Aspectos genéticos e bioquímicos da doença de Gaucher com ênfase no gene GBA1 e no metabolismo da vitamina B12

A doença de Gaucher (DG) é uma doença rara, herdada de forma autossômica recessiva, causada pela atividade reduzida da enzima lisossomal glicocerebrosidase (GCase) devido a mutações patogênicas no gene GBA1, o que leva ao acúmulo do substrato glicocerebrosídeo em macrófagos. A DG apresenta uma grande variabilidade fenotípica, sendo caracterizada principalmente por manifestações hematológicas, viscerais, ósseas e neurológicas. Embora cerca de 500 mutações já tenham sido descritas no gene GBA1, não há uma associação estabelecida entre mutações específicas e as características clínicas apresentadas pelos pacientes com DG. O acúmulo de glicocerebrosídeo pode estar afetando a função lisossômica, o que poderia levar a uma disfunção celular e a anormalidades clínicas, como a deficiência de vitamina B12 (B12). Objetivos: Caracterizar o genótipo e o metabolismo da vitamina B12 em pacientes com DG. Métodos: Foram analisados, por Multiplex ligation-dependent probe amplification (MLPA), 33 pacientes com DG para identificar a presença de deleções e inserções no GBA1 em pacientes que não apresentavam genótipo definido. Foram caracterizadas amostras de 72 pacientes com DG (n=40 da região Sul do Brasil, n=32 de outras regiões do país) utilizando o método de Sanger ou sequenciamento de nova geração e as frequências alélicas das mutações identificadas foram comparadas entre pacientes com DG de diferentes regiões do Brasil. Biomarcadores funcionais do metabolismo da B12 foram mensurados em fibroblastos de indivíduos saudáveis (n=3), pacientes com DG não tratados (tipo I=1; tipo II=1; tipo III=1) e um heterozigoto assintomático. A expressão de transcobalamina (TC) em células de indivíduos saudáveis e de pacientes com DG foi analisada por Western blot. Níveis de B12 total, holo-transcobalamina (holo-TC), homocisteína total (tHcy) e ácido metilmalônico (MMA) foram analisados retrospectivamente em 52 amostras de plasma de 10 pacientes brasileiros com DG tipo I acompanhados pelo Centro de Referência em DG do Sul do Brasil. Resultados: Foi identificada uma deleção em heterozigose de uma região do gene GBA1 em uma paciente com DG que apresentava genótipo incompleto. Trinta e uma mutações foram identificadas em pacientes brasileiros com DG, sendo duas delas não descritas na literatura. Pacientes com DG pertencentes à região Sul do Brasil apresentaram perfil alélico diferente daqueles que pertenciam às demais regiões do país. Níveis de biomarcadores funcionais do status de B12 não diferiram entre amostras de células de pacientes com DG e de indivíduos saudáveis. A análise por Western blot a partir de lisados de células indicou a presença de conteúdo normal de TC em ambas as linhagens celulares. Nenhum paciente com DG tipo I apresentou níveis 10 reduzidos de B12 total em amostras de plasmas. A análise de quatro biomarcadores do status de B12 sugere que não há uma deficiência funcional desse micronutriente em pacientes com DG tipo I. Conclusão: A técnica de MLPA é uma metodologia complementar que pode ser utilizada para analisar a presença de deleções e de inserções em pacientes com DG que não apresentam genótipo definido. Em relação à caracterização genética de pacientes brasileiros com DG, o alelo mais frequente foi o N370S. O estudo sugere que existe um perfil alélico específico no GBA1 entre pacientes com DG de diferentes regiões do Brasil, sendo que os alelos N370S, RecNciI e L444P foram mais representativos na região Sul do país. A investigação do metabolismo da B12 em pacientes com DG mostrou que as vias de transporte e de processamento da B12 estão preservadas em células de pacientes com DG. A análise de biomarcadores do status de B12 em amostras de plasmas mostrou que pacientes brasileiros com DG tipo I apresentam níveis normais de B12, o que difere de um estudo prévio que demonstrou uma alta prevalência de níveis reduzidos de B12 em pacientes com DG da população judeus Ashkenazi. Além disso, a análise de biomarcadores