RESUMEN

Parkinson's disease (PD) is a multifactorial, chronic, and progressive neurodegenerative disorder inducing movement alterations as a result of the loss of dopaminergic (DAergic) neurons of the pars compacta in the substantia nigra and protein aggregates of alpha synuclein (α-Syn). Although its etiopathology agent has not yet been clearly established, environmental and genetic factors have been suggested as the major contributors to the disease. Mutations in the glucosidase beta acid 1 (GBA1) gene, which encodes the lysosomal glucosylceramidase (GCase) enzyme, are one of the major genetic risks for PD. We found that the GBA1 K198E fibroblasts but not WT fibroblasts showed reduced catalytic activity of heterozygous mutant GCase by -70% but its expression levels increased by 3.68-fold; increased the acidification of autophagy vacuoles (e.g., autophagosomes, lysosomes, and autolysosomes) by +1600%; augmented the expression of autophagosome protein Beclin-1 (+133%) and LC3-II (+750%), and lysosomal-autophagosome fusion protein LAMP-2 (+107%); increased the accumulation of lysosomes (+400%); decreased the mitochondrial membrane potential (∆Ψm) by -19% but the expression of Parkin protein remained unperturbed; increased the oxidized DJ-1Cys106-SOH by +900%, as evidence of oxidative stress; increased phosphorylated LRRK2 at Ser935 (+1050%) along with phosphorylated α-synuclein (α-Syn) at pathological residue Ser129 (+1200%); increased the executer apoptotic protein caspase 3 (cleaved caspase 3) by +733%. Although exposure of WT fibroblasts to environmental neutoxin rotenone (ROT, 1 µM) exacerbated the autophagy-lysosomal system, oxidative stress, and apoptosis markers, ROT moderately increased those markers in GBA1 K198E fibroblasts. We concluded that the K198E mutation endogenously primes skin fibroblasts toward autophagy dysfunction, OS, and apoptosis. Our findings suggest that the GBA1 K198E fibroblasts are biochemically and molecularly equivalent to the response of WT GBA1 fibroblasts exposed to ROT.

Asunto(s)

Apoptosis , Autofagia , Fibroblastos , Glucosilceramidasa , Mitocondrias , Estrés Oxidativo , Glucosilceramidasa/metabolismo , Glucosilceramidasa/genética , Humanos , Fibroblastos/metabolismo , Autofagia/genética , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Piel/metabolismo , Piel/patología , Lisosomas/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación

RESUMEN

Gaucher Disease (GD) is an autosomal recessive, lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene, leading to the loss of β-glucocerebrosidase (GCase) enzymatic activity. Enzyme replacement therapy (ERT) with recombinant GCase is the standard of care in GD patients. Our study investigates the combined use of in silico molecular evolution, synthetic biology and gene therapy approaches to develop a new synthetic recombinant enzyme. Gaucher Disease (GD) is an autosomal recessive, lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene, leading to the loss of β-glucocerebrosidase (GCase) enzymatic activity. Enzyme replacement therapy (ERT) with recombinant GCase is the standard of care in GD patients. Our study investigates the combined use of in silico molecular evolution, synthetic biology and gene therapy approaches to develop a new synthetic recombinant enzyme. We engineered four GCases containing missense mutations in the signal peptide (SP) from four selected mammalian species, and compared them with human GCase without missense mutations in the SP. We investigated transcriptional regulation with CMV and hEF1a promoters alongside a GFP control construct in 293-FT human cells. One hEF1a-driven mutant GCase shows a 5.2-fold higher level of transcription than control GCase. In addition, this mutant exhibits up to a sixfold higher activity compared with the mock-control, and the predicted tertiary structure of this mutant GCase aligns with human GCase. We also evaluated conserved and coevolved residues mapped to functionally important positions.

RESUMEN

Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-ß-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.

Asunto(s)

Glucosilceramidasa , Enfermedad de Parkinson , Humanos , Glucosilceramidasa/genética , Rotenona/farmacología , Rotenona/metabolismo , Células HEK293 , Peróxido de Hidrógeno/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Lisosomas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo

RESUMEN

Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29 mM) and the functionalized nanoreactors (KM =0.32 mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pH 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.

