Q J Med 2001; 94: 399-402
© 2001 Association of Physicians
Editorial |
Gaucher's disease—an exemplary monogenic disorder
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge
It perhaps comes as no surprise that continued study of any defined clinical disorder will reveal diverse manifestations hitherto considered to be unimportant or rare. The paper by Goitein and colleagues from Jerusalem in this issue of QJM focuses on the life-threatening pulmonary manifestations of the inherited multi-system disorder, Gaucher's disease. Pulmonary fibrosis and other signs of lung infiltration, pulmonary arterial hypertension, and hepatopulmonary syndrome with cyanosis and intrapulmonary arterio-venous shunting, are now well-documented in this lysosomal disease.
How do these unusual manifestations come about? Earlier clinical descriptions drew attention to cells of macrophage origin as the principal pathological focus of this condition; infiltration of the spleen, liver and bone marrow has always been considered to dominate the clinical picture of the established disorder in adults, which is typically accompanied by hepatosplenomegaly, pancytopenia and recurrent episodes of bone infarction crises. Gaucher's disease is a monogenic disease but, as with most apparently simple disorders, the underlying cause of its pathological effects remains mysterious.
Conventionally, Gaucher's disease has been classified into three principal clinical types that are assigned according to the presence or absence (and severity) of neurological manifestations.1 In the absence of any neurological deficit, the term type 1 (non-neuronopathic) Gaucher's disease is used. Neurological or ‘neuronopathic’ disease may either declare itself in infancy when the accompanying systemic disease is usually inconspicuous (type 2, neuronopathic) or in childhood, with or without prominent systemic disease (type 3, neuronopathic) (online Mendelian inheritance in man, OMIM, Website nos. 23080, 23091 and 23100, respectively, www.ncbi.nlm.nih.gov/omim).
Type 2 Gaucher's disease is a cruel but easily characterized disorder in which bulbar paralysis, abnormal eye movements and opisthotonus progress rapidly, so that death in infancy is inevitable. In contrast, categorization of type 3 is very problematic: at one extreme, subtle and stable neurological disorders of ocular movement may be detected on close examination of adults and children, while on the other, progressive ataxia, myoclonus, spasticity and dementia may occur in late childhood. Type 3 Gaucher's disease thus lies outside the diagnostic exclusion zone of the type 2 syndrome, and includes all other forms of the condition that are associated with neurological signs. Inevitably, further subgroups have been introduced into this operational designation almost by stealth: type 3a (prominent hepatosplenomegaly and osseous manifestations due to marrow disease); type 3b (limited systemic manifestations); and type 3c (hepatosplenomegaly, corneal opacities, slowly progressive ataxia and dementia with prominent cardiac valve and aortic root calcification).
It is unclear how useful these intricate sub-classifications are but they pose significant questions about the pathogenesis of Gaucher's disease and have allowed at least a preliminary skirmish into the troubled field of phenotype-genotype correlations (see below).
Non-neuronopathic Gaucher's disease principally affects macrophages, and is a typical lysosomal storage disease resulting from the inborn deficiency of the acid ß-glucosidase, glucocerebrosidase (EC 3.2.1.45). The condition is inherited as a single autosomal recessive disorder. Glucocerebrosidase contributes to the degradation of naturally-occurring glycosphingolipid molecules, and is responsible for the cleavage of the ß-glucosidic bond of its principal substrate, glucosylceramide, which is derived from the breakdown of glycosphingolipids present on leucocytes, platelets and red blood cells. When glucocerebrosidase activity is lacking, N-acylsphingosyl-1–0-ß-D-glucoside and other sphingolipid metabolites, including the deacylated compound, glucosylsphingosine, accumulate. The human glucocerebrosidase gene maps to chromosome 1q21, and more than 100 causal mutations have been identified in patients with Gaucher's disease.
Glucocerebrosidase is an enzyme of the lysosomal membrane. Although it is present in nearly all cell types, deficiency of the enzyme is associated with storage of glycolipids, principally in the mononuclear phagocytes. It is thus associated with disease of the liver, spleen, bone marrow and other organs, such as the lung, that are richly endowed with resident and itinerant macrophages. The eponymous cell, identified and beautifully drawn by the medical student C.P.E. Gaucher in 1882, has all the surface marker characteristics of a mature tissue macrophage. Electron microscopy of Gaucher's cells demonstrates macromolecular aggregates of glycosphingolipid within distended lysosomal spaces.
Gaucher's disease is the most frequent lysosomal disorder, with an overall disease frequency of approximately 1:50 000 to 1:100 000 live births. Non-neuronopathic type 1 disease is by far the most common clinical form. The condition is over-represented in certain populations such as the Ashkenazim and in an unusual Swedish isolate in Norrbotten and Vesterbotten, where affected individuals, who are usually considered to have type 3 disease, have inherited two copies of a single mutant allele (L444P) of the glucocerebrosidase gene as a result of descent from a common ancestor. Homozygosity for another mutant allele (D409H) occurs in certain Arabic populations and Spain; this is associated with corneal opacities, cardiac disease and neurological deficits.
