RESUMO

Mucolipidosis II and III (MLII and MLIII) are autosomal recessive diseases caused by pathogenic variants in GNPTAB and GNPTG genes that lead to defects in GlcNAc-1-phosphotransferase. This enzyme adds mannose 6-phosphate residues to lysosomal hydrolases, which allows enzymes to enter lysosomes. Defective GlcNAc-1-phosphotransferase causes substrate accumulation and inflammation. These diseases have no treatment, and we hypothesized that the use of substrate reduction therapy and immunomodulation may be beneficial at the cell level and as a future therapeutic approach. Fibroblasts from two patients with MLIII alpha/beta and 2 patients with MLIII gamma as well as from one healthy control were treated with 10 µM miglustat, 20 µM genistein, and 20 µM thalidomide independently. ELISA assay and confocal immunofluorescence microscopy were used to evaluate the presence of heparan sulfate (HS) and the impact on substrate accumulation. ELISA assay showed HS reduction in all patients with the different treatments used (p=0.05). HS reduction was also observed by immunofluorescence microscopy. Our study produced encouraging results, since the reduction in substrate accumulation, even partial, may offer benefits to the phenotype of patients with inborn errors of metabolism.

RESUMO

Abstract GlcNAc-1-phosphotransferase is a hexameric complex formed by subunits α, β, and γ, where the first two are encoded by the GNPTAB gene and the third by the GNPTG gene. Pathogenic variants identified in the GNPTAB gene cause the diseases Mucolipidosis II and III alpha/beta, which are severe and characterized by an overflow of lysosomal hydrolases into the extracellular environment, and their absence in lysosomal compartments causes an accumulation of non-degraded macromolecules. Methodology: a retrospective study that included 32 unrelated Brazilian patients with a clinical and genetic diagnosis of Mucolipidosis II/III alpha/beta. The regional frequency of the altered alleles was determined. Results: The patients were from all regions of Brazil. The most prevalent variants were c.3503_3504del, associated with the severe form of the disease, and c.1208T>C, associated with the milder form. Variant c.3503_3504del is the most frequently found in the Midwest, Northeast, and Southeast regions of Brazil. In the South, 42.8% of the alleles present the c.1196C>T variant. Conclusions: From the perspective of all patients diagnosed with Mucolipidosis II/III in Brazil, it is possible to conclude that different regions present allelic frequencies of specific pathogenic variants, which can be explained by the occurrence of a founding effect or high inbreeding rates.

RESUMO

Mucolipidosis II and III (ML II and III) alpha/beta and ML III gamma are lysosomal diseases caused by GlcNAc-1-phosphotransferase deficiency. Previous data indicate that MLII patients have functionally impaired immune system that contributes to predisposition to infections.We evaluated the immunological phenotype of three Brazilian patients with ML III gamma. Our data suggest that the residual activity of GlcNAc-1-phosphotransferase in patients with ML III gamma is enough to allow the targeting of the lysosomal enzymes required for B-cell functions maintenance.

RESUMO

Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α/ß-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α/ß-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.

Assuntos

Mutação com Perda de Função , Mucolipidoses/enzimologia , Mucolipidoses/genética , Mutação de Sentido Incorreto , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Adulto Jovem

RESUMO

Mucolipidosis (ML) III gamma is a rare autosomal-recessive disorder caused by pathogenic mutations in the GNPTG gene. GNPTG encodes the γ-subunit of GlcNAc-1-phosphotransferase that catalyzes mannose 6-phosphate targeting signal synthesis on soluble lysosomal enzymes. ML III gamma patients are characterized by missorting of lysosomal enzymes. In this report, we describe the probable occurrence of mRNA editing in two ML III gamma patients. Patients A and B (siblings) presented at the adult age with a typical clinical picture of ML III gamma, mainly compromising bone and joints, and high levels of lysosomal enzymes in plasma and low levels in fibroblasts. Both were found to be homozygous for c.-112C>G and c.328G>T (p.Glu110Ter) mutations in genomic DNA (gDNA) analysis of GNPTG. Analysis of complementary DNA (cDNA), however, showed normal genotypes for both patients. Low GNPTG mRNA expression was observed in both patients. The mRNA editing can explain the differences found in patients A and B regarding gDNA and cDNA analysis, and the mild clinical phenotype associated with homozygosity for a nonsense mutation. Our results suggest that mRNA editing can be more frequent than expected in monogenic disorders and that GNPTG analysis should be performed on gDNA.

Assuntos

Códon sem Sentido , Homozigoto , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , RNA Mensageiro/genética , Irmãos , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Adulto , Alelos , Substituição de Aminoácidos , Biomarcadores , Variações do Número de Cópias de DNA , Feminino , Expressão Gênica , Genótipo , Humanos , Masculino , Fenótipo , Polimorfismo de Fragmento de Restrição , Edição de RNA , Análise de Sequência de DNA

RESUMO

Mucolipidosis III (ML III) gamma is a very rare autosomal-recessive disorder characterized by the abnormal trafficking and subcellular localization of lysosomal enzymes due to mutations in the GNPTG gene. The present study consists of a report of a Brazilian compound heterozygote patient with ML III gamma resulting from one mutant paternal allele and one allele that had most likely undergone a de novo or maternal germline mutation. This is the first report of a de novo mutation in ML III gamma. This finding has significant implications for genetic counseling.