Mutation analysis of Gaucher disease patients in Taiwan: high prevalence of the RecNciI and L444P mutations

Gaucher disease, the most prevalent lysosomal storage disease characterized by a remarkable degree of clinical variability, results from deleterious mutations in the beta-glucosidase gene. Although >200 mutations in the gene for human beta-glucosidase have been described, most genotype/phenotype studies have focused on screening for a few common mutations. In the present study, whole gene sequencing analysis was performed. We sequenced eight patients with type 1, five patients with type 2, and six patients with type 3 Gaucher disease in Taiwan. A total of 37 Gaucher chromosome were identified. The detection rate is 97%. For types 1 and 3 Gaucher disease, 1448 T > C (L444P) account for 53.5% Gaucher chromosome and the recombinant allele [1448 T > C, 1483 T > G, 1497 G > C] (RecNciI) has 25% prevalence rate among those patients. For type 2 Gaucher disease, all five patients carry L444P mutation, and RecNciI is found in two of the six patients. Because L444P is also present in the RecNciI mutation, all the patients in this study have a L444P mutation in their Gaucher chromosomes. The third most common mutation of type 1 Gaucher disease is 475 C > T (R120W). L444P homozygote and R120W/RecNciI genotypes are associated with non-neuronopathic Gaucher disease. RecNciI is related to neuronopathic disease, while R120W is represented as a mild mutation in Taiwan. The mutation profile of Gaucher disease in Taiwan is limited. Only four different alleles were identified in types 1 and 3 as well as in type 2 Gaucher disease.     

Organization of Gaucher disease management in France

Gaucher disease management in France is facilitated by CETG (Comitee of Evaluation and Treatment of Gaucher disease). Four hundred and fifty-six patients are enrolled in 2006 in the French Gaucher registry of CETG. Two hundred and thirty patients had treatment. Eighty five % are type 1, 10.5% type 2 and 5.5% type 3. Only 1 patient had a saposin C deficiency. Information on CETG is available on www.cetl.net.     

‘Non-neuronopathic’ Gaucher disease reconsidered. Prevalence of neurological manifestations in a Dutch cohort of type I Gaucher disease patients and a systematic review of the literature

Summary  Gaucher disease is a lysosomal storage disorder, which is classically divided into three types. Type I Gaucher disease is differentiated from types II and III disease by the absence of nervous system involvement. However, an increasing number of reports has emerged on neurological manifestations in patients with type I Gaucher disease. Whether a strict division in three different phenotypes is still valid has been the subject of debate. The main objective of this study was to provide scientific arguments whether a distinction between type I (non-neuronopathic) and types II and III (neuronopathic) Gaucher disease should be maintained. We investigated retrospectively a large Dutch cohort of type I Gaucher disease patients for the prevalence of neurological manifestations and provide an overview of the literature on this topic. A diagnosis of a neurological disease was made 34 times in 75 patients. Forty-five patients reported at least one neurological symptom during the median follow-up time of 11 years. The literature search revealed 86 studies in which type I Gaucher disease patients or carriers of a glucocerebrosidase mutation were described with a neurological disease or a condition which is known to be associated with neurological disease. In conclusion, the term non-neuronopathic Gaucher disease does not seem to be an appropriate characterization of type I Gaucher disease. However, the neurological signs and symptoms in type I Gaucher disease are of a totally different kind from and, in the majority of cases, of much less severity than the signs and symptoms associated with types II and III disease Therefore, type I disease should be classified as a separate phenotype. Competing interests: None declared     

La maladie de Gaucher : quand y penser ?