Asunto(s)

Enfermedad de Gaucher , Terapia de Reemplazo Enzimático , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Humanos , Manosa , Nanotecnología

RESUMEN

The acid ß-glucocerebrosidase (GCase) enzyme cleaves glucosylceramide into glucose and ceramide. Loss of function variants in the gene encoding for GCase can lead to Gaucher disease and Parkinson's disease. Therapeutic strategies aimed at increasing GCase activity by targeting a modulating factor are attractive and poorly explored. To identify genetic modifiers, we measured hepatic GCase activity in 27 inbred mouse strains. A genome-wide association study (GWAS) using GCase activity as a trait identified several candidate modifier genes, including Dmrtc2 and Arhgef1 (p=2.1x10-7), and Grik5 (p=2.1x10-7). Bayesian integration of the gene mapping with transcriptomics was used to build integrative networks. The analysis uncovered additional candidate GCase regulators, highlighting modules of the acute phase response (p=1.01x10-8), acute inflammatory response (p=1.01x10-8), fatty acid beta-oxidation (p=7.43x10-5), among others. Our study revealed previously unknown candidate modulators of GCase activity, which may facilitate the design of therapies for diseases with GCase dysfunction.

RESUMEN

BACKGROUND: Gaucher disease (GD) is the most prevalent lysosomal storage disorder, affecting all ethnic groups, although its prevalence is higher in Ashkenazi Jewish populations. Three clinical forms of GD have been described: Type 1 non-neuronopathic, type 2 acute neuronopathic, and type 3 subacute neuronopathic. An autosomal recessive disorder is caused by variants in the human glucocerebrosidase gene (GBA; MIM*606463) located on chromosome 1q21, resulting from deficit or lack of activity of the ß-glucocerebrosidase enzyme, leading to the accumulation of glucocerebroside substrate in the cells of the macrophage-monocyte system. The aim was to determine variants in Mexican and Spanish populations with GD. METHODS: We report the molecular analysis by a direct automatic method sequenced of both chains of the GBA gene, in 69 Mexican and 369 Spanish patients with GD. RESULTS: We detected 75 variants with pathogenic or likely pathogenic effect and, identified 3 new variants c.408_412del/p.Asn136Lysfs*15; c.820G>A/p.Glu274Lys and c.1058T>G/p*. The most frequent variants were c.1448T>C/p.Leu483Pro/L444P and c.1226A>G/p.Asn409Ser/N370S. The detected genotypes were compared with data from both GD registries to define similarities and differences in both populations. CONCLUSIONS: We defined the variant profile in patients with GD in a Mexican and a Spanish population and compared them. The screening permitted the detection of common variants and the report of three new variants, in addition to a variant associated with Parkinson disease but not with GD. Since molecular diagnosis has considerable predictive value in GD, it is important to study the genotype-phenotype correlations, establishing the severity of the variant.

Asunto(s)

Enfermedad de Gaucher , Alelos , Enfermedad de Gaucher/genética , Estudios de Asociación Genética , Genotipo , Humanos , Mutación

RESUMEN

Abstract Introduction: Gaucher disease (GD) is one of the common lysosomal storage disorder (LSD) with an estimated frequency of one in 40,000 newborns globally. GD is an autosomal recessive condition, which results from mutations in the GBA1 gene, causing partial or complete deficiency of β-glucocerebrosidase enzyme activity, which leads to the widespread accumulation of the substrate glucosylceramide. Aims: This report presents different challenges of clinical management and communication between medical specialties to reach diagnose of any rare disease in Mozambique, a low-income country, which health system has limited infrastructure, trained personnel, and budget for diagnosis and to provide treatment for rare genetic disorders such as GD. Case Presentation: The patient was a 15-year old black female patient of Mozambican nationality born from non-consanguineous parents. Three of the four patient's siblings were healthy; one sister had died of a disease with a similar clinical features. Our patient presented with abdominal distention and hepatosplenomegaly. Blood tests revealed pancytopenia and a high level of ferritin. Liver biopsy and histologic examination revealed infiltration of the splenic parenchyma and portal area of the liver as well as enlarged histiocytic cells with granular cytoplasm. Magnetic resonance imaging showed liver enlargement, changes in the femoral heads without osteonecrosis, a pathological fracture of the third thoracic vertebrae (T3), with absence of brain and spinal cord neurological abnormalities. The biochemical investigation disclosed low levels of β-glucocerebrosidase (0.223 nmol/h/ml; normal: above 0.98) and increased levels of lyso-Gb1 (0.43 µg/ml; normal: up to 0.003). Genotyping of the GBA1 gene indicated the presence of the pathogenic variant p.Arg87Trp (R48W) in homozygosis. Discussion and Conclusion: To the best of our knowledge, this report describes the first case of GD type 1 confirmed via biochemical and molecular genetic testing in Mozambique. As awareness of the GD and rare genetic diseases among Mozambican health professionals is very limited, and resources for diagnosis are scarce in the national health system, it is possible that other cases remain undiagnosed in this low-income country.

