Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Department of Neurology, Brain Research Institute, Niigata University, Japan
Type 1 (Chronic non-neuronopathic form): The ages of onset can vary from infancy to adulthood. Hepatosplenomegaly is the principal manifestation and is not associated with neurological signs or symptoms. The career frequency among Ashkenazic Jewish population has been estimated to be one in 12-13 (2).
Type 2 (acute neuronopathic): The age of onset is usually before 6 months of age. Clinical presentations are characterized by severe mental retardation, trismus, strabismus and dysphagia, in addition to hepatosplenomegaly. The clinical features are so severe that most patients die within the first 2 years.
Type 3 (chronic neuronopathic form): Systemic signs are similar to those of type 1, but type 3 is associated with neurological signs such as myoclonus, seizure or eye movement disorder of supranuclear palsy type.
Recent advances in studies on molecular genetics of Gaucher disease has revealed that these clinical phenotypes are a result of mutations in the glucocerebrosidase gene. Furthermore, it has also been demonstrated that there is a substantial correlation between genotype and clinical phenotypes of Gaucher disease. The presence of some exceptional cases, however, has made it difficult, from a practical point of view, to apply the genotype analysis for the prediction of clinical phenotypes of Gaucher disease.
Recent dramatic improvement in enzyme replacement therapy has promised that severe hepatosplenomegaly, anaemia and thrombocytopenia can be improved by the enzyme replacement therapy. On the other hand, however, the extremely expensive cost for the production of human glucocerebrosidase has brought another serious issue to patients and families suffering from Gaucher disease.
With this background I will attempt to summarize the results of recent advances of molecular genetic analysis of Gaucher disease, together with the implications, and limitations, for clinical application of molecular diagnosis of Gaucher disease. I will also discuss the recent achievement of enzyme replacement therapy for Gaucher disease and serious problems caused by the extremely high expense of making human glucocerebrosidase.
1. Molecular genetics of Gaucher disease
Molecular cloning of cDNA for human glucocerebrosidase was first described by Ginns et al. (3). Detailed nucleotide sequence analyses revealed that human mature glucocerebrosidase contains 497 amino acids, which is generated from its precursor protein containing a 19 amino acid signal peptide (4). The signal peptide consists of 19 amino acids, which contains a hydrophobic core consisting of Gly-Leu-Leu-Leu-Leu. There is a glycine residue at the signal peptide cleavage site. These characteristics of the signal peptide are quite similar to those identified in secretory proteins.
The glucocerebrosidase gene has been shown to contain 11 exons (5) with the first 2 exons coding for the signal peptide and the rest 9 exons coding for mature enzyme. The locus for human glucocerebrosidase gene has been located to chromosome 1q21 by in situ hybridization (6). Interestingly, a pseudogene, which contains highly homologous sequences and exon-intron structures consisting of 11 exons, has been identified. As the pseudogene carries numerous mutations compared to the functional glucocerebrosidase gene, we need to pay precautions in the analysis of mutations of the functional gene, especially if the genes are analyzed by polymerase chain reaction (PCR).
To identify mutations causing Gaucher disease, we first generated genomic DNA libraries from Gaucher patients. After the mutant genes were cloned using a phage vector, the nucleotide sequences of exons, flanking introns as well as the promoters were analyzed in detail. With this strategy we have succeeded in identifying the two most common missense mutations, which are 444Leuto Pro and 370Asnto Ser. The development of PCR has made the mutation analysis very efficient, and so far, as many as 38 mutations have been identified in the glucocerebrosidase gene (10, 11).
Extensive analysis of the correlation between genotypes and phenotypes has revealed that there is substantial correlation, if not complete, between the genotypes and the phenotypes. The above mentioned 444Leuto Pro and 370Asnto Ser are associated predominantly with neuronopathic and non-neuronopathic forms, respectively (7-10).