Gaucher disease is a rare autosomal recessive genetic disease, caused by a deficiency of the lysosomal enzyme, glucocerebrosidase that leads to the accumulation of its substrate (glucosylceramide) in lysosomal macrophages. In the general population, its incidence varies between 0.4 and 5.8/100,000 inhabitants. Type 1 Gaucher disease is the most frequent and is characterized by its extreme heterogeneity including asymptomatic or more severe presentations. The most frequent symptoms are anemia, thrombocytopenia, splenomegaly, and/or hepatomegaly, and a potentially severe bone involvement. Type 2 and type 3 Gaucher diseases are associated with neurological involvement that can be severe. Diagnosis is confirmed by demonstrating a deficiency of glucocerebrosidase activity in leucocytes, and by the identification of biallelic pathogenic variants in GBA1 gene. Type 1 Gaucher disease is associated with a higher risk of Parkinson disease, some solid cancers, and hematologic diseases in particularly multiple myeloma. Specific treatment, such as enzyme replacement therapy or substrate reduction therapy is indicated in symptomatic type 1 Gaucher disease. Only enzyme replacement therapy is indicated in type 3 Gaucher disease. Treatment improves quality of life and prognosis. The rarity of Gaucher disease and its wide variability in clinical presentations lead to diagnosis delays. There is a strong need for a better knowledge of its symptoms among physicians, to reduce irreversible complications.     

Improvement of Splenomegaly and Pancytopenia by Enzyme Replacement Therapy Against Type 1 Gaucher Disease: A Report of Sibling Cases

Gaucher disease is a genetic lipid storage disease and represents a potentially serious health problem. It arises from a deficiency of glucocerebrosidase activity with secondary accumulation of large quantities of glucocerebroside. Symptoms are usually multisystemic, often debilitating or disabling, and sometimes disfiguring, and they can lead to death. We report objective clinical response's to repeated infusion of human placental and recombinant glucocerebrosidase in 2 patients with type 1 Gaucher disease and increased hemoglobin levels and platelet counts. Splenic volume decreased during the period of enzyme administration. Enzyme replacement therapy has improved the treatment of type 1 Gaucher disease by safely and effectively arresting, decreasing, or normalizing many of its major signs and symptoms. Consideration by physicians must be given to Gaucher disease, and appropriate treatments must be given when confronted with cryptogenic pancytopenia or hepatosplenomegaly.     

Management of non-neuronopathic Gaucher disease with special reference to pregnancy, splenectomy, bisphosphonate therapy, use of biomarkers and bone disease monitoring

Summary  Enzyme replacement was introduced as treatment for non-neuronopathic Gaucher disease more than 15 years ago. To ensure the best use of this costly ultra-orphan agent, a systematic disease management approach has been proposed by an international panel; this includes the development, by consensus, of achievable treatment goals. Here we critically review these goals and monitoring guidelines and incorporate emerging experience of the disease in the therapeutic era, as well as contemporary clinical research. This review makes recommendations related specifically to the management of pregnancy; the appropriate use of splenectomy and bisphosphonate treatment; the relevance of biochemical markers to disease monitoring; and the use of semi-quantitative methods for assessing bone marrow infiltration. In addition, we identify key areas for development, including the requirement for a validated index of disease severity; the need to correlate widely used biomarkers with long-term disease outcomes, and the desirability of establishing agreed standards for monitoring of bone disease particularly in infants and children with Gaucher disease. Competing interests: None (see Acknowledgements) References to electronic databases: Non-neuronopathic Gaucher disease, type I: OMIM 230800. Acute neuronopathic Gaucher disease, type 2: OMIM 230900. Chronic neuronopathic Gaucher disease, type 3: OMIM 231000. Glucocerebrosidase: EC 3.2.1.45.     

Increased glucocerebrosidase (GBA) 2 activity in GBA1 deficient mice brains and in Gaucher leucocytes

Lysosomal glucocerebrosidase (GBA1) deficiency is causative for Gaucher disease. Not all individuals with GBA1 mutations develop neurological involvement raising the possibility that other factors may provide compensatory protection. One factor may be the activity of the non-lysosomal β-glucosidase (GBA2) which exhibits catalytic activity towards glucosylceramide and is reported to be highly expressed in brain tissue. Here, we assessed brain GBA2 enzymatic activity in wild type, heterozygote and GBA1 deficient mice. Additionally, we determined activity in leucocytes obtained from 13 patients with Gaucher disease, 10 patients with enzymology consistent with heterozygote status and 19 controls. For wild type animals, GBA2 accounted for over 85 % of total brain GBA activity and was significantly elevated in GBA1 deficient mice when compared to heterozygote and wild types (GBA1 deficient; 92.4?±?5.6, heterozygote; 71.5?±?2.4, wild type 76.8?±?5.1 nmol/h/mg protein). For the patient samples, five Gaucher patients had GBA2 leucocyte activities markedly greater than controls. No difference in GBA2 activity was apparent between the control and carrier groups. Undetectable GBA2 activity was identified in four leucocyte preparations; one in the control group, two in the carrier group and one from the Gaucher disease group. Work is now required to ascertain whether GBA2 activity is a disease modifying factor in Gaucher disease and to identify the mechanism(s) responsible for triggering increased GBA2 activity in GBA1 deficiency states.     