RESUMEN

Resumen: La enfermedad de Gaucher (GD) es el trastorno de almacenamiento lisosomal que se caracteriza por la deficiencia en la actividad enzimática de la β-glucosidasa (BGLU), lo que produce la acumulación de glucosilceramida en las células. Su diagnóstico se orienta a la valoración de la enzima en los leucocitos afectados. Se han realizado estudios en DBS para la actividad de BGLU en el seguimiento de poblaciones de alto riesgo; sin embargo, presentan interferencias relacionadas a leucopenias severas o expresión aumentada de la isoforma neutra de la enzima BGLU, molécula no relacionada con GD. El objetivo de este estudio fue la estandarización de un método de tamizaje en DBS (punch: 5 mm) con el uso de 4-metilumbeliferil-β-D-glucósido y conduritol-β-epóxido. Se analizaron muestras de DBS de 395 individuos con sospecha clínica (población de alto riesgo o AR), 151 controles y 16 pacientes afectados, usando la elución de un corte de 5 mm (≈10 μl de sangre) en 300 μl de Tritón X-100/(0,5 %). Como resultados, se obtuvieron los rangos, AR: 0,84-26,92 nmol/ml/h, controles: 3,56- 8,92 nmol/ml/h (M = 5,56, DS = 1,15) y pacientes confirmados con GD: 0,82- 2,88 nmol/ml/h (M = 1,64, DS = 0,57). El punto de corte entre deficientes y controles fue 3,22 nmol/ml/h, obtenido a partir de análisis ROC (99 % confianza, 100 % sensibilidad y 100 % especificidad). El protocolo permitió evidenciar la deficiencia en todos los casos de GD, confirmados mediante el análisis en paralelo de la enzima en aislamiento leucocitario. Se recomienda el uso del CBE y realizar la elución del corte a 5 mm, a fin de llevar a cabo la valoración enzimática con un volumen mayor aproximado de sangre y en ausencia de la actividad generada por la isoforma neutra.

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Abstract: Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency in the enzymatic activity of β-glucosidase (BGLU), resulting in the accumulation of glucosylceramide in cells. Its diagnosis is aimed at checking the enzyme in the affected leukocytes. Studies have been conducted on dried blood spots (DBS) for bglu activity to monitor high-risk populations; however, they exhibit interferences related to severe leukopenias or increased expression of the neutral bglu isoform, a molecule not related to gd. This study intends to standardize a screening method on dbs (punch: 5 mm) using 4-methylumbelliferyl-β-D-glucoside and conduritol-β-epoxide (CβE). dbs samples from 395 individuals clinically suspected of gd (high-risk or hr population), 151 controls, and 16 affected patients were analyzed using the elution of 5 mm punches (≈10 μl of blood) in 300 μl of Triton X-100/ (0.5 %). As a result, the following ranges were obtained; HR: 0.84-26.92 nmol/ml/h, controls: 3.56-8.92 nmol/ml/h (M = 5.56, SD = 1.15), and patients with confirmed GD: 0.82-2.88 nmol/ml/h (M = 1.64, SD = 0.57). The cut-off point between patients with gd and controls was 3.22 nmol/ml/h, obtained from roc analysis (99 % ci, 100 % sensitivity, and 100 % specificity). The protocol revealed a deficiency in all gd cases, confirmed by parallel bglu analysis in isolated leukocytes. The use of cbe and the elution of 5 mm punches are recommended for enzymatic evaluation with a higher approximate volume of blood and in the absence of neutral isoform activity.

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Resumo: A doença de Gaucher (GD) é o trastorno de armazenamento lisosomal caracterizado pela deficiência na atividade enzimática da β-glucosidase (BGLU), o que produz a acumulação de glucossilceramida nas células. Seu diagnóstico está orientado à avaliação da enzima nos leucócitos afetados. Foram realizados estudos em dbs para a atividade de BGLU no seguimento de populações de alto risco; contudo, são apresentadas interferências relacionadas a leucopenias graves ou a expressão aumentada da isoforma neutra da enzima BGLU, molécula não relacionada com GD. O objetivo deste estudo foi a padronização de um método de tamisação emdbs (punch: 5 mm) com o uso de 4-metilumbeliferil-β-D- glicosídeo e conduritol-β-epóxido. Foram analisadas amostras de dbs de 395 indivíduos com suspeita clínica (população de alto risco ou ar), 151 controles e 16 pacientes afetados, usando a eluição de um corte de 5 mm (≈10 μl de sangue) em 300 μl de Tritão X-100/(0,5 %). Como resultados, foram obtidos os intervalos: AR: 0,84-26,92 nmol/ml/h, controles: 3,56-8,92 nmol/ml/h (M = 5,56, DS = 1,15) e pacientes confirmados com GD: 0,82- 2,88 nmol/ml/h (M = 1,64, DS = 0,57). O ponto de corte entre deficientes e controles foi 3,22 nmol/ml/h, obtido a partir de análise ROC (99 % confiança, 100 % sensibilidade e 100 % especificidade). O protocolo permitiu evidenciar a deficiência em todos os casos de GD, confirmados mediante a análise em paralelo da enzima em isolamento leucocitário. É recomendado o uso do cbe e a realização da eluição do corte a 5 mm, a fim de implementar a avaliação enzimática com um volume maior aproximado de sangue e em ausência da atividade gerada pela isoforma neutra.

