Genotype-phenotype pitfalls in Gaucher disease

Gaucher disease (GD), caused by inherited deficiency of β-glucocerebrosidase (β-Glc, EC 3.1.2.45), is classified type I if the CNS is not involved (non-neuronopathic), type II if CNS involvement is early and rapidly progressive (acute neuronopathic), and type III if CNS involvement occurs later and is slowly progressive (subacute neuronopathic). The clinical course is not predictable by measurement of residual β-Glc activity. Patient classification by identification of specific mutations is more promosing: homozygosity for the common A⁵⁸⁴¹ ->(N370S) mutation invariably predicts type I; homozygosity for the T⁶⁴³³ ->C (L444P) mutation usually indicates type III (Norbottnian). Type II disease patients often carry the T⁶⁴³³ ->C allele together with a complex allele derived in part from the downstream pseudogene by crossover or gene conversion, producing a T⁶⁴³³ ->C substitution, plus 2 or 3 additional single base substitutions (fusion gene). Employing selective PCR amplification of the structural gene, we detected homozygous T6433C (L444P) point mutations in a Caucasian boy, initially classified as having GD type I, who succumbed to severe visceral GD before age 3 years. A second novel PCR procedure for discriminating between the normal gene and the fusion gene confirmed the homozygous point mutation results. Post mortem neuropathological findings showed neuronal complex lipid accumulation consistent with late-onset type III disease. Although in Norbottnian patients it is generally accepted that onset of neurological findings is delayed, patients with the L444P/L444P genotype can only be initially classified as type Ill with this ancestry. Other patients described sporadically elsewhere are invariably considered type I until neurological findings arise. This is the first actual demonstration of a transition from type I to type Ill based on medical findings, not geography. © 1994 Wiley-Liss, Inc.     

Enzyme replacement therapy for Gaucher disease

Gaucher disease is the most common lysosomal storage disease, and the first lysosomal storage disease for which a specific therapy has been developed. Enzyme replacement therapy, with glucocerebrosidase purified from human placentae, was introduced in 1991. Recombinant human glucocerebrosidase, produced by Chinese hamster ovary cells in tissue culture, became available in 1994 and has replaced the placenta-derived product. These therapies have revolutionized the care of patients with type 1 Gaucher disease, reversing many of the pathological consequences of this disease, and preventing further progression. Furthermore, they have served as a model for the treatment of other lysosomal storage diseases and inborn errors of metabolism.     

Toward gene therapy for Gaucher disease

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomarkers in Serbian patients with Gaucher disease

Objectives

The aim of the study was to evaluate the efficiency of the biomarkers chitotriosidase (Chito), total acid phosphatase (TACP), angiotensin converting enzyme (ACE) and ferritin in the diagnosis of Gaucher disease (GD) and to assess the utility of biomarkers for monitoring the effects of enzyme replacement therapy (ERT).

Design and methods

Forty treatment-naive Gaucher patients were studied. 27/40 GP were put on ERT and monitored every 6 months.

Results

The baseline median values of Chito, TACP, ACE and ferritin were highly elevated in GP: 10216 nmol/mL/h, 26.1 U/L, 253 U/L, 515 μg/L, and 555 μg/L, respectively. The only significant difference between mild and moderate GP subgroups is observed for Chito activity (p = 0.0116). During ERT, Chito showed the steepest decrease in regard to TACP and ACE, mainly within the first year (71.4%).

Conclusions

Among these biomarkers, Chito proved to be the most useful biomarker for diagnosing GD and monitoring the ERT.     

Decreased salivary output in patients with Gaucher disease

BACKGROUND: Gaucher disease, the most common sphingolipid storage disease, results in accumulation of glucocerebroside in macrophages or "Gaucher cells". In a preliminary screening of 109 patients with type I disease, when asked specifically about dry mouth, approximately one quarter claimed to suffer from this symptom. AIM: To ascertain whether decreased salivary output is a feature of Gaucher disease. DESIGN: Prospective case-control study. METHODS: Salivary output was measured in 65 adult patients and 65 healthy controls using the Saxon test with Hochberg's modification. RESULTS: Mean salivary output was 1.91+/-1.19 g/min in the patient group vs. 2.74+/-1.17 g/min in the control group (p<0.001). This difference was greater among males. These results were not improved in the patients receiving enzyme replacement therapy, which is effective in ameliorating most Gaucher-related signs and symptoms. DISCUSSION: Recent studies have implicated an association between sicca syndrome and viral hepatitis C infection, which may imply an immunological trigger for these findings, but in this specific cohort, only three patients were reactive for hepatitis C. Follow-up of patients, both untreated and receiving enzyme therapy, is needed to delineate the association with salivary hypofunction, and ascertain whether enzyme therapy may induce sicca symptoms.     

