John A. Barranger

Fabry Disease, an Under-Recognized Multisystemic Disorder: Expert Recommendations for Diagnosis, Management, and Enzyme Replacement Therapy

Fabry disease is an X-linked recessive lysosomal storage disorder that is caused by the deficient activity of -galactosidase A (-Gal A, also termed ceramide trihexosidase) (1, 2) and the resultant accumulation of globotriaosylceramide (also termed ceramide trihexoside) and related glycosphingolipids (3-5). In patients with the classic phenotype, levels of -Gal A activity are very low or undetectable. Patients with detectable -Gal A activity have a milder, variant phenotype (6-8). In classically affected males, the progressive glycosphingolipid accumulation, particularly in the vascular endothelium (Figure 1), leads to renal, cardiac, and cerebrovascular manifestations and early death. The disease is panethnic, and estimates of incidence range from about 1 in 40 000 to 60 000 males (5, 9). Fabry disease predominantly affects males, although carrier (heterozygous) females also can be affected to a mild or severe degree because of random X-chromosomal inactivation (5). Figure 1. Distinctive laboratory findings in Fabry disease. A B In the absence of a family member who has already received a diagnosis of the disorder, many cases are not diagnosed until adulthood (average age, 29 years) (9, 10), when the pathology of the disorder may already be advanced. Although clinical onset occurs in childhood, disease presentation may be subtle, and its signs and symptoms are often discounted as malingering or are mistakenly attributed to other disorders, such as rheumatic fever, erythromelalgia, neurosis, the Raynaud syndrome, multiple sclerosis, chronic intermittent demyelinating polyneuropathy, lupus, acute appendicitis, growing pains, or petechiae (5, 11). Fabry disease was first identified a century ago, but until now, no disease-specific treatment has been available. Patients have been managed with supportive, nonspecific treatment for pain management, cardiac and cerebrovascular complications, and end-stage renal disease. These interventions may prolong life, but their utility is limited because they do not address the underlying cause of the disease, that is, the lack of -Gal A and the progressive accumulation of globotriaosylceramide. Recently, enzyme replacement with human -Gal A has been shown to safely reverse the pathogenesis of the major clinical manifestations, to decrease pain, and to stabilize renal function in patients with Fabry disease (12, 13). The European Agency for the Evaluation of Medicinal Products has approved the treatment, and the U.S. Food and Drug Administration is currently reviewing it. Thus, recommendations for the diagnosis and treatment of Fabry disease are timely. Formation of Expert Panel and Basis of Recommendations In June and July of 2001, two groups of investigators published randomized, placebo-controlled trials that demonstrated that enzyme replacement therapy in Fabry disease can reverse the major pathologic consequences and improve outcomes (12, 13). With a disease-specific therapy finally available, the need for prompt and accurate diagnosis of this devastating, progressive disease became paramount so that patients could be identified and treated before incurring irreversible organ damage. Recognizing the need for initial guidelines for diagnosis, management, and the use of enzyme replacement therapy, Dr. Robert Desnick and Dr. Roscoe Brady (senior authors of the enzyme replacement trials) assembled an international panel of experts with extensive clinical experience and diverse subspecialty expertise in Fabry disease and lysosomal storage disorders. Panelists met face-to-face to identify and discuss salient issues. An independent coordinator conducted numerous global and specific searches of the MEDLINE database (19912001), including a global search of the recent literature on Fabry disease. The coordinator then interviewed each panelist in detail and, with the first author, prepared a draft statement. In a second face-to-face session, the draft was reviewed, revised, and finalized by the panel. A teleconference was convened to revise the manuscript after journal review. Support for the expert panel process was obtained from the Genzyme Corporation (Cambridge, Massachusetts), which had no formative role in the literature review, the formulation of recommendations, or the drafting and revising of the manuscript. As would be expected for a rare, under-recognized disease, the literature on Fabry disease mostly consists of single or small case studies and reviews in addition to book chapters written by Fabry experts. The few larger studies focus on disease manifestations and mechanisms of disease rather than the effectiveness of interventions or disease management. The literature on enzyme replacement therapy is limited to the clinical trials published in the last 2 years. Thus, clinical experience and expertise played an important role in the formulation of these recommendations. Disease Pathophysiology