RESUMEN

Gaucher disease (GD) is caused by a genetic deficiency of the lysosomal enzyme glucocerebrosidase (GCase) leading to the accumulation of glucocerebroside in the liver, spleen, and bone marrow. The early diagnosis allows a prompt enzyme replacement therapy reversing cytopenias and visceromegaly and preventing irreversible bone lesions. Current diagnostic algorithms are based on well-recognized hematological manifestations. Although bone symptoms are present in 25-32% of the patients, they are not usually suspected as associated with Gaucher disease at clinical presentation. We designed an educational program aimed to give advice on the skeletal involvement in GD and a new diagnostic algorithm that considers bone symptoms to facilitate its early diagnosis (BIG project: Bone Involvement in Gaucher Disease). The study aims at describing the BIG project and the results of its application in our clinic in various cities in Argentina. Within the frame of this project, between March 2017 and December 2018, 38 dry blood spot samples from patients with bon e manifestations suspected of having GD were submitted to quantification of GCase activity. One sample did not meet the inclusion criteria. Deficient GCase activity was detected in three of the remaining 37 samples. The diagnosis of GD was confirmed in two girls who presented bone manifestations of 4 and 2 years of evolution, respectively, without hematological alterations. The third patient with low enzyme activity had normal leukocyte GCase. The two newly diagnosed cases of GD show the efficacy of our dual strategy aimed to facilitate the early diagnosis of this rare disease.

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La enfermedad de Gaucher (EG) es causada p or una deficiencia genética de la glucocerebrosidasa (GCasa) que provoca acumulación de glucocerebrósido en hígado, bazo y médula ósea. La terapia temprana de reemplazo enzimático revierte citopenias, visceromegalias y previene lesiones óseas irreversibles, por lo cual el diagnóstico precoz es fundamental. Los algoritmos diagnósticos en uso apuntan a manifestaciones hematológicas clásicas. Los síntomas óseos están presentes en 25-32% de los pacientes pero no suelen despertar sospecha de EG. Diseñamos un programa educativo sobre la afecta ción ósea de la EG y un algoritmo focalizado en la presentación con manifestaciones óseas para facilitar su diagnóstico precoz (proyecto BIG: Bone Involvement in Gaucher Disease). El objetivo del trabajo es describir el proyecto BIG y los resultados de su aplicación en nuestra consulta. Entre marzo de 2017 y diciembre de 2018 se recibieron 38 muestras de sangre seca de pacientes con alguna manifestación ósea sospechosa de EG para cuantificar la actividad de GCasa. Una muestra no cumplía los criterios de inclusión y en 3 de las 37 restantes se observó actividad deficiente de GCasa. El diagnóstico de EG se confirmó por medición de GCasa en leucocitos en dos niñas con manifestaciones óseas de 4 y 2 años de evolución, respectivamente, sin citopenia ni visceromegalia clínicamente evidentes. En el otro paciente con baja actividad la medición en leucocitos fue normal. Los casos detectados muestran la efectividad de un programa educacional de difusión y la utilidad de un algoritmo de detección precoz basado en síntomas óseos que facilitaría el diagnóstico de EG.

Asunto(s)

Enfermedad de Gaucher/diagnóstico , Glucosilceramidasa , Argentina , Diagnóstico Precoz , Terapia de Reemplazo Enzimático , Femenino , Humanos

