Jane M. Quirk

Replacement Therapy for Inherited Enzyme Deficiency: Use of Purified Ceramidetrihexosidase in Fabry's Disease

Abstract The effect of intravenous administration of glucocerebrosidase isolated from human placenta was investigated in two patients with Gauchers disease who are deficient in this enzyme. The first received one injection of 1.5 x 106 units of glucocerebrosidase, and the second an injection of 1.65 x 106 units on two successive days. Liver biopsies were obtained before and 24 hours after injection of enzyme. Glucocerebroside in the liver of the first patient decreased from 702 to 519 μg per gram wet weight and from 1634 to 1214 μg per gram in the second after infusion of glucocerebrosidase. The quantity of glucocerebroside in erythrocytes of the two patients before infusion was 7.4 and 6.2 μg per milliliter of cells respectively and 2.9 and 2.6 μg per milliliter of cells 72 hours afterward. These findings indicate that exogenous glucocerebrosidase causes definite decreases in the quantity of accumulated lipid in patients with Gauchers disease. (N Engl J Med 291:989–993, 1974)

Pediatric Fabry Disease

Background. Fabry disease is an underdiagnosed, treatable, X-linked, multisystem disorder. Objectives. To test the hypothesis that quality of life and sweating are decreased among pediatric patients with Fabry disease, compared with control subjects, and to provide quantitative natural history data and novel clinical end points for therapeutic trials. Design. Prospective, cross-sectional, observational study. Setting. Referral to the National Institutes of Health. Participants. Twenty-five male children with Fabry disease (mean age: 12.3 ± 3.5 years) and 21 age-matched control subjects. Main Outcome Measures. Quality of life (measured with the Child Health Questionnaire) and sweating (assessed with the quantitative sudomotor axon reflex test). Results. Quality of life scores for pediatric patients <10 years of age with Fabry disease, compared with published normative values, were 55 ± 17 vs 83 ± 19 for bodily pain and 62 ± 19 vs 80 ± 13 for mental health. Bodily pain scores for patients ≥10 years of age were 54 ± 22 vs 74 ± 23. Sweat volume in the Fabry disease group was 0.41 ± 0.46 μL/mm2, compared with 0.65 ± 0.44 μL/mm2 in the control group. Renal function, urinary protein excretion, and cardiac function and structure were normal for the majority of patients. The 3 patients with residual α-galactosidase A activity ≥1.5% of normal values were free of cornea verticillata and had normal serum and urinary globotriaosylceramide levels. All other children had glycolipid levels comparable to those of adult patients with Fabry disease. Acroparesthesia and cardiac abnormalities were generally present before anhidrosis and proteinuria. Mapping of the missense mutations on the crystallographic structure of α-galactosidase A revealed that the mutations were partially surface-exposed and distal to the active site among individuals with residual enzyme activity. Mutations associated with left ventricular hypertrophy (defined as left ventricular mass index of >51 g/m2.7) were localized near the catalytic site of the enzyme. Conclusions. Despite the absence of major organ dysfunction, Fabry disease demonstrates significant morbidity already in childhood. We have identified important, potentially correctable or preventable, outcome measures for future therapeutic trials. Prevention of complications involving major organs should be the goal for long-term specific therapy.

Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of alpha-galactosidase A resulting in globotriaosylceramide (Gb(3)) storage in cells. The pathogenic role of Gb(3) in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb(3) in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb(3)-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb(3) also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb(3) by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb(3). This study provided direct evidence that excess intracellular Gb(3) induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patients plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

Physiological characterization of neuropathy in Fabry's disease.