Gaucher disease in childhood

Gaucher disease is well-known in adult patients and must be regarded as a pediatric disease, two thirds of the patients manifesting before the age of 20. Three clinical forms have been defined based on the presence of neurological involvement. Gaucher disease type 1, without neurological signs, generally begins before the five years age with splenomegaly as the main symptom. The bone crises are more frequent than in adulthood. Gaucher disease type 2 or acute neuronopathic form begins between three and six months and do not have any treatment. Type 3 or chronic neuronopathic form appears like a type 1 with progressive horizontal saccade-initiation failure and developmental delay. Onset in childhood is predictive of a severe and progressive phenotype. The presence of neurological symptoms induces important consequences for treatment, prognosis and genetic counselling.     

Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease.

Fibroblasts from normal subjects and patients with the three types of Gaucher disease were labeled with [3H]leucine. Glucocerebrosidase antigen was immunoprecipitated using affinity-purified Sepharose-bound antibody. Normal cells initially formed a 60-kDa polypeptide antigen that was gradually replaced by a broad band of antigen averaging 63 kDa. This position corresponds with that of mature fibroblast and placental enzyme. Processing of glucocerebrosidase in six unrelated patients with type I Gaucher disease and one patient with type III Gaucher disease was exactly the same as normal. In contrast, three patients with the severe infantile (type II) form of the disease manifested a very unstable enzyme; the 60-kDa band appeared transiently and the mature 63-kDa band was never seen. These results indicate that type II Gaucher disease may well be distinguishable from type I disease by virtue of the very unstable enzyme precursor. Contrary to some earlier reports, processing of glucocerebrosidase in type I disease appears to be entirely normal.     

Tartrate-resistant acid phosphatase staining of monocytes in Gaucher disease

Cytochemical studies were performed on peripheral blood from 30 patients with type 1 Gaucher disease. In 29 of the patients, peripheral blood monocytes stained positively for tartrate-resistant acid phosphatase, whereas monocytes from 18 normal individuals and 14 patients with monocytosis did not. In the Gaucher patients, the percentage of monocyte positivity for tartrate-resistant acid phosphatase ranged from 2 to 97. There was no correlation between the percent monocyte staining and the degree of disease severity, as measured by hepatosplenomegaly, pancytopenia, or extent of bone disease, for the group as a whole. In Gaucher patients who had not undergone splenectomy, however, there was a significant correlation between percent monocyte staining and the degree of hepatosplenomegaly, anemia, and thrombocytopenia. The presence of tartrate-resistant acid phosphatase may be secondary to the lysosomal accumulation of glucosyl ceramide within these monocytes, although this remains to be confirmed. If so, these circulating cells may represent precursors of the Gaucher cells in tissues. Tartrate-resistant acid phosphatase staining of peripheral blood monocytes may be useful as a diagnostic marker for Gaucher type 1 disease and for further studies on the pathogenesis of the disease.     

Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease.

Multiple molecular forms of beta-glucocerebrosidase that permit discrimination between neurologic and non-neurologic phenotypes of Gaucher disease have been identified radioimmunologically in fibroblasts and human brain tissue. In normal human fibroblasts these forms have been shown by NaDodSO4/polyacrylamide gel electrophoresis to have apparent Mr of 63,000 (form A1), 61,000 (form A2), and 56,000 (form B). The Mr 63,000 form may be a precursor of the Mr 56,000 form. Non-neurologic Gaucher disease (type 1) fibroblasts and normal brain tissue are characteristic in that they contain only one major immunoreactive protein, the Mr 56,000 form. In contrast, fibroblast extracts and brain tissue from neurologic Gaucher disease phenotypes contain only the higher molecular weight forms A1 and A2. These data and the low residual activity of the enzyme in all the variants of Gaucher disease suggest that the mutations of beta-glucocerebrosidase are allelic and involve the active site.     

