Effects of a Shortage of Imiglucerase on Three Patients with Type I Gaucher Disease

Background: Children with Gaucher disease type I (GD1) are usually treated with enzyme replacement therapy (ERT) at a dose of 30-60U/Kg/2W. Recently, due to an acute shortage supply of imiglucerase, a reduced dose or a reduced infusion frequency was recommended. Objective: To evaluate the effects of a reduced infusion frequency of imiglucerase over 15 months of follow-up. Patients and Methods: Three patients (1M:2F) were treated with ERT since a median age of 7 years (range 5-12). Only one had bone crisis and Erlenmeyer deformations. Median duration of treatment before dose reduction was 3 years (range 1-8). ERT resulted in total regression of symptoms, normalization of hematological parameters and progressive improvement of chitotriosidase in all patients. In August 2009 infusion schedule was changed from a media 45U/Kg every two weeks to every four weeks. Results: All patients remained asymptomatic and with no major change on hematological parameters except for the patient with bone crisis who presented subnormal platelet count. All patients showed an upward trend in chitotriosidase values. Comments: Although a longer follow-up is needed, is probable that even children completely stabilized can probably not be kept on lower doses even though the reduction of frequency of the infusions represent a lower social burden.

High frequency of mutation G377S in Brazilian type 3 Gaucher disease patients

Gaucher disease (GD), the most prevalent lysosome storage disorder, presents an autosomal recessive mode of inheritance. It is a paradigm for therapeutic intervention in medical genetics due to the existence of effective enzyme replacement therapy. We report here the analysis of GD in 262 unrelated Brazilian patients, carried out in order to establish the frequency of the most common mutations and to provide prognostic information based on genotype-phenotype correlations. Among 247 type 1 GD patients, mutation N370S was detected in 47% of all the alleles, but N370S/N370S homozygosity was found in only 10% of the patients, a much lower frequency than expected, suggesting that most individuals presenting this genotype may not receive medical attention. Recombinant alleles were detected at a high frequency: 44% of the chromosomes bearing mutation L444P had other mutations derived from the pseudogene sequence, present in 25% of patients. Three neuronopathic type 2 patients were homozygous for L444P, all presenting additional mutations (E326K or recombinant alleles) that probably lead to the more severe phenotypes. Six children, classified as type 1 GD patients, had a L444P/L444P genotype, showing that neuronopathic symptoms may only manifest later in life. This would indicate the need for a higher treatment dose during enzyme replacement therapy. Finally, mutation G377S was present in 4 homozygous type 1 patients and also in compound heterozygosity in 5 (42%) type 3 patients. These findings indicate that G377S cannot be unambiguously classified as mild and suggest an allele-dose effect for this mutation.

Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.

We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease.

The effect of chaperone compounds on glucocerebrosidase stability and activity in fibroblasts from Gaucher Disease patients with different genotypes

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Using induced pluripotent stem cells to investigate the mechanistic link between Gaucher disease and Parkinson related synucleinopathies

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Dentomaxillofacial manifestations of Gaucher's disease: preliminary clinical and radiographic findings

Objectives: A wide variety of manifestations is presented in patients with Gaucher's disease (GD), including bone, haematology and visceral disturbances. This study was conducted to ascertain the main maxillofacial abnormalities by means of clinical survey, panoramic and cone beam CT (CBCT); to compare the patient's group with an age-sex matched control group; and to correlate clinical and radiological data. Methods: Ten patients previously diagnosed with GD were submitted to clinical and radiological surveys (CBCT and panoramic radiographs). The examination consisted of anamnesis, extra- and intraoral examinations and analyses of each patient's records. Imaging data were collected from the point of view of 3 observers, and the results compared with a healthy group (20 individuals) by means of statistical analysis (Fisher's exact test). Results: Gaucher patients had significantly more manifestations than otherwise healthy carriers. The most prevalent findings were enlarged marrow spaces, generalized osteopenia and effacement of jaw structures (mandibular canal, lamina dura and mental foramen). Here we describe a case in which thickening of the maxillary sinus mucosa was observed on CBCT rather than opacification of the sinus as seen on panoramic radiographs. Pathological fractures, root resorption and delay on tooth eruption were not observed. Conclusions: A poor relationship could be observed between clinical and radiological data. Patients showed important bone manifestations, which require careful diagnostic and surgical planning whenever necessary. Although panoramic radiographs have shown significant differences, CBCT is more effective in pointing out differences between patients and a control group, thus showing it as an important tool for evaluation of Gaucher patients. Dentomaxillofacial Radiology (2012) 41, 541-547. doi: 10.1259/dmfr/143023353

Association between Parkinson`s disease and glucocerebrosidase mutations in Brazil

Objective: To evaluate the association between parkinsonism and mutations in the glucocerebrosidase gene (GBA) in Brazilian patients. Methods: We searched for three GBA common mutations (N370S, L444P and G377S) in 65 Brazilian patients affected by PD with disease onset before the age of 55 and compared the results to 267 age- and sex-matched controls. Results: GBA mutations were detected at a significantly higher frequency among Parkinson`s disease patients (2/65 = 3%), when compared to the control group (0/267): P = 0.0379. Conclusion: These results provide further evidence for GBA mutations being a possible hereditary risk factor for PD. (C) 2007 Elsevier Ltd. All rights reserved.