Blood oxidative stress markers in Gaucher disease patients

BACKGROUND: Gaucher disease (GD) is the most common glycosphingolipidosis resulting in accumulation of glucoceramide. The most effective treatment for this disease is enzyme replacement therapy (ERT) which involves recombinant enzyme infusion. Enzymatic deficiency in GD patients may induce a cascade of events culminating in secondary effects such as the production of reactive oxygen species (ROS). We investigated the relationship between ROS and GD by analyzing blood oxidative stress markers in GD patients submitted to ERT at different stages during the treatment. METHODS: Blood were collected before and just after enzyme infusion. Red blood cell catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (tGSH), and plasma thiobarbituric acid reactive substances (TBARS) were assayed by spectrophotometry. Homocysteine concentrations and related polymorphisms were also studied. Control individuals matched for sex and age were also analyzed. RESULTS: Concentrations of homocysteine and TBARS, and GPx enzyme activity were not different in ERT-treated GD patients. CAT activity was higher while SOD was lower in patients compared to controls. No variations in any of these parameters were found before and just after ERT. Regarding tGSH, a significant increase was observed in GD patients after infusion. Genotypic frequencies studied did not differ from controls or other Brazilian samples. CONCLUSION: ERT-treated GD patients show an improvement in antioxidant capacity, which is further increased just after recombinant enzyme infusion.     

Modelling long-term evolution of chitotriosidase in non-neuronopathic Gaucher disease

Chitotriosidase, an enzyme secreted by activated macrophages, is widely used as a biomarker for therapeutic monitoring and patient follow-up in Gaucher disease (GD), a lysosomal disorder caused by an inherited deficiency of glucocerebrosidase. We analyzed the long-term evolution of chitotriosidase aiming to establish an accurate model that describes the influence of enzyme replacement therapy (ERT) and the impact of several covariates. A total of 55 patients with non-neuronopathic (type 1) GD were followed for almost 17 years (during a maximum of 7.57 and 8.96 years, before and after the onset of ERT, respectively). Plasma chitotriosidase activity, measured yearly before the onset of ERT and at 6-month intervals after the initiation of ERT, was analyzed as a function of several covariates (age at diagnosis and at ERT initiation, nature of the most frequent genotypes, spleen status and the occurrence of bone complications). The evolution of chitotriosidase was approximated by a sigmoidal function, which allows the calculation of predicted values, based on several parameters inferred from our data. Splenectomy and the occurrence of bone complications significantly delayed the decline in chitotriosidase activity and induced higher mean residual values after long-term (4–9?years) ERT. Likewise, patients who started ERT infusions under 15 years of age had significantly higher mean residual chitotriosidase activities. The influence of other covariates did not reach statistical significance. In conclusion, we propose a novel model describing the evolution of chitotriosidase, allowing more accurate treatment adjustments, according to the variations of this biomarker.     

Marked variation in blood beta-hexosaminidase in Gaucher disease.

Gaucher disease is due to a primary deficiency of acid beta-glucosidase activity and is associated with secondary elevations of several plasma/serum lysosomal enzyme activities, including beta-hexosaminidase. We analyzed plasma and serum beta-hexosaminidase A & B activities in 55 patients with enzyme-documented Gaucher disease. The mean beta-hexosaminidase activity was increased and the percent of the A isozyme decreased, consistent with earlier studies. Gaucher disease patients had 2,067 +/- 1,491 nmol ml-1 h-1 units of beta-hexosaminidase activity with 51.9 +/- 15.5% beta-hexosaminidase A compared to 1,086 +/- 260 nmol ml-1 h-1 and 67.8 +/- 4.0% beta-hexosaminidase A in normal controls and 965 +/- 261 nmol ml-1 h-1 and 43.6 +/- 5.5% beta-hexosaminidase A in Tay-Sachs disease heterozygotes. Contrary to previous reports, marked heterogeneity of both total plasma/serum enzyme activity and isozyme pattern was noted, as some patients had normal enzyme levels and others had severe reductions in the percent of hexosaminidase A. These data argue against the suggestions of recent studies that routine serum beta-hexosaminidase testing done in Tay-Sachs disease heterozygote detection programs can be effectively used to screen for patients with Gaucher disease.     

