Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

A Drosophila Model of Neuronopathic Gaucher Disease Demonstrates Lysosomal-Autophagic Defects and Altered mTOR Signalling and Is Functionally Rescued by Rapamycin.

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. Here we describe a novel Drosophila model of GD that lacks the two fly GBA1 orthologs. This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. We also demonstrate a block in autophagy flux in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, our results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD SIGNIFICANCE STATEMENT: We developed a Drosophila model of neuronopathic GD by knocking-out the fly orthologs of the GBA1 gene, demonstrating abnormal lysosomal pathology in the fly brain. Functioning lysosomes are required for autophagosome-lysosomal fusion in the autophagy pathway. We show in vivo that autophagy is impaired in dGBA-deficient fly brains. In response, mechanistic target of rapamycin (mTOR) activity is downregulated in dGBA-deficient flies and rapamycin ameliorates the lifespan, locomotor, and oxidative stress phenotypes. dGBA knock-out flies also display an upregulation of the Drosophila ortholog of mammalian TFEB, Mitf, a response that is unable to overcome the autophagy block. Together, our results suggest that rapamycin may have potential benefits in the treatment of GD, as well as PD linked to GBA1 mutations.

Prevention is the Best Therapy: The Geneticist's Approach.

Abstract During the last two decades prenatal genetic screening and diagnosis has become the cornerstone of medical care for family planning to prevent genetic disease. Carrier screening programs for genetic disorders that are prevalent in various populations identify couples and pregnancies at risk of having an affected child. These couples can proceed with a choice of invasive prenatal diagnosis tests of the fetus (chorionic villous sampling and amniocentesis), or non-invasive prenatal testing of free fetal DNA circulation in the maternal blood which has emerged within the last few years and is currently available for fetal sexing for X Linked disorders. Despite the advances in prenatal diagnosis, couples found to have a fetus affected with a genetic disorder may need to face the dilemma of pregnancy termination. Preimplantation genetic diagnosis (PGD) is an alternative to preempt risk of having a child affected with a life-altering genetic disorder. This technique allows biopsy and genetic diagnosis of embryos obtained from in vitro fertilization by analysis of the genetic material from one or a few embryonic cells. Only unaffected embryos are returned to the mother to establish the pregnancy. We present our experience using PGD for four Lysosomal storage disorders: Tay Sachs, Gaucher type 1, Hunter and Fabry disease with some of the couples being carriers of more than one genetic disorder. PGD is applicable to most disorders for which the gene and the familial mutation are known and should be presented to couples as an alternative to invasive prenatal testing.

Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease.

Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP(+) (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP(+) induced toxicity in vitro. However, only CBE-induced enhancement of MPP(+) toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.

[Gaucher disease--guidelines for diagnosis and management of adult patients]. / Gaucherova bolest--smjernice za dijagnozu i lijecenje odraslih bolesnika.

Gaucher disease is an autosomal recessive disorder, characterized by decreased levels of the lysosomal enzyme glucocerebrosidase. This deficiency results in a decreased breakdown of this glycosphingolipid glucocerebroside, which accumulates in the lysosomes of the monocyte-macrophage system. It is the most common form of sphingolipidosis. Clinically, the principle signs of Gaucher's disease are hepatosplenomegaly, bone involvement, hematological changes and CNS involvement. The diagnosis of Gaucher disease has to be confirmed by the measurement of the activity of the enzyme glucocerebrosidase in leukocytes or fibroblasts and genetic testing. An effective therapy for Gaucher disease has now been available for more than 10 years. It consists of life-long intravenous replacement of the deficient enzyme--glucocerebrosidase. If enzyme replacement therapy is started early enough, it leads to significant improvement in patient's general condition and quality of life. The aim of this document is to provide to the Croatian medical audience the guidelines for diagnosis and management of adult patients with Gaucher disease. These guidelines are produced by specialists who have long lasting experience with patients with rare metabolic diseases working in the Division of Metabolic Diseases, Department of Internal Medicine, University Hospital Center Zagreb which is the Referral Center for Rare and Metabolic diseases of the Ministry of Health, Republic of Croatia. They were endorsed by the Croatian Society for Rare Diseases, Croatian Medical Association. These are the first guidelines published in Croatia on diagnosis, treatment and follow-up of Gaucher disease.

High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

Carrier screening for Gaucher disease: lessons for low-penetrance, treatable diseases.