Substrate reduction therapy of glycosphingolipid storage disorders

Summary In the last 15 years enormous progress has been made regarding therapy of type I Gaucher disease, a severely disabling disorder characterized by intralysosomal storage of glucosylceramide in tissue macrophages. Effective enzyme replacement therapy of type I Gaucher disease, based on chronic intravenous administration of mannose-terminated recombinant human glucocerebrosidase, has been available since 1990 and has been applied in several thousand patients without serious adverse effects. An alternative therapeutic approach, so-called substrate reduction therapy, is based on partial reduction of the synthesis of glucosylceramide and hence of subsequent metabolites. Oral administration of an inhibitor of glucosylceramide synthesis (N-butyldeoxynojirimycin, registered in Europe since 2002 as miglustat (Zavesca)), is effective in reversing clinical symptoms in type I Gaucher patients with mild to moderate disease manifestations. The growing long-term experience with substrate reduction therapy indicates that this treatment is also without major adverse effects. Substrate reduction therapy, in conjunction with enzyme replacement therapy, may play an important role in the future clinical management of patients suffering from type I Gaucher disease. Clinical trials are under way that should reveal the value of substrate reduction for maintenance therapy of type I Gaucher disease and for treatment of neuronopathic variants of Gaucher disease, Niemann–Pick disease type C, late-onset Tay–Sachs disease and Sandhoff disease. Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9 September 2005 Competing interests: None declared     

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.     

Physical and kinetic properties of β-glucosidase in Gaucher disease

Membrane-bound 'acid' beta-glucosidase of human spleen was solubilized with either sodium cholate or 'Cutscum'. The solubilized enzyme in type 1 (adult) Gaucher disease was less heat-stable than the normal enzyme, and when precipitated by ammonium sulphate it had a higher apparent molecular weight than the corresponding normal enzyme. The normal beta-glucosidase was activated by taurocholate, whereas the Gaucher enzyme was inhibited. The decrease in 'acid' beta-glucosidase activity in Gaucher disease was associated with a profound deficiency of that form of the enzyme which bound to Concanavalin A. The results are consistent with faulty processing of newly synthesized 'acid' beta-glucosidase in type 1 Gaucher disease.     

Biochemical markers of bone turnover as tools in the evaluation of skeletal involvement in patients with type 1 Gaucher disease

Gaucher disease is caused by the inherited deficiency of glucocerebrosidase, a lysosomal enzyme responsible for the cleavage of glucosylceramide. In addition to organomegaly and hematological abnormalities, type 1 (nonneuronopathic) disease is characterized by insidiously progressive and often severe skeletal involvement. We investigated the applications of biochemical markers of bone turnover in the evaluation of skeletal lesions in type 1 Gaucher disease patients. Serum osteocalcin, a marker of osteoblastic bone formation, and type I collagen C-terminal telopeptide, a marker of osteoclastic bone resorption, were measured in 16 type 1 Gaucher disease patients and in 29 age-matched controls. Our results indicate a significant decrease of both osteocalcin (11.13 +/- 2.27 ng/ml) and type I collagen C-terminal telopeptide (3617.62 +/- 536.69 ng/ml) values in patients with Gaucher disease, compared to the unaffected controls (38.67+/-6.24 ng/ml and 6808.57 ng/ml +/- 865.66, respectively). These low values were observed in all patients, irrespective of their age, indicating a marked failure of both osteoblastic and osteoclastic functions in Gaucher disease. Significant differences in the mean serum values of biochemical bone turnover markers were found in different evolutional stages of bone involvement. We conclude that measurement of serum biochemical markers of bone turnover can be used as an auxiliary tool in the diagnosis, staging, and monitoring the skeletal lesions in Gaucher disease, in conjunction with imaging methods and bone densitometry measurements.     

Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in the type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is approximately equal to 80% informative in all Gaucher patients studied.     