and Clinical Manifestations The major debilitating manifestations of Fabry disease result from the progressive accumulation of globotriaosylceramide in the vascular endothelium (Figure 1), leading to ischemia and infarction, especially in the kidney, heart, and brain. The ischemia and infarction of small vessels are primarily due to vascular occlusion (5); however, evidence for a prothrombotic state has recently been published (14). In addition, early and substantial deposition of globotriaosylceramide occurs in podocytes, leading to proteinuria and, with age, in cardiomyocytes, causing cardiac hypertrophy and conduction abnormalities (Figure 1). Patients are generally divided into two major groups on the basis of the absence or presence of residual -Gal A activity: classic disease and milder, later-onset, atypical variants (5). Presentation and clinical course can vary within these phenotypes, and an intermediate phenotype has also been described (15-17). The Classic Phenotype Males with classic disease have no or very low -Gal A activity, resulting in severe renal, cardiac, and cerebrovascular disease manifestations. Before treatment of uremia became available, the average lifespan of affected males was about 40 years (18). With the advent of renal dialysis or transplantation, the median survival was about 50 years (19). Clinical manifestations (Figures 1 and 2), which usually begin in childhood or adolescence, include intermittent pain in the extremities (acroparesthesias); episodic Fabry crises of acute pain lasting hours to days; characteristic skin lesions (angiokeratomas); a corneal opacity that does not affect vision; hypohidrosis; heat, cold, and exercise intolerance; mild proteinuria; and gastrointestinal problems. By adulthood, the renal involvement inevitably results in end-stage renal disease, which requires dialysis or transplantation (20, 21). Cardiac manifestations include left ventricular hypertrophy, valvular disease (especially mitral insufficiency), ascending aortic dilatation, coronary artery disease, and conduction abnormalities (Figure 1), leading to congestive heart failure, arrhythmias, and myocardial infarction (5, 22-24). Cerebrovascular manifestations include early stroke, transient ischemic attacks, white matter lesions, hemiparesis, vertigo or dizziness, and complications of vascular disease (such as diplopia, dysarthria, nystagmus, tinnitus, hemiataxia, memory loss, and hearing loss) (5, 25). Figure 2. Distinctive clinical features of Fabry disease. A B C Clinical manifestations in carrier females range from asymptomatic to full-blown disease as severe as that in affected males (5, 26-28). Although many carriers will be relatively asymptomatic and have a normal lifespan, carriers may experience symptoms in childhood and adolescence (such as pain and proteinuria) and adulthood (such as cardiac or, more rarely, renal manifestations). In late adulthood, some carriers develop left ventricular hypertrophy and substantial cardiomyopathy. Data on carriers are limited. A recent study of obligate carrier females found significant disease manifestations in 20 of 60 women, including 17 of whom who had experienced transient ischemic attacks or cerebrovascular accidents (28). Atypical Variants Atypical male variants have a milder, later-onset phenotype (5-7, 17, 29). Because of low residual -Gal A levels, these patients do not have the early major clinical manifestations of classic Fabry disease. For example, cardiac variants present with cardiomegaly and mild proteinuria usually after 40 years of age, when patients with classic Fabry disease would be severely affected or would have died (6, 7, 29). Two recent studies have suggested that the cardiac variant of Fabry disease may be an important cause of idiopathic left ventricular hypertrophy (7) or late-onset hypertrophic cardiomyopathy (30). Tissue biopsies or autopsy studies of cardiac variants reveal globotriaosylceramide accumulation in the myocardium and not in the vascular endothelium throughout the body (5, 6, 29). These findings suggest that even low levels of -Gal A can prevent globotriaosylceramide accumulation in the microvasculature and that this lack of accumulation is associated with the absence or attenuation of disease manifestations. Thus, reversal of the underlying vascular endothelial pathology by enzyme replacement therapy will probably be clinically therapeutic in patients with classic Fabry disease. Enzymatic and Molecular Diagnosis In affected males with the classic or variant phenotype, the disease is readily diagnosed by determining the -Gal A activity in plasma or peripheral leukocytes. In contrast, female carriers can have normal to very low -Gal A activity; therefore, their specific family mutation in the -Gal A gene must be demonstrated. Most kindreds have family-specific or private mutations; to date, more than 300 mutations have been identified, of which most are missense (amino acid substitutions) or nonsense (causing premature truncation of the amino acid sequence) mutations. Spli