RESUMEN

Resumen La enfermedad de Gaucher (EG) es causada por una deficiencia genética de la glucocerebrosidasa (GCasa) que provoca acumulación de glucocerebrósido en hígado, bazo y médula ósea. La terapia temprana de reemplazo enzimático revierte citopenias, visceromegalias y previene lesiones óseas irreversibles, por lo cual el diagnóstico precoz es fundamental. Los algoritmos diagnósticos en uso apuntan a manifestaciones hematológicas clásicas. Los síntomas óseos están presentes en 25-32% de los pacientes pero no suelen despertar sospecha de EG. Diseñamos un programa educativo sobre la afectación ósea de la EG y un algoritmo focalizado en la presentación con manifestaciones óseas para facilitar su diagnóstico precoz (proyecto BIG: Bone Involvement in Gaucher Disease). El objetivo del trabajo es describir el proyecto BIG y los resultados de su aplicación en nuestra consulta. Entre marzo de 2017 y diciembre de 2018 se recibieron 38 muestras de sangre seca de pacientes con alguna manifestación ósea sospechosa de EG para cuantificar la actividad de GCasa. Una muestra no cumplía los criterios de inclusión y en 3 de las 37 restantes se observó actividad deficiente de GCasa. El diagnóstico de EG se confirmó por medición de GCasa en leucocitos en dos niñas con manifestaciones óseas de 4 y 2 años de evolución, respectivamente, sin citopenia ni visceromegalia clínicamente evidentes. En el otro paciente con baja actividad la medición en leucocitos fue normal. Los casos detectados muestran la efectividad de un programa educacional de difusión y la utilidad de un algoritmo de detección precoz basado en síntomas óseos que facilitaría el diagnóstico de EG.

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Abstract Gaucher disease (GD) is caused by a genetic deficiency of the lysosomal enzyme glucocerebrosidase (GCase) leading to the accumulation of glucocerebroside in the liver, spleen, and bone marrow. The early diagnosis allows a prompt enzyme replacement therapy reversing cytopenias and visceromegaly and preventing irreversible bone lesions. Current diagnostic algorithms are based on well-recognized hematological manifestations. Although bone symptoms are present in 25-32% of the patients, they are not usually suspected as associated with Gaucher disease at clinical presentation. We designed an educational program aimed to give advice on the skeletal involvement in GD and a new diagnostic algorithm that considers bone symptoms to facilitate its early diagnosis (BIG project: Bone Involvement in Gaucher Disease). The study aims at describing the BIG project and the results of its application in our clinic in various cities in Argentina. Within the frame of this project, between March 2017 and December 2018, 38 dry blood spot samples from patients with bone manifestations suspected of having GD were submitted to quantification of GCase activity. One sample did not meet the inclusion criteria. Deficient GCase activity was detected in three of the remaining 37 samples. The diagnosis of GD was confirmed in two girls who presented bone manifestations of 4 and 2 years of evolution, respectively, without hematological alterations. The third patient with low enzyme activity had normal leukocyte GCase. The two newly diagnosed cases of GD show the efficacy of our dual strategy aimed to facilitate the early diagnosis of this rare disease.

Asunto(s)

Humanos , Femenino , Enfermedad de Gaucher/diagnóstico , Glucosilceramidasa , Argentina , Diagnóstico Precoz , Terapia de Reemplazo Enzimático

RESUMEN

La enfermedad de Gaucher es un trastorno metabólico autosómico recesivo crónico y progresivo que se caracteriza por depósito lisosomal con deficiencia de la enzima glucocerebrosidasa ácida produciendo causando daño celular y disfunción orgánica; se asocia a enfermedades neoplásicas hematológicas; sin embargo, su asociación con linfomas es rara. El linfoma hidroa vacciniforme like es una enfermedad rara per se pero afecta en más casos a niños y adolescentes; está caracterizado por lesiones vesiculares cutáneas, adenopatías y visceromegalias. Presentamos el caso de una niña proveniente de una comunidad andina de Cusco de 12 años que presentó durante siete años vesículas costrosas, fiebre, edema facial con ulcera palpebral, ganglios palpables, hepatoesplenomegalia, acompañado de pancitopenia. Se realizó un estudio enzimático y genético observándose deficiencia de β-glucosidasa y del gen GBA; en la biopsia de piel se encontró un infiltrado linfoide dérmico con pleomorfismo nuclear compatible con linfoma de células T tipo hidroavacciniforme like, posteriormente la paciente presentó leve mejoría con el tratamiento de reemplazo enzimático pero falleció debido al shock hipovolémico tras dos episodios de hemorragia digestiva baja.