CONTEXT: The aim of carrier screening is to prevent severe, untreatable genetic disease by identifying couples at risk before the birth of an affected child, and providing such couples with options for reproductive outcomes for affected pregnancies. Gaucher disease (GD) is an autosomal recessive storage disorder, relatively frequent in Ashkenazi Jews. Carrier screening for GD is controversial because common type 1 GD is often asymptomatic and effective treatment exists. However, screening is offered to Ashkenazi Jews worldwide and has been offered in Israel since 1995. OBJECTIVE: To examine the scope and outcomes of nationwide GD screening. DESIGN, SETTING, AND PARTICIPANTS: All Israeli genetic centers provided data on the number of individuals screened for GD, the number of carriers identified, the number of carrier couples identified, and the mutations identified in these couples between January 1, 1995, and March 31, 2003. Carrier couples were interviewed via telephone between January 21, 2003, and August 31, 2004, using a structured questionnaire for relevant outcome measures. MAIN OUTCOME MEASURES: Screening scope (number of testing centers, tested individuals, and carrier couples), screening process (type of pretest and posttest consultations), and screening outcomes (utilization of prenatal diagnosis and pregnancy terminations). RESULTS: Between January 1, 1995, and March 31, 2003, 10 of 12 Israeli genetic centers (83.3%) offered carrier screening. Carrier frequency was 5.7%, and 83 carrier couples were identified among an estimated 28,893 individuals screened. There were 82 couples at risk for offspring with type 1 GD. Seventy of 82 couples (85%) were at risk for asymptomatic or mildly affected offspring and 12 of 82 couples (15%) were at risk for moderately affected offspring. At postscreening, 65 interviewed couples had 90 pregnancies, and prenatal diagnosis was performed in 68 pregnancies (76%), detecting 16 fetuses with GD (24%). Pregnancies were terminated in 2 of 13 fetuses (15%) predicted to be asymptomatic or mildly affected and 2 of 3 fetuses (67%) with predicted moderate disease. There were significantly fewer pregnancy terminations in couples who in addition to genetic counseling had medical counseling with a GD expert (1 of 13 [8%] vs 3 of 3 with no medical counseling [100%], P = .007). CONCLUSIONS: In this study of GD screening among Ashkenazi Jewish couples in Israel, most couples did not terminate affected pregnancies, although screening was associated with a few pregnancy terminations. The main possible benefit was providing couples with knowledge and control. The divergence of these outcomes from stated goals of screening programs is likely to confront carrier screening programs for low-penetrance diseases.

Effective cell and gene therapy in a murine model of Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9-11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5-6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD in vivo. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.

Prepregnancy testing for single-gene disorders by polar body analysis.

Preventive measures for single-gene disorders are currently based on carrier screening in pregnancy and prenatal diagnosis. Although this has been extremely effective for preventing new cases of common inherited conditions, the major limitation is still termination of 25% of wanted pregnancies following detection of affected fetuses. To overcome this important problem, we developed a method for prepregnancy genetic testing that involves DNA analysis of the first and second polar bodies, which are extruded during maturation and fertilization of oocytes. We offered this option to 28 couples at risk for having children with single-gene disorders. Fifty clinical cycles were performed from these patients for the following conditions: 20 for cystic fibrosis, 18 for thalassemia, 6 for sickle cell disease, 2 each for Gaucher disease and LCHAD (long-chain 3-hydroxyacyl-COA dehydrogenase deficiency), and 1 each for hemophilia B and phenylketonuria. Oocytes obtained from these patients using in vitro fertilization procedures (IVF) were tested by a sequential multiplex nested PCR analysis of the first and second polar body to detect the gene involved simultaneously with linked polymorphic markers. A total of 191 of 399 oocytes with predicted genotype were mutation free and preselected for fertilization and transfer. In all but three cycles, one to three unaffected embryos with predicted unaffected genotypes were transferred, resulting in 20 pregnancies, from which 19 healthy children have been born. The follow-up analysis of embryos resulting from oocytes with predicted affected genotype, confirmed the diagnosis in 97% of cases, demonstrating the reliability of prepregnancy diagnosis of single-gene defects by polar body analysis.

Identification of the second common Jewish Gaucher disease mutation makes possible population-based screening for the heterozygous state.

Gaucher disease is an autosomal recessive glycolipid storage disease characterized by a deficiency of glucocerebrosidase. The disease is most common in persons of Ashkenazi Jewish ancestry and the most common mutation, accounting for about 75% of the mutant alleles in this population, is known to be an A----G substitution at cDNA nucleotide (nt) 1226. Screening for this disease has not been possible because nearly 25% of the mutant alleles had not been identified, but linkage analysis led to the suggestion that most of these could be accounted for by a single mutation. We now report the discovery of this mutation. The insertion of a single nucleotide, a second guanine at cDNA nt 84 (the 84GG mutation), has been detected in the 5' coding region of the glucocerebrosidase gene. The amount of mRNA produced is shown to be normal but since the frameshift produced early termination, no translation product is seen. This finding is consistent with the virtual absence of antigen found in patients carrying this mutation. The 84GG mutation accounts for most of the previously unidentified Gaucher disease mutations in Jewish patients. The common Jewish mutation at nt 1226, the 84GG mutation, and the less-common mutation at nt 1448 accounted for 95% of all of the Gaucher disease-producing alleles in 71 Jewish patients. This now makes it possible to screen for heterozygotes on a DNA level with a relatively low risk of missing couples at risk for producing infants with Gaucher disease.