A Mutation in the Human Glucocerebrosidase Gene in Neuronopathic Gaucher's Disease

To search for a genetic marker for type 2 Gauchers disease (acute neuronopathic form), we compared the nucleotide sequence of a cloned glucocerebrosidase gene from a patient with Gauchers disease with a normal gene. We found only a single base substitution (T----C) in exon X. This mutation results in the substitution of proline for leucine in position number 444 and produces a new cleavage site for the NciI restriction endonuclease. We analyzed NciI enzymatic digests of genomic DNA from 20 patients with type 1, 5 with type 2, and 11 with type 3 Gauchers disease, and 29 normal controls for a restriction-fragment-length polymorphism (RFLP). Four of 5 patients with type 2 disease and all 11 with type 3 disease had at least one allele with the mutation. Two of 5 patients with type 2 disease and 7 of 11 with type 3 were homozygous for this mutation. Only 4 of 20 patients with type 1 Gauchers disease had the mutant allele and were heterozygous for it. None of the 29 normal controls had the mutant allele. The high frequency of this mutation (444leucine----proline) in patients with neuronopathic Gauchers disease, detectable by the NciI RFLP, may be of value in the identification of patients who will have the neurologic sequelae of Gauchers disease.

Centrifugal enhancement of retroviral mediated gene transfer

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

Uptake and distribution of placental glucocerebrosidase in rat hepatic cells and effects of sequential deglycosylation

The clearance of native human placental glucocerebrosidase by rat liver shows the presence of two distinct enzyme forms with different recognition characteristics. The clearance and uptake of native enzyme by liver cells was compared to that of glucocerebrosidase sequentially treated with neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. The initial rate of clearance of infused enzyme was increased greater than 10-fold for the asialo-, agalacto- and ahexoenzymes over that of native glucocerebrosidase. Incorporation of asialo enzyme was increased in hepatocytes over that of native enzyme, while the distribution of agalacto- and ahexoenzyme preparations was increased in non-parenchymal cells. This observation is consistent with the identification of a galactose receptor on hepatocytes and N-acetylglucosamine/mannose receptors on Kupffer cells. These data and inhibition studies by specified monosaccharide-terminal glycoprotein derivatives demonstrate the importance of these sugars in the uptake of this lysosomal enzyme by receptor-mediated endocytosis. Modification of the enzyme to expose certain monosaccharide moieties results in increased delivery to specific cell types. Therefore, naturally occurring receptors can be utilized for targeting glucocerebrosidase to the non-parenchymal cell in liver.

Skeletal Complications of Gaucher Disease

Gaucher disease is a collection of related disorders of sphingolipid catabolism caused by the deficiency of a specific beta-glucosidase. The inefficiency of this enzyme, glucocerebrosidase, to degrade its natural substrate leads to the accumulation of the complex lipid glucocerebroside in tissue macrophages. The pathogenesis of the disease is, as yet, poorly understood. The manifestations of the disease are protean with hepatosplenomegaly and bone deterioration frequently the predominating signs. The disease most frequently causes disability because of its effects on the skeleton. This review seeks to summarize the current clinical understanding of these complications. Experience with 327 patients reveals that the bone disease in this disorder is extremely variable. The severity of the problems range from asymptomatic persons with neither radiographic, scintigraphic, nor histologic evidence of bone involvement to those whose skeleton is completely devastated by a process of osteopenia, osteonecrosis, and osteosclerosis. These severely affected individuals show the most bizarre deformities in their bones and are subject to pathologic fracture. Most patients fortunately, are less profoundly affected, but many are plagued by bone pain of an arthritic nature or by an acute prostrating bone crisis probably best described as a bone infarction. The accepted etiology that these crises are a result of vascular compromise produced by occlusion of vessels by Gaucher cells is not supported by scintigraphic or histologic studies. Moreover, the vascular hypothesis does not explain the variety of lesions of the skeleton seen in this multifocal bone disease. Preliminary metabolic and endocrinologic studies suggest that this is not a systemic disorder of metabolism which affects bone uniformly. On the contrary, the lesions are multiple and localized, and sometimes much of the skeleton is preserved. These observations suggest that bone is affected because of collections of Gaucher cells scattered throughout its substance and may be the result of a toxic process around these foci. Alternatively, the storage of glucocerebroside in tissue macrophages may disturb the generation of competent osteoclasts and thus result in a failure to maintain a healthy skeleton. Further research is needed to delineate the pathogenesis of this disorder before any effective therapy can be developed.

Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages: I. Effects of various agents

Abstract The relationship between the production and release of lymphocyte-activating factor (LAF, or interleukin 1) by cultured murine peritoneal macrophages (Mφ) was investigated. Unstimulated Mφ produce high levels of intracellular LAF within a few hours after culturing, but release little of this activity into their culture medium. Addition of various agents was found to increase significantly the production and release of LAF, with three different patterns: (a) Both intracellular and extracellular LAF activities were increased in response to latex beads, (b) A marked increase of intracellular LAF, with just a minimal elevation of extracellular activity, was stimulated by LPS. (c) Sharp increases in LAF release, with small increments of the intracellular activity, were induced by silica and glucocerebroside (GL 1 ). Silica and GL 1 damaged the cultured Mφ, as indicated by the increased release of lactate dehydrogenase. It is significant, therefore, that silica and GL 1 increased both intracellular and extracellular LAF levels, suggesting that damage of Mφ may stimulate total LAF production. A combination of LPS with silica or GL 1 acted synergistically on Mφ to release very high levels of LAF, which far exceeded those released by the individual agents. The agents were also tested on Mφ which were precultured, to deplete their LAF content. Latex, LPS, or silica increased LAF production and release by precultured Mφ, but the levels were lower than those obtained with freshly cultured Mφ. The results of this study thus show that the level of LAF release does not necessarily reflect the level of total LAF production by cultured Mφ and suggest that injurious agents may promote LAF production.

Evaluation of Gaucher disease using magnetic resonance imaging.

Magnetic resonance imaging was used to study the skeletal involvement in a series of twenty-four patients with Gaucher disease. Many sites in the marrow of these patients were characterized by an abnormally low signal intensity that reflected shortened T1 and markedly shortened T2 values in the replaced marrow. The abnormality was non-homogeneous in distribution. In the lower extremity, the proximal (femoral) areas were more frequently affected than the distal (tibial) sites. The epiphyses were generally spared unless the involvement of bone was extensive. Lack of epiphyseal involvement on the magnetic resonance images generally precluded any suspicion of osteonecrosis. The extent of involvement, as suggested by the magnetic resonance data, appeared to correlate well with the occurrence of musculoskeletal complications. Magnetic resonance imaging is more sensitive than computerized tomography in demonstrating the extent of abnormalities in patients with Gaucher disease, and it may have prognostic value.

Improvement of bone disease by imiglucerase (Cerezyme) therapy in patients with skeletal manifestations of type 1 Gaucher disease: results of a 48-month longitudinal cohort study.

