RESUMO
PIGT-CDG, an autosomal recessive syndromic intellectual disability disorder of glycosylphosphatidylinositol (GPI) anchors, was recently described in two independent kindreds [Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (OMIM, #615398)]. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of GPI to proteins. GPI facilitates attachment (anchoring) of proteins to cell membranes. We describe, at ages 7 and 6 years, two children of non-consanguineous parents; they had hypotonia, severe global developmental delay, and intractable seizures along with endocrine, ophthalmologic, skeletal, hearing, and cardiac anomalies. Exome sequencing revealed that both siblings had compound heterozygous variants in PIGT (NM_015937.5), i.e., c.918dupC, a novel duplication leading to a frameshift, and c.1342C > T encoding a previously described missense variant. Flow cytometry studies showed decreased surface expression of GPI-anchored proteins on granulocytes, consistent with findings in previous cases. These siblings further delineate the clinical spectrum of PIGT-CDG, reemphasize the neuro-ophthalmologic presentation, clarify the endocrine features, and add hypermobility, low CSF albumin quotient, and hearing loss to the phenotypic spectrum. Our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in subjects with early onset severe seizure disorders and dysmorphic facial features, even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our case reaffirms that the use of flow cytometry and genetic testing is essential for diagnosis in this group of disorders.
Assuntos
Aciltransferases/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Aciltransferases/química , Aciltransferases/genética , Criança , Deficiências do Desenvolvimento/metabolismo , Fibroblastos , Mutação da Fase de Leitura , Heterozigoto , Humanos , Hipotonia Muscular/metabolismo , Mutação de Sentido Incorreto , Pele/citologiaRESUMO
BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).
Assuntos
Síndrome de Fanconi/genética , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Enzima Bifuncional do Peroxissomo/genética , Sequência de Aminoácidos , Animais , População Negra , Cromossomos Humanos Par 3 , Modelos Animais de Doenças , Síndrome de Fanconi/etnologia , Feminino , Ligação Genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Linhagem , Enzima Bifuncional do Peroxissomo/química , Enzima Bifuncional do Peroxissomo/metabolismo , Fenótipo , Análise de Sequência de DNARESUMO
Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive, quadriceps sparing type commonly referred to as HIBM but also termed h-IBM or Inclusion Body Myopathy 2 (IBM2). The clinical manifestations begin with muscle weakness progressing over the next 10-20 years uniquely sparing the quadriceps until the most advanced stage of the disease. Histopathology of an HIBM muscle biopsy shows rimmed vacuoles on Gomori's trichrome stain, small fibers in groups and tubulofilaments without evidence of inflammation. In affected individuals distinct mutations have been identified in the GNE gene, which encodes the bifunctional enzyme uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase/N-acetyl-mannosamine (ManNAc) kinase (GNE/MNK). GNE/MNK catalyzes the first two committed steps in the biosynthesis of acetylneuraminic acid (Neu5Ac), an abundant and functionally important sugar. The generation of HIBM animal models has led to novel insights into both the disease and the role of GNE/MNK in pathophysiology. Recent advances in therapeutic approaches for HIBM, including administration of N-acetyl-mannosamine (ManNAc), a precursor of Neu5Ac will be discussed.
Assuntos
Complexos Multienzimáticos/genética , Miosite de Corpos de Inclusão/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Miosite de Corpos de Inclusão/patologia , Ácido N-Acetilneuramínico/metabolismoRESUMO
Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.
Assuntos
Alelos , Ácido N-Acetilneuramínico do Monofosfato de Citidina/farmacologia , Fibroblastos/enzimologia , Genes Dominantes , Complexos Multienzimáticos/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Doença do Armazenamento de Ácido Siálico/enzimologia , Substituição de Aminoácidos , Células Cultivadas , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação de Sentido Incorreto , Doença do Armazenamento de Ácido Siálico/tratamento farmacológico , Doença do Armazenamento de Ácido Siálico/genéticaRESUMO
Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.
Assuntos
Hexosaminas/uso terapêutico , Nefropatias/genética , Nefropatias/metabolismo , Ácido N-Acetilneuramínico/biossíntese , Proteinúria/genética , Proteinúria/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Feminino , Humanos , Nefropatias/tratamento farmacológico , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica , Modelos Biológicos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Gravidez , Proteinúria/tratamento farmacológicoRESUMO
Aberrant glycosylation of dystroglycan occurs in certain muscular dystrophies, including hereditary inclusion body myopathy (HIBM). HIBM harbors a widely varying clinical severity and age of onset, which raised the suspicion of the presence of disease modifier genes. We considered the highly polymorphic dystroglycan gene (DAG1) as a feasible candidate modifier gene. DAG1 genomic DNA was sequenced for 32 HIBM patients, mainly of Persian-Jewish descent. Five novel DAG1 single nucleotide polymorphisms (SNPs) were identified, bringing the total number of SNPs to 19. However, no direct correlation between DAG1 SNPs and clinical severity of HIBM could be detected. Several identified SNPs substitute an amino acid and might modulate dystroglycan function or glycosylation status, and deserve further research. These data are valuable for future studies on the role of DAG1 in HIBM and other muscular dystrophies, especially those dystrophies that involve abnormal glycosylation of dystroglycan.
Assuntos
Distroglicanas/genética , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/patologia , Polimorfismo de Nucleotídeo Único , Adulto , Idade de Início , Sequência de Bases , Primers do DNA , Humanos , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Índice de Gravidade de DoençaRESUMO
Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients, with HIBM harboring missense mutations in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription-translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and it revealed that mutations in one enzymatic domain (in GNE, G135V, V216A, and R246W; in MNK, A631V, M712T) affected not only that domain's enzyme activity, but also the activity of the other domain. Moreover, studies of the residual enzyme activity associated with specific mutations revealed a discrepancy between the fibroblasts and the cell-free systems. Fibroblasts exhibited higher residual activities of both GNE and MNK than the cell-free system. These findings add complexity to the tightly regulated system of sialic acid biosynthesis. This cell-free approach can be applied to other glycosylation pathway enzymes that are difficult to evaluate in whole cells because their substrate specificities overlap with those of ancillary enzymes.
Assuntos
Miosite de Corpos de Inclusão/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adulto , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Sistema Livre de Células , Células Cultivadas , Análise Mutacional de DNA/métodos , Ativação Enzimática/fisiologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Mutação , Miosite de Corpos de Inclusão/complicações , Miosite de Corpos de Inclusão/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Congenital disorders of glycosylation (CDG) are a group of metabolic disorders with multisystemic involvement characterized by abnormalities in the synthesis of N-linked oligosaccharides. The most common form, CDG-Ia, resulting from mutations in the gene encoding the enzyme phosphomannomutase (PMM2), manifests with severe abnormalities in psychomotor development, dysmorphic features and visceral involvement. While this disorder is panethnic, we present the first cases of CDG-Ia identified in an African American family with two affected sisters. The proband had failure to thrive in infancy, hypotonia, ataxia, cerebellar hypoplasia and developmental delay. On examination, she also exhibited strabismus, inverted nipples and an atypical perineal fat distribution, all features characteristic of CDG-Ia. Direct sequencing demonstrated that the patient had a unique genotype, T237M/c.565-571 delAGAGAT insGTGGATTTCC. The novel deletion-insertion mutation, which was confirmed by subcloning and sequencing of each allele, introduces a stop codon 11 amino acids downstream from the site of the deletion. The presence of this deletion-insertion mutation at cDNA position 565 suggests that this site in the PMM2 gene may be a hotspot for chromosomal breakage.