funcionais (tHcy e MMA) não indicou deficiência de B12 em pacientes com DG. Portanto, o acúmulo de glicocerebrosídeo nos lisossomos parece não afetar o processamento e o tráfego da vitamina B12, não ocasionando uma deficiência funcional de B12 em pacientes com DG tipo I.Gaucher disease (GD) is a rare autosomal recessive disease caused by the reduced activity of the lysosomal enzyme glucocerebrosidase (GCase) due to pathogenic mutations in GBA1 gene, which leads to an accumulation of its substrate, glucocerebroside, in the macrophages. GD presents a considerable phenotypic variation, and it is characterized mainly by hematological, visceral, skeletal and neurological manifestations. Although around 500 mutations have been described in the GBA1, there is no clear association between mutation and clinical features presented by patients with GD. The accumulation of glucocerebroside may be affecting the lysosomal function and leads to cellular dysfunction and clinical abnormalities, such as a vitamin B12 deficiency. Objectives: To characterize the genotype and vitamin B12 metabolism in GD patients. Methods: Thirty-three patients with GD were analyzed by Multiplex ligation-dependent probe amplification (MLPA) method to detect deletions or insertions in the GBA1 in patients with uncharacterized alleles. Samples from 72 GD patients (n=40 from South Brazil, n=32 from other regions of Brazil) were analyzed by Sanger or next-generation sequencing, and allele frequencies were compared in GD from different regions of Brazil. Functional biomarkers of B12 metabolism were measured in fibroblasts from healthy individuals (n=3), GD untreated patients (type I=1; type II=1; type III=1) and an asymptomatic heterozygous. The expression of transcobalamin (TC) in cells from healthy individuals and GD patients was analyzed by Western blot. Total B12, holo-transcobalamin (holo-TC), total homocysteine (tHcy) and methylmalonic acid (MMA) were prospectively measured in 52 plasma samples from 10 Brazilian patients with GD type I followed by Reference Center for GD from south of Brazil. Results: A deletion in a region of the GBA1 gene was identified in one GD patient presenting incomplete genotype. Thirty-one mutations were identified in Brazilian patients with GD, two of them not described in the literature. GD patients from the southern region of Brazil had different allelic profile than those from other regions of the country. B12 biomarkers levels did not differ between cells from GD patients and healthy individuals. Western blot analysis of whole cell lysates indicated standard content of TC in both cell lines. No patient with GD type I presented low levels of total B12 in plasma samples. Four biomarkers of B12 status suggests that there is no functional deficiency of this micronutrient in GD type I patients. Conclusion: The MLPA technique is a complementary methodology that can be used to analyze the presence of deletions and insertions in patients with GD that do not have defined genotype. Regarding the genetic characterization of Brazilian GD patients, the most frequent allele was N370S. The 12 study suggests that there is a specific allele profile in GBA1 among patients with GD from different regions of Brazil, whereas the N370S, RecNciI, and L444P alleles are more representative in the southern region of the country. The investigation of B12 metabolism in GD patients showed that transport and processing pathways of B12 are overall preserved in GD cells. Analysis of B12 status biomarkers in plasma samples showed that Brazilian GD type I patients present normal levels of B12, in contrast with the previous study that demonstrated high prevalence of low B12 in untreated patients with GD from the Ashkenazi Jewish population. Furthermore, the analysis of functional biomarkers (tHcy and MMA) was not suggestive of B12 deficiency in GD patients. Therefore, glucocerebroside accumulation in lysosomes seems not affect the processing and trafficking of B12, and it not causing a functional impairment of B12 in patients with GD type I