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Gaucher disease is a chronic and progressive autosomal recessive metabolic disorder that is characterized by lysosome depots with deficiency of acid glucocerebrosidase enzyme, which leads to cell damage and organic dysfunction. This condition is associated with some hematological malignancies; however, its association with lymphomas is rare. Hydroa vacciniform-like lymphoma is a rare condition per se, but it is becoming increasingly frequent in children and adolescents. It is characterized by the presence of cutaneous vesicular lesions, adenopathy, and visceromegaly. We present the case of a 12-year old girl from an Andean community in Cusco who presented with crusting vesicles, fever, face edema with eyelid ulceration, palpable lymph nodes, and hepatosplenomegaly, accompanied by pancytopenia. An enzymatic and genetic study was carried out, and both β-glucosidase deficiency and GBA gene deficiency were found. Skin biopsies revealed a dermal lymphoid infiltrate with nuclear pleomorphism compatible with hydroa vacciniform like T-cell lymphoma. Subsequently, the patient developed slight improvement with the enzyme replacement therapy, but she died because of hypovolemic shock after two episodes of low gastrointestinal hemorrhage.

RESUMEN

BACKGROUND: Mutations in the glucocerebrosidase (GBA) gene are an important risk factor for Parkinson's disease (PD). However, most GBA genetic studies in PD have been performed in patients of European origin and very few data are available in other populations. METHODS: We sequenced the entire GBA coding region in 602 PD patients and 319 controls from Colombia and Peru enrolled as part of the Latin American Research Consortium on the Genetics of Parkinson's disease (LARGE-PD). RESULTS: We observed a significantly higher proportion of GBA mutation carriers in patients compared to healthy controls (5.5% vs 1.6%; OR = 4.3, p = 0.004). Interestingly, the frequency of mutations in Colombian patients (9.9%) was more than two-fold greater than in Peruvian patients (4.2%) and other European-derived populations reported in the literature (4-5%). This was primarily due to the presence of a population-specific mutation (p.K198E) found only in the Colombian cohort. We also observed that the age at onset was significantly earlier in GBA carriers when compared to non-carriers (47.1 ±â€¯14.2 y vs. 55.9 ±â€¯14.2 y; p = 0.0004). CONCLUSION: These findings suggest that GBA mutations are strongly associated with PD risk and earlier age at onset in Peru and Colombia. The high frequency of GBA carriers among Colombian PD patients (∼10%) makes this population especially well-suited for novel therapeutic approaches that target GBA-related PD.

Asunto(s)

Glucosilceramidasa/genética , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Colombia/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Perú/epidemiología , Adulto Joven

RESUMEN

In the last years, lysosomal storage diseases appear as a bridge of knowledge between rare genetic inborn metabolic disorders and neurodegenerative diseases such as Parkinson's disease (PD) or frontotemporal dementia. Epidemiological studies helped promote research in the field that continues to improve our understanding of the link between mutations in the glucocerebrosidase (GBA) gene and PD. We conducted a review of this link, highlighting the association in GBA mutation carriers and in Gaucher disease type 1 patients (GD type 1). A comprehensive review of the literature from January 2008 to December 2018 was undertaken. Relevance findings include: (1) There is a bidirectional interaction between GBA and α- synuclein in protein homeostasis regulatory pathways involving the clearance of aggregated proteins. (2) The link between GBA deficiency and PD appears not to be restricted to α⁻synuclein aggregates but also involves Parkin and PINK1 mutations. (3) Other factors help explain this association, including early and later endosomes and the lysosomal-associated membrane protein 2A (LAMP-2A) involved in the chaperone-mediated autophagy (CMA). (4) The best knowledge allows researchers to explore new therapeutic pathways alongside substrate reduction or enzyme replacement therapies.

RESUMEN

RESUMEN Introducción y Objetivo: La enfermedad de Gaucher es una condición autosómica recesiva de baja prevalencia, de difícil diagnóstico y manejo, especialmente en embarazo. Reportamos una gestante con enfermedad de Gaucher manejada desde la semana 15,3 hasta el término del embarazo con Taliglucerasa en la Unidad de Alta Complejidad Obstétrica, en la Fundación Valle del Lili en Cali, Colombia. Métodos: Reporte de caso de gestante con diagnóstico de enfermedad de Gaucher diagnosticada durante el embarazo, con exacerbación de síntomas, quien presento severa pancitopenia y esplenomegalia. Resultados: El manejo medico interdiscilplinario permitió el control del severo deterioro clínico de la paciente durante el parto, presentó hemorragia postparto con choque hipovolémico, con adecuada respuesta al manejo médico. La madre y el neonato fueron dados de alta sin otras complicaciones asociadas. Conclusión: El manejo interdisciplinario es indispensable en gestantes con esta entidad para lograr buenos resultados maternos y perinatales.