Fabrys disease is commonly associated with a painful, debilitating neuropathy. Characterization of the physiological abnormalities is an important step in evaluating response to specific therapies. Twenty‐two patients with Fabrys disease, and with relatively preserved renal function, underwent conventional and near‐nerve conduction studies, electromyography, sympathetic skin responses, and quantitative sensory testing (QST). Nerve conduction studies were mostly normal except for an increased frequency of median nerve entrapment at the wrist in 6 (27%) patients. Sympathetic skin responses were preserved in 19 of 20 (95%) of the patients. The QST showed increased or immeasurable cold and warm detection thresholds in patients, significantly different from controls (n = 28) in the hand (P < 0.001, P = 0.04, respectively) and foot (P < 0.001 for both). Cold thresholds were more often abnormal than were warm thresholds. Vibration thresholds were normal in the feet and, in some patients, elevated in the hand only, probably due to frequent median nerve entrapment at the wrist. Our findings suggest that the neuropathy of Fabrys disease is characterized by an increased prevalence of median nerve entrapment at the wrist and by thermal afferent fiber dysfunction in a length‐dependent fashion, with greater impairment of cold than warm sensation.

Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer

Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb3) caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). The condition is associated with an increased morbidity and mortality due to renal failure, cardiac disease, and early onset of stroke. Hemizygous males are primarily affected clinically with variable expression in heterozygous females. Gene-therapy trials have been initiated recently in α-gal A knockout mouse models of Fabry disease by using a variety of viral vectors. In the present investigation we administered single i.v. injections of 1 × 1010 genomes of recombinant adeno-associated virus (rAAV) encoding the human α-gal A gene driven by a modified chicken β-actin (CAG) promoter to α-gal A knockout (Fabry) mice. Transgenic mice were analyzed for expression of α-gal A activity and Gb3 levels in liver, kidney, heart, spleen, small intestine, lung, and brain. Administration of the rAAV-CAG-hAGA vector resulted in stable expression of α-gal A in organs of the Fabry mice for >6 months. α-Gal A activity in the organs became equal to or higher than that of wild-type mice. Accumulated Gb3 in the liver, heart, and spleen was reduced to that of wild-type mice with lesser but significant reductions in kidney, lung, and small intestine. Injection of the rAAV-CAG-hAGA construct into skeletal muscle did not result in expression of α-gal A in it or in other tissues. This study provides a basis for a simple and efficient gene-therapy approach for patients with Fabry disease and is indicative of its potential for the treatment of other lysosomal storage disorders.

Delivery of active hexosaminidase across the blood‐brain barrier in rats

The ability to deliver enzymatically active human hexosaminidase A across the blood-brain barrier and into brain cells of the normal rat was examined. Following osmotic blood-brain barrier modification in the rat, intraarterially administered human hexosaminidase A and B were shown to cross the barrier and enter brain cells. Subcellular fractionation studies demonstrated that most of the human enzyme delivered across the barrier was functionally active and appeared to be inside a subcellular organelle. These studies provide evidence that blood-brain barrier modification permits delivery of functionally active hexosaminidase A into subcellular organelles consistent with that known to be the appropriate site of physiologic activity.

Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.

Objective To examine the relationship between types and locations of mutations of the enzyme &agr;-galactosidase (Gal) A in Fabry disease and the response to the pharmacological chaperone 1-deoxygalactonojirimycin (DGJ). Methods T cells grown from normal individuals or from patients with Fabry disease were tested for response to treatment with DGJ by increased activity of &agr;-Gal A. Results Cells from normal controls responded with a 28% increase in &agr;-Gal A activity, whereas response in Fabry individuals was mutation dependent ranging from no increase to fully normal activity. Nine truncation mutations (all nonresponsive) and 31 missense mutations were tested. Three groups of missense mutations were categorized: responders with activity more than 25% of normal, nonresponders, with less than 7% and an intermediate response group. In normal cells and in responders an increase in the mature lysosomal form of &agr;-Gal A was observed after DGJ treatment. Nonresponders showed little or no protein with or without DGJ. The intermediate response group showed an increase in band intensity but incomplete processing of the enzyme to the mature form. Conclusion Mapping the missense mutations to the structure of &agr;-Gal A identified several factors that may influence response. Mutations in regions that are not in &agr;-helix or &bgr;-sheets, neither involved in disulfide bonds nor with an identified functional or structural role were more likely to respond. Predictability is, however, not precise and testing of each mutation for response to pharmacological chaperone therapy is necessary for Fabry disease and related lysosomal storage disorders.