Genotype +/+ +/- -/-
239 0 0
Gaucher type 1 16 4 0
Gaucher type 2 1 2 2
Gaucher type 3 0 4 7
It has long been puzzling to see type 2 and type 3 patients having apparently identical genotypes of homozygous state for 444Leu to Pro. Detailed analyses made by Lathram et al. , however, have revealed that type 3 patients are truly homozygous for the 444Leu to Pro mutation. They found that type 2 Gaucher patients carry 456Ala to Pro mutation in addition to 444Leu to Pro. Interestingly, both of these mutations are present in the pseudogene, raising a possibility that the two mutations might have been brought to the functional gene by a mechanism of either gene conversion or unequal crossing over (12).
In contrast to 444Leu to Pro mutation, 370Asn to Ser is exclusively present in type 1 Gaucher patients (Table 2), suggesting that the presence of 370Asn to Ser mutation may somehow prevent patients from developing neurological symptoms.
Genotype +/+ +/- -/-;
Control 12 0 0
Gaucher type 1 6 15 3
Gaucher type 2 6 0 0
Gaucher type 3 0 0 0
For the mutation analysis of Gaucher disease, because of the presence of pseudogene with high homology, we must be very cautious on the analysis for the possibility that there might be more than one missense mutations on the same allele. If the mutations are a result of gene conversion or unequal crossing over, it might well be the case that more than one mutations are brought to the functional gene.
2. Problems associated with molecular diagnosis of Gaucher disease
As mentioned above, there is a good correlation of the genotypes and phenotypes in most cases as far as 444Leuto Pro and 370Asnto Ser are concerned. The presence of exceptional cases as shown in Table 3 indicates that there is some limitation for the prediction of clinical features based on genotype analysis. Tay-Sachs disease, another form of hereditary sphingolipidosis, usually presents with a rather stereotypic phenotype. In Gaucher disease, however, we have observed considerable variations in the clinical phenotypes even if they share identical genotypes. The results imply that we need to understand the limitation of the prediction of clinical phenotypes. Furthermore, the results also suggest that factors other than mutations might well be involved in the modification of the clinical phenotypes.
Although most of frequently occurring mutations have now been identified, there are still many more rare unidentified mutations. For those carrying as yet undetermined mutations, it would be still laborious to identify mutations in those patients, and furthermore, it would be difficult to predict clinical prognosis based on genotypes because we do not have sufficient information on the correlation between genotypes and phenotypes for such mutations.
Total; Ser370 /Ser370; Ser370 /Pro444; Ser370 /cys463; Ser370 /x; Pro444 /Pro444; Pro444 /cys463; Pro444 /x; cys463 /x; x/x;
Type 1 39 3 5 2 18 1 0 7 2 1
Ashkenazic 18 3 0 2 11 0 0 1 0 1
Non-Ashkenzic or black 13 0 5 0 5 0 0 1 2 0
1/2 Ashkenazic 3 0 0 0 2 1 0 0 0 0
Blacks 5 0 0 0 0 0 0 5 0 0
Type 2 7 0 0 0 0 2 0 4 0 1
Type 3 16 0 0 1 0 7 4 2 2 0
TOTAL 62 3 5 3 18 10 4 13 4 2
In order to develop effective therapeutic measures, we need to transduct active glucocerebrosidase into the most relevant cells, macrophages, by means of various strategies. Among these strategies, we can consider, in principle, the following three strategies: 1. give glucocerebrosidase purified from human placenta. 2. introduce active macrophages into patients, which can be accomplished by allogenic bone marrow transplantation. 3. try to transduct wild type glucocerebrosidase gene using vectors such as retrovirus into bone marrow stem cells.
Among these therapies, the gene therapy is still at the stage of development, because efficient integration of foreign gene into human bone marrow stem cells, which can be maintained for stable expression, is still one of the most difficult tasks and needs development of further efficient methods.
Allogenic bone marrow transplantation has already been demonstrated to be effective, although it is still not clear how effective it would be for the alleviation of neurologic signs and symptoms. Considerable mortality rate (10%) even for uncomplicated patients still suggests that other therapeutic measures without serious mortality rate, if available, would be a choice from a practical point of view.