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ABSTRACT Introduction and Objective: Gaucher disease is a low prevalence autosomal recessive condition, difficult to diagnose and manage especially during pregnancy. We reported a pregnant woman with Gaucher disease managed with Taliglucerase in a critical care obstetric unit from week 15.3 until the end of her pregnancy, at the Fundación Valle del Lili, Cali, Colombia. Methods: A case report of a pregnant woman diagnosed during her pregnancy with Gaucher disease, who presented severe pancytopenia and splenomegaly. Results: The interdisciplinary medical management allowed the control of the severe clinical deterioration of the patient. During the delivery, she presented postpartum hemorrhage with hypovolemic shock, which resolved with medical management. The mother and the newborn were discharged without other associated complications. Conclusion: Interdisciplinary management is essential for handling a critically ill obstetric patient with Gaucher disease, and to achieve good maternal and perinatal outcomes.

Asunto(s)

Humanos , Femenino , Embarazo , Adulto , Adulto Joven , Trombocitopenia , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/etiología , Enfermedad de Gaucher/terapia , Complicaciones del Embarazo , Resultado del Embarazo , Resultado del Tratamiento , Colombia , Terapia de Reemplazo Enzimático/métodos , Enfermedad de Gaucher/complicaciones , Hepatomegalia

RESUMEN

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Asunto(s)

Terapia de Reemplazo Enzimático , Enfermedad de Gaucher/genética , Glucosilceramidasa/biosíntesis , Proteínas Recombinantes/administración & dosificación , Adenoviridae/genética , Animales , Femenino , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/patología , Glucosilceramidasa/administración & dosificación , Glucosilceramidasa/genética , Glucosilceramidas/metabolismo , Cabras/genética , Humanos , Glándulas Mamarias Animales/enzimología , Leche/metabolismo

RESUMEN

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. On the other hand it is well known that inflammation is a key player in GD pathology. In this work, we revealed increased levels of the proinflammatory CD14(+)CD16(+) monocyte subset and increased inflammatory cytokine production by monocytes and T cells in the circulation of GD patients. We showed increased levels of osteoclast precursors in PBMC from patients and a higher expression of RANKL in the surface of T cells. PBMC from patients presented higher osteoclast differentiation compared to healthy controls when cultured in the presence of M-CSF alone or in combination with RANKL. In vitro treatment with Velaglucerase reduced osteoclast levels to control levels. On the other hand THP-1 derived osteoclast precursors cultured in the presence of conditioned media from PBMC of GD patients presented higher differentiation to active osteoclasts. This induction involved TNF-α and RANKL.

Asunto(s)

Resorción Ósea , Enfermedad de Gaucher/metabolismo , Enfermedad de Gaucher/patología , Leucocitos Mononucleares/metabolismo , Osteoclastos/metabolismo , Adolescente , Adulto , Anciano , Antígenos de Superficie , Huesos/metabolismo , Huesos/patología , Diferenciación Celular , Niño , Citocinas/metabolismo , Femenino , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/genética , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Osteoclastos/citología , Adulto Joven

RESUMEN

Lysosomal integral membrane protein 2 (LIMP-2, SCARB2) is directly linked to ß-glucocerebrosidase enzyme (ßGC) and mediates the transport of this enzyme from the Golgi complex to lysosomes. Active ßGC cleaves the ß-glycosidic linkages of glucosylceramide, an intermediate in the metabolism of sphingoglycolipids, generating ceramide. In this study we used mouse embryonic fibroblasts (MEFs) deficient for LIMP-2 and observed that these cells were more susceptible to infection by extracellular amastigotes of the protozoan parasite Trypanosoma cruzi when compared to wild-type (WT) fibroblasts. The absence of LIMP-2 decreases the activity of ßGC measured in fibroblast extracts. Replacement of ßGC enzyme in LIMP-2 deficient fibroblasts restores the infectivity indices to those of WT cells in T. cruzi invasion assays. Considering the participation of ßGC in the production of host cell ceramide, we propose that T. cruzi extracellular amastigotes are more invasive to cells deficient in this membrane component. These results contribute to our understanding of the role of host cell lysosomal components in T. cruzi invasion.

Asunto(s)

Antígenos CD36/inmunología , Antígenos CD36/metabolismo , Glucosilceramidasa/inmunología , Glucosilceramidasa/metabolismo , Estadios del Ciclo de Vida/fisiología , Proteínas de Membrana de los Lisosomas/inmunología , Proteínas de Membrana de los Lisosomas/metabolismo , Trypanosoma cruzi/patogenicidad , Animales , Antígenos CD36/genética , Línea Celular , Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/parasitología , Proteínas de Membrana de los Lisosomas/genética , Ratones , Ratones Noqueados