Genetic defects that impair acid ß-glucosidase activity below a critical level first disable the phagosome/lysosome system of macrophages so that it is unable completely to degrade the glycosphingolipid derived from the membranes of ingested red cells and leucocytes. Thus type 1 Gaucher's disease is associated with missense mutations that give rise to partial enzyme deficiency. Homozygosity for the widely distributed mutant N370S allele is associated only with type 1 disease; the presence of one allele N370S protects against the development of neurological disease. Indeed, even asymptomatic N370S homozygotes may be found in the population at large.
In contrast, inactivating point mutations, recombinant alleles and intragenic deletions of the glucocerebrosidase gene are associated with the accumulation of endogenously supplied glycolipid breakdown products in neural tissue, including neurones, adventitial cells and perivascular macrophages. Thus neuronopathic variants of Gaucher's disease are caused by such mutations: homozygosity for the widespread L444P missense allele is strongly, but not invariably associated with type 3 disease. Homozygosity for the D409H missense mutation is also associated with a neuronopathic Gaucher's disease variant; this variant is also accompanied by cardiac calcification and corneal clouding. Absent glucocerebrosidase activity also has unusual effects and rare infants with this die shortly after birth with desquamation of the skin and dehydration—an appearance attributed to loss of the product of the glucocerebrosidase reaction, ceramide, which appears to be critical for cutaneous integrity.
The clinical diversity of Gaucher's disease thus cannot be explained directly by simple molecular analysis of the glucocerebrosidase gene: examples of twins, including monozygotic twins homozygous for the N370S allele, with striking differences in disease expression have been reported, and homozygosity for the L444P allele has been recorded in patients with all three clinical variants of Gaucher's disease. Of particular note is the unique phenotype associated with homozygosity for a single missense mutation, D409H: the occurrence of corneal opacification, aortic root disease and mitral valve calcification resembles the clinical manifestations of another group of lysosomal disorders, the mucopolysaccharidoses, e.g. Hurler's disease. This immediately suggests that glucocerebrosidase is a lysosomal enzyme with as yet uncharacterized activities on glycoprotein or mucopolysaccharide substrates; it seems likely that the D409 residue may participate critically in a unique functional domain of a multi-functional enzyme whose existence has been revealed by a mutational experiment in the laboratory of nature.
Gaucher's disease was the first lysosomal disorder for which an effective treatment was developed as a result of the uncompromising application of molecular cell biology to human therapeutics. Christian de Duve realised that one of the organelles that he had discovered, the lysosome, would be susceptible to the presentation of proteins and other chemical agents delivered through the fluid phase.2 The organelle has proved to be a fascinating pathological resource.
With the recognition of more than 40 or so lysosomal storage diseases, the theoretical basis for enzymatic complementation was ably demonstrated in vitro by studies of fibroblasts cultured from patients with the mucopolysaccharidoses by Elizabeth Neufeld and colleagues.3 These studies showed that functional complementation by delivery of lysosomal hydrolases that were missing in a particular cell line could occur as a result of protein uptake from the culture medium, thus providing experimental support for de Duve's theories.
In relation to Gaucher's disease, initial results of enzymatic complementation were disappointing, but the reason for this was soon established. Crude enzyme preparations obtained from human tissues such as the placenta were not taken up by non-parenchymal hepatic cells. This occurred because the enzyme preparations did not harbour the appropriate glycoprotein targeting sequences for uptake by membrane receptors leading to delivery to the nascent organelle. Mannose-6-phosphate residues are typically found on nascent lysosomal membranes, and are components of the pathway for endogenous and exogenous delivery of a large class of lysosomal proteins. This pathway is not in fact the relevant pathway for delivery of nascent glucocerebrosidase, which is a lysosomal membrane protein. Later, with the discovery of the macrophage mannose receptor, deglycosylation of purified preparations of human tissue glucocerebrosidase to reveal terminal mannose residues was successful in facilitating uptake of the enzyme molecules by enriched populations of macrophages. Mannosylated human glucocerebrosidase, like other high-mannose glycoproteins, is taken up by a saturable process by macrophages. It presumably enters the phagolysosome compartment, where it may encounter the storage material that accumulates in the pathological macrophage that is the Gaucher's cell.4,5
These studies provided the basis for the early trials of successful enzyme therapy of Gaucher's disease, leading ultimately to the use of mannose-terminated human placental glucocerebrosidase (alglucerase, Ceredase, and the recombinant human modified human glucocerebrosidase, imiglucerase, Cerezyme). The modified enzyme preparations have been subject to extensive clinical trials, and have been licensed through the Orphan Drug legislation, leading to impressive clinical results in the treatment of type 1 Gaucher's disease as well as the more indolent neuronopathic variants. There have been many prescriptions issued worldwide and significant commercial success for the Genzyme Company; several thousand patients are receiving this agent. This success was predicated on an understanding of the molecular cell biology of lysosomal disorders and Gaucher's disease in particular; there have been further consequential endeavours since. Fabry's disease, Pompe's disease, Hurler-Scheie disease, and Maroteaux-Lamy disease are all lyosomal storage diseases in which there have been successful trials of enzyme replacement therapy. These successes have been based on the commercial and clinical triumphs so prominently displayed in Gaucher's disease.