Factors that influence the uptake of β-hemosaminidase a by rat peritoneal macrophages

Summary Cultured rat peritoneal macrophages contain a receptor-mediated system for the uptake of the lysosomal enzyme [ 125 I]β-hexosaminidase A (E.C.3.2.1.30). The uptake process is saturable (4.5 ng/10 6 cells) and has a Kuptake of 5.9 × 10 −8 M. The uptake is inhibited 68% by unlabeled β-hexosaminidase A, 66% by mannan, and 24% by ahexosaminofetuin (mannose terminated). Methylamine, ammonium ion, (both 20 mM) mepacrine and chloroquine (both 0.1 mM) strongly inhibit the specific uptake of [ 125 I]β-hexosaminidase A while cytochalasin B (5 μg/ml) and colchicine (10- 5 M) inhibit specific uptake by 35 and 28% respectively. Mepacrine inhibits phagocytosis of latex beads 76%; methylamine chloroquine, and cytochalasin B inhibit approximately 50%; colchicine and ammonium ion have negligible effect. These results suggest that adsorptive pinocytosis and phagocytosis function independently of each other. Extrapolation of these different effects may help to elucidate the mechanism of lysosomal enzyme uptake by macrophages.

Enzyme Replacement Therapy for the Sphingolipidoses

The greatest progress in the field of inheritable disorders during the past decade was made in the understanding and control of lipid storage diseases. Since original demonstrations in 1965 and 1966 of the metabolic defects in Gaucher’s disease (6,7) Niemann-Pick disease (8), Fabry’s disease (9), and metachromatic leukodystrophy (17), specific enzyme deficiencies were demonstrated in ten heritable disorders of lipid metabolism. These discoveries paved the way for the development of facile procedures for the diagnosis of homozygotes, the detection of heterozygous carriers, and the monitoring of pregnancies at risk for any of these diseases (1–4). These applications of basic research endeavors have provided much relief from human anguish and suffering and they are in wide use at this time. However, there still remains much to be done for affected patients and compassionate physicians continue to seek further help in the treatment of hundreds of patients with these diseases. In particular, the therapy of patients with Type I (adult) Gaucher’s disease in whom skeletal involvement causes severe bone and joint pains, and patients with Fabry’s disease with intractable pains in arms and legs and progressive renal impairment deserves intensive attention. During the past three years much of our research efforts have been devoted to these two disorders.

Skin-impedance in Fabry Disease: A prospective, controlled, non-randomized clinical study

BackgroundWe previously demonstrated improved sweating after enzyme replacement therapy (ERT) in Fabry disease using the thermo-regularity sweat and quantitative sudomotor axon reflex tests. Skin-impedance, a measure skin-moisture (sweating), has been used in the clinical evaluation of burns and pressure ulcers using the portable dynamic dermal impedance monitor (DDIM) system.MethodsWe compared skin impedance measurements in hemizygous patients with Fabry disease (22 post 3-years of bi-weekly ERT and 5 ERT naive) and 22 healthy controls. Force compensated skin-moisture values were used for statistical analysis. Outcome measures included 1) moisture reading of the 100th repetitive reading, 2) rate of change, 3) average of 60–110th reading and 4) overall average of all readings.ResultsAll outcome measures showed a significant difference in skin-moisture between Fabry patients and control subjects (p < 0.0001). There was no difference between Fabry patients on ERT and patients naïve to ERT. Increased skin-impedance values for the four skin-impedance outcome measures were found in a small number of dermatome test-sites two days post-enzyme infusions.ConclusionThe instrument portability, ease of its use, a relatively short time required for the assessment, and the fact that DDIM system was able to detect the difference in skin-moisture renders the instrument a useful clinical tool.