BackgroundEnzyme replacement therapy is currently the only approved therapy for Fabry disease. From June 2009 on, viral contamination of Genzymes production facility resulted in a worldwide shortage of agalsidase beta leading to involuntary dose reductions (approved dose 1 mg/kg/eow, reduced dose 0.5 mg/kg/m), or switch to agalsidase alpha (administered dose 0.2 mg/kg/eow). An assessment report from the European Medicines Agency (EMA) raised serious concerns about an increase in adverse events at lower dosages of agalsidase beta. We determined the influence of the shortage on clinical event incidence and the most sensitive biochemical marker (lysoGb3) in Dutch Fabry patients.MethodsThe incidence of clinical events per person per year was calculated from start of agalsidase beta treatment until the shortage, and was compared to the incidence of clinical events during the shortage period. In addition, plasma lysoGb3, eGFR, quality of life (SF-36) and brief pain inventory (BPI) questionnaires were analysed.ResultsAll thirty-five Dutch Fabry patients using agalsidase beta (17 males) were included. Mean clinical event incidence was unchanged: 0.15 events per person per year before versus 0.15 during the shortage (p = 0.68). In total 28 clinical events occurred in 14 patients during 4.6 treatment years, compared to 7 events in 6 patients during the 1.3 year shortage period. eGFR and BPI scores were not significantly altered. Two SF-36 subscales were significantly but minimally reduced in females. In males, lysoGb3 increased with a median of 8.1 nM (range 2.5 - 29.2) after 1 year of shortage (p = 0.001). Increases in lysoGb3 were found in both patients switching to agalsidase alpha and on a reduced agalsidase beta dose. Antibody status, treatment duration or clinical event incidence showed no clear correlation to lysoGb3 increases.ConclusionsNo increase in clinical event incidence was found in the adult Dutch Fabry cohort during the agalsidase beta shortage. Increases in lysoGb3, however, suggest recurrence of disease activity.

The value of estimated GFR in comparison to measured GFR for the assessment of renal function in adult patients with Fabry disease

BACKGROUND Renal disease is one of the major complications in Fabry disease, an X-linked lysosomal storage disease due to deficiency of the enzyme alpha-galactosidase A. The aim of our study was to determine the value of creatinine-, cystatin C- and beta-trace-based formulas for the estimation of glomerular filtration rate (eGFR) in Fabry patients. For comparison, the gold standard method (125)I-labelled iothalamate/(131)I-labelled hippuran [measured GFR (mGFR)] was used. METHODS GFR was estimated by using 11 different formulas based on creatinine, cystatin C and beta-trace protein. Accuracy and precision, detection of early decline of renal function and follow-up of renal function by eGFR was compared to mGFR. RESULTS One hundred and thirty-six GFR measurements and plasma samples were available from 36 (20 male) Fabry patients, treated with agalsidase alpha or beta with a median follow-up of 3.1 (range 1.5-5.2) years. Median mGFR was 97.3 (15.5-148.6) ml/min/1.73 m(2) in males and 84.4 (23.0-131.0) ml/min/1.73 m(2) in females at the start of follow-up. CONCLUSIONS Although none of the investigated endogenous markers proved to be an equivalent substitute for mGFR in Fabry patients, the Stevens equation, a creatinine- and cystatin C-based formula, most closely approximated the mGFR. When a creatinine-based formula is preferred, considering that there is no standardized method available for cystatin C, the abbreviated Modification of Diet in Renal Disease (aMDRD) and the recently developed Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formulas had the best performance. In male Fabry patients, the aMDRD may overestimate GFR, especially in the higher ranges. In these cases, CKD-EPI may perform better.

Vascular Aspects of Fabry Disease in Relation to Clinical Manifestations and Elevations in Plasma Globotriaosylsphingosine

Fabry disease is an X-linked hereditary lysosomal storage disorder attributed to a deficiency of &agr;-galactosidase A leading to increased plasma levels of globotriaosylsphingosine (lysoGb3). The disease presents as a vascular disease, with cerebral, cardiac, and renal complications. Carotid intima-media thickness (IMT), brachial flow-mediated dilation (FMD), pulse wave velocity, and advanced glycation end products were measured in 57 classically affected patients (22 men and 35 women), 55 healthy matched controls (20 men and 35 women), and 10 atypical Fabry disease patients (5 men and 5 women). Most patients received enzyme replacement therapy. In classically affected male patients, brachial FMD was decreased (2.9% [95% CI, 0.8% to 7.9%] versus 5.9% [2.1% to 8.5%] in controls; P=0.01), and carotid IMT was increased (0.67 mm [95% CI, 0.50–0.96 mm] versus 0.59 mm [95% CI, 0.40–0.76 mm] in controls; P=0.01). In women and atypical patients these vascular parameters were comparable with controls. Pulse wave velocity was not different; advanced glycation end products were only slightly increased in atypical patients. In classically affected women, a small increase in lysoGb3 was associated with an increase in IMT independent of age. In the classically affected men, all with increased IMT and high levels of plasma lysoGb3, lysoGb3 levels did not add to a higher IMT, suggestive of a ceiling effect. For FMD, elevated lysoGb3 levels (>7 nmol/L) contributed to a 2.9% lower FMD independent of age and sex (P=0.02). Increased carotid IMT and decreased brachial FMD occur in classic Fabry disease, which is associated with plasma lysoGb3 level independent of age and sex. These observations still exist despite enzyme replacement therapy.