It is recognised that in some way the build-up of the glycolipid storage material leads to disease pathology; at the same time unrelated work on the transmission and propagation of the human immune deficiency virus showed that certain iminosugars selectively inhibited the biosynthesis of human glycosphingolipids.6 These tentative findings led to the practical development of substrate deprivation strategies, which have now advanced to the level of clinical trials. For example, it has recently been shown that the use of the oral agent, N-butyldeoxynojirimycin improved clinical parameters of Gaucher's disease activity when administered to a group of patients with Gaucher's disease not receiving enzyme therapy.7 These findings were accompanied by a reduction in substrate composition in blood cell membranes and a decrease in the plasma concentrations of storage lipid glucosylceramide. Since N-butyldeoxynojirimycin and its cogeners are orally active and may enter the brain, it may be that these agents will have therapeutic application in the intractable glycosphingolipidoses such as Tay-Sachs disease and Sandhoff disease, as well as Fabry's disease.
Gaucher's disease has become the prototype of the glycosphingolipidoses in several ways. We have entered an era, after the initial success and publication of the draft sequence of the human genome, in which the combined study of gene and protein expression information is recognized as the best means to understand the complex functional networks that lead to disease. The challenge now is to provide a convincing intellectual link between the profiles of tissue gene expression, the analysis of protein expression (proteomics) and to relate this to complex clinical phenotypes. The systematic large-scale investigation of cellular proteins, and the high through-put methods of cDNA micro-analysis represent competing and intensely promising avenues of research which have much promise for the combinatorial molecular analysis of disease.
In relation to functional genomics and proteomics, Gaucher's disease—and the glycosphingolipidoses generally—present a relatively simple challenge. These disorders are, after all, defined by the pathological accumulation of a restricted family of aggregated lipids within the lysosome which ultimately generate a now well-documented clinical phenotype. Gene profiling studies, combined with the later development of tissue proteomics, are thus likely to reveal a great deal about the altered cell metabolism and pathways of disease that result in clinical disability.
One pathological cell, one pathological organelle and one class of storage product pose a compelling set of temptations for investigators. Initial studies using a newly-described subtractive procedure based on the polymerase chain reaction to identify genes whose transcriptional products are increased in target tissues has been used to identify a group of over-expressed cDNAs that demonstrate disease specificity and whose products are found in excess in the blood of patients with Gaucher's disease. These studies, reported from the author's laboratory, also identified overexpression of several cysteine proteases already implicated in cell signalling and tissue modelling processes. Expression in the plasma correlated with Gaucher's disease activity and decreased during the course of favourable responses to enzyme replacement therapy.8 Hitherto, no systematic studies of the proteome have been reported in Gaucher's disease but in combination with gene expression profiling, it is to be hoped that novel disease biomarkers that reflect aspects of disease activity may be identified.
The Gaucher's cell is derived from a professional phagocyte, but until now the molecular control of phagocytosis and the genesis of the phagosome have been areas of scientific obscurity. Now the emergence of high through-put methods for identifying intracellular proteins that originate from specific organelles offers the hope of a better understanding of the pathological macrophage that is the focus of the condition.
In Gaucher's disease the spleen may weigh 3–4 kg, only a minute fraction of which is accounted for by pathological glycolipid. One is immediately struck by our complete inability to provide a pathological link between storage and the inflammatory phenotype that represents the established storage disease. Early studies have pointed to the release of certain cytokines and other systemic manifestations of Gaucher's disease not readily explained by intracellular glycolipid storage. Recently, Garin et al. reported the characterization of mouse phagosome proteins isolated after exposure of their parent macrophages to indigestible latex beads that in some ways resemble the accumulated glycolipid that is refractory to digestion in the glycosphingolipidoses such as Gaucher's disease. By using tandem mass spectroscopy techniques to characterize tryptic fragments of phagosome proteins resolved by two-dimensional electrophoretic techniques, these authors identified more than 140 species of phagosome-specific proteins, including several cathepsins, thus paving the way for a systematic analysis of macrophage activation and differentiation.9
The path ahead for understanding pathogenesis is far from easy and with our potential for the acquisition of a vast amount of expression data using high throughput multiplex analytical methods, there will be an increasing need to solve combinatorial problems in biology and medicine and to improve computational methods for resolving data clusters. Again, Gaucher's disease offers an opportunity for continued investigations into the basic understanding of complex cellular phenotypes and for the exploration of experimental agents to arrest pathology.
Gaucher's disease has a long record of translational medical research, and serves as an inspiring model of utility and progress. One day we may understand how pulmonary hypertension develops in some patients with Gaucher's disease, and why osteolytic lesions and bone infarction crises cripple others. Like Goiten and colleagues, we need to continue to document the protean clinical manifestations of this disorder even more thoroughly to provide the clinical base for relevant and informative research.
Welcome to the post-genomic era!
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