Multicenter Analysis of Glucocerebrosidase Mutations in Parkinson`s Disease

Background Recent studies indicate an increased frequency of mutations in the gene encoding glucocerebrosidase (GBA), a deficiency of which causes Gaucher`s disease, among patients with Parkinson`s disease. We aimed to ascertain the frequency of GBA mutations in an ethnically diverse group of patients with Parkinson`s disease. Methods Sixteen centers participated in our international, collaborative study: five from the Americas, six from Europe, two from Israel, and three from Asia. Each center genotyped a standard DNA panel to permit comparison of the genotyping results across centers. Genotypes and phenotypic data from a total of 5691 patients with Parkinson`s disease (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews) were analyzed, with multivariate logistic-regression models and the Mantel-Haenszel procedure used to estimate odds ratios across centers. Results All 16 centers could detect two GBA mutations, L444P and N370S. Among Ashkenazi Jewish subjects, either mutation was found in 15% of patients and 3% of controls, and among non-Ashkenazi Jewish subjects, either mutation was found in 3% of patients and less than 1% of controls. GBA was fully sequenced for 1883 non-Ashkenazi Jewish patients, and mutations were identified in 7%, showing that limited mutation screening can miss half the mutant alleles. The odds ratio for any GBA mutation in patients versus controls was 5.43 across centers. As compared with patients who did not carry a GBA mutation, those with a GBA mutation presented earlier with the disease, were more likely to have affected relatives, and were more likely to have atypical clinical manifestations. Conclusions Data collected from 16 centers demonstrate that there is a strong association between GBA mutations and Parkinson`s disease.

Rastreamento populacional para Doença de Gaucher em Tabuleiro do Norte-CE

Background. Gaucher Disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid β-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using Dried Blood Spots on Filter Paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. Methods. Between June 1, 2007 and May 31, 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity <2.19 nmol/h/mL were referred to analysis of GBA and chitotriosidase activity in peripheral leukocytes and in plasma, respectively. Subjects at highest risk for GD (GBA activity in peripheral leukocytes <5.6 nmol/h/mg protein) were submitted to molecular analysis to confirm diagnosis. Results. Screening with DBS-FP identified 135 subjects (18.2%) with GBA activity <2.19 nmol/h/mL, 131 of whom remained in the study. In 10 of these (7.6%), GBA activity in leukocytes was 2.6 5.5 nmol/h/mg protein. Subsequent molecular analysis confirmed 6 cases of heterozygosity and 4 normals for GD. Conclusion. DBS-FP assay was shown to be an effective initial GD screening strategy for high-prevalence populations in developing regions. Diagnosis could not be established from GBA activity in leukocytes alone, but required confirmation with molecular analysis

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the β-subunit of β-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Consanguinidade e efeito fundador da mutação G377S da doença de Gaucher em população de Tabuleiro do Norte – Ceará – Brasil

Gaucher’s disease (GD) is caused by a β-glucocerebrosidase deficiency, leading to the accumulation of glucocerebroside in the reticuloendothelial system. The prevalence of GD in Tabuleiro do Norte (TN) (1:4000) is the highest in Brazil. The purpose of this study was to present evidence of consanguinity and founder effect for the G377S mutation (c.1246G>A) among GD patients in TN based on enzyme, molecular and genealogical studies. Between March 2009 and December 2010, 131 subjects at risk for GD (GC in dried blood ≤2.19 nmol/h/ml) and 5 confirmed GD patients from the same community were submitted for molecular analysis to characterize the genetic profile of the population. Based on the enzymatic and molecular analysis, the subjects were classified into three categories: affected (n=5), carrier (n=20) and non-carrier (n=111). All carriers were (G377S/wt). Affected subjects were homozygous (G377S/G377S). The identification of a single mutation in carriers and homozygotes from different generations, the history of the community and the genealogy study suggest that the high prevalence of GD in this population may be due to a combination of consanguinity and founder effect for the G377S mutation

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Nouvelles thérapies pour les maladies osseuses de l'enfant [New therapies for children affected by bone diseases].

Considerable progress has been achieved in recent years in treating children affected by bone diseases. Advances in the understanding of the molecular pathophysiology of genetic bone diseases have led to the development of enzyme replacement therapies for various lysosomal storage diseases, following the breakthrough initiated in treating Gaucher disease. Clinical studies are underway with tailored molecules correcting bone fragility and alleviating chronic bone pain and other manifestations of hypophosphatasia, or promoting growth of long bones in achondroplasia patients. We further report our very encouraging experience with intravenous bisphosphonate treatment in children suffering from secondary osteopenia and the high prevalence of calcium and vitamin D deficits in these severely disabled children.