Hepatopulmonary syndrome: a complication of type 1 Gaucher disease

Gaucher's disease is a not exceptional lysosomial disease in Tunisia. Type 1 is by far the most common one. Pulmonary involvement is considered to be rare in type 1 Gaucher's disease. Pulmonary hypertension, infiltration of the lungs with Gaucher cells, and severe hypoxemia due to intrapulmonary arterial-venous shunts, have been described in case reports and small case series. We reported the case of hepatopulmonary syndrome in a 14-year-old boy with type 1 Gaucher disease. The diagnosis of Gaucher disease was established, at 2 years age, by enzyme assay of leucocyte β-glucosidase. The patient presented dyspnoea, digital clubbing and cyanosis of the lips. The arterial blood gas found severe hypoxaemia with PaO(2) at 56.9 mmHg. The diagnosis of hepatopulmonary syndrome, in our patient, was confirmed by demonstration of the intrapulmonary shunting using contrast-enhanced echocardiography and the technetium-99m-labeled macroaggregated albumin. The patient was treated by symptomatic measure, long term oxygen therapy because the insufficiency of the enzyme replacement therapy. Screening for hypoxemia in children with liver disease should be considered.     

Molecular characterization of type 3 (neuronopathic) Gaucher disease in Thai patients

Gaucher disease is an autosomal recessive lysosomal storage disorder due to deficiency of the lysosomal enzyme glucocerebrosidase. Three clinical phenotypes, type 1, nonneuronopathic; and types 2 and 3, acute and subacute neuronopathic are recognized. The incidence of Gaucher disease in the Thai population is unknown, but likely under-diagnosed. We performed molecular analysis in four patients, from three sibships, with type 3 Gaucher disease. Four mutant glucocerebrosidase (GBA) alleles were identified including two novel splice site mutations, IVS6-1G>C and IVS9-3C>G; both are predicted to result in truncated protein products, p.F255fsX256, and p.K464fsX487 and p.S463fsX480, respectively. One patient, homozygous for the L444P point mutation, had a "Norbottnian-like" phenotype, with more severe visceral involvement, kyphosis, barreled chest, and no neurological involvement other than supranuclear gaze palsy. These molecular studies of neuronopathic Gaucher disease will provide additional genotype-phenotype correlation particularly in non-Caucasian population.     

Recommendations for the management of the haematological and onco-haematological aspects of Gaucher disease

Current knowledge of the haematological and onco-haematological complications of type 1 Gaucher disease has been reviewed with the aim of identifying best clinical practice for treatment and disease management. It was concluded that: (i) Awareness of typical patterns of cytopenia can help clinicians distinguish haematological co-morbidities. (ii) Red blood cell studies and complete iron metabolism evaluation at baseline are recommended. (iii) Haemoglobin levels defining anaemia should be raised and used in Gaucher disease treatment and monitoring. (iv) Surgeons should be aware of potential bleeding complications during surgery in Gaucher patients. The higher incidence of multiple myeloma in Gaucher disease suggests that Gaucher patients should have their immunoglobulin profile determined at diagnosis and monitored every 2 years (patients <50 years) or every year (patients >50 years). If monoclonal gammopathy of undetermined significance (MGUS) is found, general MGUS guidelines should be followed. Future studies should focus on the utility of early treatment to prevent immunoglobulin abnormalities and multiple myeloma.     

Therapeutic goals in Gaucher disease

Evidence-based therapeutic goals have been developed by several European and American experts in Gaucher disease in order to attempt to reverse the entire Type 1 Gaucher phenotype, prevent complications, improve quality of life and prevent life-threatening complications. Patients with Gaucher disease will benefit by maximum enzyme replacement treatment (ERT) efficiency if it is given at the optimal time i.e. generally during the asymptomatic phase of the disease. Currently, Cerezyme is the standard of care for all severities of type 1 Gaucher disease due to its high efficiency at ameliorating bleeding tendencies, reversing organomegaly and part of skeletal damages and eliminating bone crises. ERT has also demonstrated a remarkable safety profile with tolerability at 3 years greater than 99%. Treatment of Gaucher disease is a long-life treatment that the patient should not interrupt without a careful monitoring of the disease evolution.