Gaucher disease of the spleen: CT and MR findings

We present a 26-year-old male patient with Gaucher disease who presented with epigastric pain and a palpable mass in the left abdomen. Ultrasound, abdominal computed tomography, and magnetic resonance imaging showed massive splenomegaly with multiple splenic nodules up to 7 cm in diameter. Splenic nodules should be included in the differential diagnosis of splenic masses. Follow-up is necessary because of the increased incidence of hematologic malignancies in Gaucher disease. Received: 28 September 1999/Accepted: 20 October 1999     

Heterozygote detection of type I Gaucher disease using blood platelets

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of beta-glucosidase and 4-methylumbelliferyl-beta-D-glucoside as substrate. Platelet lysates have at least two identifiable beta-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the beta-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of beta-hexosaminidase to beta-glucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of beta-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of beta-glucosidase at pH 5.6 in the presence of sodium taurocholate with the ratio of beta-hexosaminidase to beta-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.     

Mutations in the gene encoding cytosolic beta-glucosidase in Gaucher disease

Patients with Gaucher disease have a deficiency of the lysosomal acid beta-glucosidase. The phenotypes of genotypically identical patients with Gaucher disease may differ markedly. We have examined the possibility that polymorphisms in another beta-glucosidase are responsible for this variability in the phenotype. Sequence analysis of the gene encoding cytosolic beta-glucosidase (GBA3) from 4 chromosomes revealed the presence of 4 single-nucleotide substitutions: c.316 G -->A (D106N), c.1353A-->G (Y451Y), c.1368T-->A (Y456X), and c.1540 to 1541AG -->T in the 3' untranslated region. We examined the DNA from 62 patients with Gaucher disease who were homozygous for the 1226A-->G (N370S) mutation and from 542 control subjects from various populations for these polymorphisms. Six of the possible 16 haplotypes were found, and none was over- or underrepresented among patients with the severe Gaucher disease phenotypes compared with those from patients with mild phenotypes.     

Effect of miglustat on bone disease in adults with type 1 Gaucher disease: a pooled analysis of three multinational, open-label studies

BACKGROUND: Bone manifestations are a source of disability among patients with Gaucher disease (GD) and a focus of disease management. The effect of enzyme replacement therapy (ERT) on GD bone disease can be limited and may take up to 8 years to become manifest. Miglustat, a glucosylceramide synthase inhibitor, may have a positive influence on GD bone disease. OBJECTIVES: The aim of this analysis was to evaluate the effects of miglustat on bone manifestations and bone mineral density (BMD) in patients with type 1 GD. METHODS: This was a pooled analysis of data collected prospectively over an observation period of 2 years from patients who participated in 3 multinational, open-label clinical trials evaluating the efficacy and tolerability of miglustat 100 mg TID (the currently approved therapeutic dose). Bone manifestations were assessed qualitatively and in relation to treatment and spleen status. The effects of miglustat on BMD were assessed by dual-energy x-ray absorptiometry at the lumbar spine and/or femoral neck. Bone response was defined as a positive change in BMD, based on the change in BMD Z-score from baseline to months 6, 12, and 24. Changes in BMD were also analyzed according to spleen status and baseline severity of osteopenia. RESULTS: The analysis involved 72 patients, including 41 (57%) who had received previous ERT and 20 (28%) who had undergone splenectomy. Patients' mean (SD) age was 41.2 (13.1) years. The most frequent bone-related manifestations at study entry were osteoporosis (43/63 [68%] patients) and bone pain (41/65 [63%] patients). At 2 years, 54/65 (83%) patients reported no bone pain. The reductions in bone pain were comparable among all subgroups, including high-risk patients (ie, splenectomized). No new cases of bone crisis, avascular necrosis, or pathologic fractures were reported. BMD Z-scores were improved from baseline at both the lumbar spine and femoral neck at each time point (months 6, 12, and 24) (P < 0.001). As early as 6 months after the initiation of miglustat monotherapy, significant increases from baseline in the BMD Z-score were observed at both the lumbar spine (mean, 0.15; P = 0.022) and femoral neck (0.23; P < 0.001); the increases remained significant at 12 months (0.19 [P = 0.012] and 0.21 [P = 0.017], respectively) and 24 months (0.21 [P = 0.015] and 0.18 [P = 0.039]). Significant increases in BMD Z-scores were observed at the femoral neck in splenectomized patients (P < 0.001) and at both sites in osteoporotic patients (lumbar spine: P < 0.001; femoral neck: P = 0.006). CONCLUSION: This pooled analysis of 3 open-label studies of miglustat 100 mg TID suggests that miglustat monotherapy may reduce the incidence of bone pain and improve BMD in patients with type 1 GD, including those with a history of splenectomy and/or osteoporosis.