Assessment of cellular cobalamin metabolism in Gaucher disease

Background: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. Methods: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. Results: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. Conclusions: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease

Rare GBA1 genotype associated with severe bone disease in Gaucher disease type 1

Introduction:Gaucher disease (GD) type 1 is a lysosomal disease characterised by hepatosplenomegaly, anemia,thrombocytopenia, bone changes, and bone marrow infiltration. The disease is caused by biallelic pathogenicvariants inGBA1which codes for glucocerebrosidase, an enzyme involved in the catabolic pathway of complexlipids.Aims:To report on the case of two sisters with GD type 1 who bear a genotype never reported in the literature.Case report:Patient 1 is a 47-year-old female diagnosed at 42 years of age with chronic lumbar pain, mildsplenomegaly, slightly reduced platelets and normal hemoglobin values, severe Bone Marrow Burden (BMB)score, high chitotriosidase activity, and low glucocerebrosidase. Patient 2 is a 50-year-old female, sister of pa-tient 1, who was diagnosed after familial screening. At 45 years of age, she had osteonecrosis of the left femurand a total hysterectomy because of uncontrollable bleeding. Atfirst evaluation, she had bone pain with a highBMB score, mild splenomegaly, normal hemoglobin, normal platelets count, elevated chitotriosidase activity,and low glucocerebrosidase activity. Both patients were found to be compound heterozygotes for thep.Glu388Lys and the p.Ser405Asn variants inGBA1.Conclusions:This is thefirst family with GD and this combination of variants which causes a phenotype re-markable for severe bone disease with no or mild hematological manifestations

Liver involvement in patients with Gaucher disease types I and III

Background & aims Gaucher disease (GD) is a multisystemic disease. Liver involvement in GD is not well characterised and ranges from hepatomegaly to cirrhosis and hepatocellular carcinoma. We aim to describe, and assess the effect of treatment, on the hepatic phenotype of a cohort of patients with GD types I and II. Methods Retrospective study based on the review of the medical files of the Gaucher Reference Centre of the Hospital de Clínicas de Porto Alegre, Brazil. Data from all GD types I and III patients seen at the centre since 2003 were analysed. Variables were compared as pre- (“baseline”) and post-treatment (“follow-up”). Results Forty-two patients (types I: 39, III: 3; female: 22; median age: 35 y; enzyme replacement therapy: 37; substrate reduction therapy: 2; non-treated: 3; median time on treatment-MTT: 124 months) were included. Liver enzyme abnormalities, hepatomegaly, and steatosis at baseline were seen in 19/28 (68%), 28/42 (67%), and 3/38 patients (8%), respectively; at follow-up, 21/38 (55%), 15/38 (39%) and 15/38 (39%). MRI iron quantification showed overload in 7/8 patients (treated: 7; MTT: 55 months), being severe in 2/7 (treated: 2/2; MTT: 44.5 months). Eight patients had liver biopsy (treated: 6; MTT: 58 months), with fibrosis in 3 (treated: 1; time on treatment: 108 months) and steatohepatitis in 2 (treated: 2; time on treatment: 69 and 185 months). One patient developed hepatocellular carcinoma. Conclusions GD is a heterogeneous disease that causes different patterns of liver damage even during treatment. Although treatment improves the hepatocellular damage, it is associated with an increased rate of steatosis. This study highlights the importance of a follow-up of liver integrity in these patients

Elevated holo-transcobalamin in Gaucher disease type II : a case report

Gaucher disease (GD), one of the most common lysosomal disorders, is caused by deficiency of β-glucocerebrosidase. Based on the presence and severity of neurological complications, GD is classified into types I, II (the most severe form), and III. Abnormalities in systemic markers of vitamin B12 (B12) metabolism have been reported in GD type I patients, suggesting a higher prevalence of B12 deficiency in these patients. A 2-month-old male with GD type II was admitted to the hospital presenting jaundice, hepatosplenomegaly, and ichthyosis. At admission, cholestasis and ascites, abnormal liver function enzymes, prolonged prothrombin time, and high levels of B12 were confirmed. Analysis of biomarkers of B12 status revealed elevated B12 and holo-transcobalamin (holo-TC) levels. The B12 profile found in our patient is the opposite to what is described for GD type I patients. Holo-TC may increase in inflammatory states or due to liver diseases. In GD, the accumulation of glucocerebroside may be a trigger that initiates a systemic inflammatory reaction, characterized by macrophage activation. We suggest higher levels of holo-TC could be associated with a more severe (neuronopathic) GD, and be a biomarker of GD type II