Enzyme replacement therapy for Gaucher disease has long been tried by Brady (12). Although initial trials for the enzyme replacement therapy were not promising, the development of mannose-terminated glucocerebrosidase has made this strategy to be extremely promising, which is designed in such a way that the mannose-terminated glucocerebrosidase can be effectively uptaken by macrophages by the receptors on macrophages (13, 14). It has been confirmed from a number of groups that the mannose-terminated glucocerebrosidase is effective for reducing the sizes of liver and spleen, and for increasing the hemoglobin and platelet counts, though it is not certain at this point if it is effective for neurologic signs and symptoms as well.
A serious problem associated with the enzyme replacement therapy is its cost. Currently the protocol requires intravenous injection of 60 units/kg every other two weeks. The cost for treating a patient with a body weight of 70 kg is estimated to be $380,000 (10). Even higher costs are estimated for other inborn errors of metabolism (10). Although it is surprisingly exciting to be able to develop such a successful enzyme replacement therapy, we have to overcome the problems to make the enzyme replacement therapy more acceptable for our society. To accomplish this, further development of our technologies for the production of huge amount of enzymes at much lower cost or development of safe and effective gene therapy would be the next urgent issues.
1. Barranger, J.A. & Ginns, E, Glucosylceramide lipidosis: Gaucher disease, pp.1677-1720 in The metabolic basis of inherited disease, 6th ed., eds. C.R. Scriver, A.L. Beaudet, W.S. Sly & D. Valle (New York: McGraw-Hill 1989).
2. Kolodny, E.H., et al., Phenotypic manifestations of Gaucher disease: clinical features in 48 biochemically verified type 1 patients and comment on type 2 patients, pp.23-65 in Gaucher disease: a century of delineation and research: proceedings of the First International Symposium on Gaucher disease, held in New York City, July 22-24, 1981, eds.R.J. Desnick, S. Gatt & G.A. Grabowski (New York: Alan R Liss 1982).
3. Ginns, E, et al. (1984) Isolation of cDNA clones for human beta-glucocerebrosidase using the lgt11 expression system. Biochem. Biophys. Res. Commun. 123: 574-580.
4. Tsuji, S., et al. (1986) Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase. J. Biol. Chem. 261: 50-53.
5. Horowitz, M., et al. (1989) The human glucocerebrosidase gene and pseudogene: Structure and function. Genomics 4: 87-96.
6. Ginns, E, et al. (1985) Gene mapping and leader polypeptide sequence of human glucocerebrosidase: implications for Gaucher disease. Proc. Natl. Acad. Sci. U.S.A. 82: 7101-7105.
7. Tsuji, S., et al. (1987) A mutation in the human glucocerebrosidase gene in neuronopathic Gaucher's disease. New Engl. J. Med. 316: 570-575.
8. Tsuji, S., et al. (1988) Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals. Proc. Natl. Acad. Sci. U.S.A. 85: 2349-2352.
9. Beutler, E. (1992) Gaucher disease: New molecular approaches for diagnosis and treatment", Science 256: 794-799.
10. Beutler, E. (1993) Gaucher disease as a paradigm of current issues regarding single gene mutations of humans. Proc. Natl. Acad. Sci. U.S.A. 90: 5384-5390.
11. Sidransky, E., et al. (1992) DNA mutation analysis of Gaucher Patients", Amer. J. Med. Genet. 42: 331-336.
12. Latham, T., et al. (1990) Complex alleles of the acid beta-glucosidase gene in Gaucher disease", Amer. J. Hum. Genet. 47: 79-86.
13. Brady, R.O., et al. (1974) Replacement therapy for inherited enzyme deficiency - Use of purified glucocerebrosidase in Gaucher's disease. New Engl. J. Med. 291, 989-993.
14. Barton, N.W., et al. (1990) Therapeutic response to intravenous infusions of gluco-cerebrosidase in a patient with Gaucher disease", Proc. Natl. Acad. Sci. U.S.A. 87: 1913-1916.
15. Barton, N.W. et al.(1991) Replacement therapy for inherited enzyme deficiency - Macrophage - targeted glucocerebrosidase for Gaucher's disease. New Engl. J. Med. 324: 1464-1470.