Progressive skeletal disease accounts for some of the most debilitating complications of type 1 Gaucher disease. In this 48‐month, prospective, non‐randomized, open‐label study of the effect of enzyme replacement therapy on bone response, 33 imiglucerase‐naïve patients (median age 43 years with one or more skeletal manifestations such as osteopenia, history of bone crisis, or other documented bone pathology) received imiglucerase 60 U/kg/2 weeks. Substantial improvements were observed in bone pain (BP), bone crises (BC), and bone mineral density (BMD). Improvements in BP were observed at 3 months (p < 0.001 vs baseline) and continued progressively throughout the study, with 39% of patients reporting pain at 48 months vs 73% at baseline. Eleven of the 13 patients with a pre‐treatment history of BC had no recurrences. Biochemical markers for bone formation increased; markers for bone resorption decreased. Steady improvement of spine and femoral neck BMD, measured using dual‐energy X‐ray absorptiometry was noted. Mean Z score for spine increased from −0.72 ± 1.302 at baseline to near‐normal levels (−0.09 ± 1.503) by month 48 (p = 0.042) and for femoral neck from −0.59 ± 1.352 to −0.17 ± 1.206 (p = 0.035) at month 36. This increase was sustained at 48 months. With imiglucerase treatment, patients should anticipate resolution of BC, rapid improvement in BP, increases in BMD, and decreased skeletal complications.

Imiglucerase (Cerezyme) improves quality of life in patients with skeletal manifestations of Gaucher disease.

Health‐related quality of life (HRQOL) can be diminished in patients with type 1 Gaucher disease (GD) owing to the debilitating clinical manifestations of this chronic disease. This study investigates the impact of imiglucerase treatment on HRQOL of patients with type 1 GD and bone involvement. Thirty‐two previously untreated type 1 GD patients with skeletal manifestations including bone pain, medullary infarctions, avascular necrosis, and lytic lesions received biweekly imiglucerase (at 60 U/kg). The Short Form‐36 Health Survey (SF‐36) was administered at regular intervals to assess HRQOL. Mean baseline SF‐36 physical component summary (PCS) scores were diminished relative to US general population norms. Low PCS scores were more common in patients with medullary infarction, lytic lesions, and higher bone pain severity scores. Statistically significant improvements were observed for all eight SF‐36 subscales after 2 years of treatment. Mean PCS and mental component summary (MCS) scores increased to within the normal range after 2 years of treatment and were maintained through year 4. Large HRQOL gains were observed even in patients with the most advanced disease and lowest baseline PCS scores. Imiglucerase treatment has a significant positive impact on HRQOL of type 1 GD patients with skeletal disease, including those with bone infarctions, lytic lesions, and avascular necrosis.

Selective effects of glucocerebroside (Gaucher's storage material) on macrophage cultures.

Although the enzymatic lesion in Gauchers disease is well established, little is known concerning the pathogenic mechanisms involved in the clinical manifestations of the disease. In order to obtain insight into this unexplored aspect of Gauchers disease, we examined the effects of glucocerebroside (GL(1)) at the cellular level in monolayers of cultured murine macrophages. The addition of GL(1) to these cultures stimulated the macrophages to release increased amounts of lymphocyte-activating factor (LAF) and lysosomal enzymes into the medium. These responses were proportional to the amount of GL(1) added to the culture. At higher levels of GL(1) (>/=20 mug/ml), lactic dehydrogenase, a cytoplasmic enzyme was also released indicating cellular damage at these doses. Intracellular LAF also increased in macrophages incubated with the high doses of GL(1), demonstrating an increase in total LAF production by these cells. Lipopolysaccharide acted synergistically with GL(1) and stimulated the release of exceedingly high levels of LAF which had a molecular weight profile similar to that of LAF released by exposure to lipopolysaccharide alone. Unlike GL(1), galactocerebroside, sphingomyelin, and ceramidetrihexoside, exerted little or no effect on the release of macrophage products. The effect of GL(1) was selective for macrophages since addition of this material to mouse lens epithelial cells had no detectable cytotoxic effect and it was only slightly toxic to lymphocytes or P815 cells in concentrations at which macrophages were clearly affected. A direct relationship was observed between the cytotoxicity of the sphingolipids and their accumulation in various cells. Macrophages accumulated large amounts of GL(1) but not sphingomyelin, whereas the other cells examined in this investigation did not accumulate either of these lipids. Human monocytes, like murine macrophages, also release increased amounts of LAF when incubated with GL(1). The effect of GL(1) was dose-responsive and synergy was found with lipopolysaccharide. The relevance of these findings to the pathogenesis of Gauchers disease is considered.