RESUMEN

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. The homeostasis of bone tissue is maintained by the balanced processes of bone resorption by osteoclasts and formation by osteoblasts. We decided to test whether bone resorption and/or bone formation could be altered by the use of a chemical in vitro murine model of Gaucher disease. We used two sources of cells from monocyte/macrophages lineage isolated from normal mice, splenocytes (S) and peritoneal macrophages (PM), and were exposed to CBE, the inhibitor of GCase (S-CBE and PM-CBE, respectively). Addition of both conditioned media (CM) from S-CBE and PM-CBE induced the differentiation of osteoclasts precursors from bone marrow to mature and functional osteoclasts. TNF-α could be one of the factors responsible for this effect. On the other hand, addition of CM to an osteoblast cell culture resulted in a reduction in expression of alkaline phosphatase and mineralization process. In conclusion, these results suggest implication of changes in both bone formation and bone resorption and are consistent with the idea that both sides of the homeostatic balance are affected in GD.

Asunto(s)

Enfermedad de Gaucher/patología , Osteoblastos/metabolismo , Osteoclastos/fisiología , Animales , Antígenos de Diferenciación/metabolismo , Células de la Médula Ósea/fisiología , Calcificación Fisiológica , Diferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados , Enfermedad de Gaucher/inducido químicamente , Enfermedad de Gaucher/metabolismo , Inositol/análogos & derivados , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteólisis/metabolismo , Factor de Necrosis Tumoral alfa/fisiología

RESUMEN

A doença de Gaucher (DG) foi a primeira doença de armazenamento lisossomal descrita e a mais encontrada. Caracteriza-se pela deficiência hereditária da atividade da enzima lisossomal glucocerebrosidase, que bloqueia o metabolismo do glicocerebrosídeo. A proposta deste trabalho foi estudar as características clínicas, laboratoriais e radiológicas, as principais mutações encontradas, relacionando-as com as formas clínicas e avaliar a resposta à terapia de reposição enzimática (TRE) nos pacientes com DG em Santa Catarina. Foram estudados dez pacientes com DG no Hospital Universitário, no período entre 1998 e 2003, após confirmação diagnóstica da doença pela dosagem da enzima beta-glicosidase em leucócitos. Pesquisa das mutações foi realizada em amostras de sangue e de mucosa oral. A média de idade ao diagnóstico foi de 19,6 anos. A DG tipo 1 foi diagnosticada em 80 por cento dos casos, e a tipo 2 em 20 por cento. Quatro pacientes tiveram história familiar de DG. Hepatoesplenomegalia foi a manifestação clínica mais comum. Anemia e trombocitopenia ocorreram em 100 por cento dos casos. Dores ósseas foram relatadas por 75 por cento dos pacientes. Os alelos mutantes encontrados foram N370S e L444P. Houve elevação dos níveis de hemoglobina em todos os pacientes com DG tipo 1. Concluímos que a DG tipo 1 é a forma clínica mais comum. Anemia, trombocitopenia, hepatoesplenomegalia e osteopenia são as características mais freqüentes dos pacientes com DG. O alelo N370S é o mais freqüente, estando relacionado com o tipo 1. O alelo L444P em homozigose sugere letalidade precoce. A TRE é segura e efetiva para a DG tipo 1.

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Gaucher Disease (GD) was the first described and is the most common lysosomal deposit disease. It is characterized byahereditary deficiency of glucocerebrosidase lysosomal enzyme activity which blocks the metabolism of glucocerebrosideo. The aim of this work was to study the clinical, laboratorial and radiological characteristics, the main mutations correlating them with the clinical form of the disease and evaluating the response to enzymatic replacement therapy in patients with GD in Santa Catarina. Ten GD patients were studied at a University Hospital between 1998 and 2003. The disease was diagnosed by measurement of the beta-glucosidase enzyme in leukocytes. Investigation of mutations used samples of blood and oral mucus. The average age at diagnosis was 19.6 years. Type 1 GD was diagnosis in 80 percent of the cases and type 2 in 20 percent. Four patients had a family history of GD. Hepatosplenomegaly was the most common clinical manifestation. Anemia and thrombocitopenia occurred in all cases. Bone pain was reported by 75 percent of the patients. The mutanted alleles identified were N370S and L444P. The hemoglobina levels were elevated in all patients with type 1 GD. In conclusion, type 1 GD is the most common clinical form and anemia, thrombocitopenia, hepatosplenomegaly and osteopenia were the most common characteristics of GD patients. The N370S allele is the most common mutation and is related with type 1 GD 1. Homozygosity of the L444P allele suggests early death. Enzyme replacement therapy is safe and efficacious in type 1 GD.

Asunto(s)

Humanos , Enfermedad de Gaucher , Genotipo , Glucosilceramidasa , Lipidosis