RESUMO
Pathogenic, biallelic variants in SORD were identified in 2020 as a novel cause for autosomal-recessive Charcot-Marie-Tooth disease (CMT) type 2, an inherited neuropathy. SORD codes for the enzyme sorbitol dehydrogenase. Loss of this enzyme's activity leads to an increase of sorbitol in serum. We retrospectively screened 166 patients with axonal neuropathy (predominantly CMT type 2, but including intermediate form of CMT and distal hereditary motor neuropathy (dHMN)) without identified genetic etiology for SORD mutations at a single large German neuromuscular center. Clinical and electrophysiology exam findings were analyzed for genotype-phenotype correlation. Five patients of the total cohort of 166 patients harbored pathogenic variants in SORD (3%). The homozygous frameshift variant c.757delG (p.Ala253Glnfs*27) was the most common (4/5). One additional case carried this variant on one allele only and an additional pathogenic missense variant c.458C > A (p.Ala153Asp) on the other allele. Age of onset ranged from early infancy to mid-twenties, and phenotypes comprised axonal CMT (4) and dHMN (1). Our findings strengthen the importance of screening for pathogenic variants in SORD, especially in patients with genetically unconfirmed axonal neuropathy, especially CMT type 2 and dHMN.
Assuntos
Doença de Charcot-Marie-Tooth , Fenótipo , Humanos , Doença de Charcot-Marie-Tooth/genética , Feminino , Masculino , Adulto , Mutação , Estudos Retrospectivos , Estudos de Associação Genética/métodos , Criança , Adolescente , Axônios/patologia , Adulto Jovem , Pré-EscolarRESUMO
SCN2A (sodium channel 2A) encodes the Nav1.2 channel protein in excitatory neurons in the brain. Nav1.2 is a critical voltage-gated sodium channel of the central nervous system. Mutations in SCN2A are responsible for a broad phenotypic spectrum ranging from autism and developmental delay to severe encephalopathy with neonatal or early infantile onset. SCN2A can be spliced into two different isoforms, a neonatal (6N) and an adult (6A) form. After birth, there is an equal or higher amount of the 6N isoform, protecting the brain from the increased neuronal excitability of the infantile brain. During postnatal development, 6N is gradually replaced by 6A. In an infant carrying the novel SCN2A mutation c.643G > A (p.Ala215Thr) only in the neonatal transcript, seizures started immediately after birth. The clinical presentation evolved from a burst-suppression pattern with 30-50 tonic seizures per day to hypsarrhythmia. The first exome analysis, focusing only on common transcripts, missed the diagnosis and delayed early therapy. A reevaluation including all transcripts revealed the SCN2A variant.
Assuntos
Epilepsia , Espasmos Infantis , Epilepsia/genética , Humanos , Lactente , Recém-Nascido , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Isoformas de Proteínas/genética , Convulsões/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/genéticaRESUMO
Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.
Assuntos
Retículo Endoplasmático Rugoso/genética , Retardo do Crescimento Fetal/genética , Glicoproteínas/genética , Fosfatase Ácida Resistente a Tartarato/genética , Criança , Pré-Escolar , Diarreia/genética , Diarreia/patologia , Insuficiência de Crescimento/genética , Insuficiência de Crescimento/patologia , Feminino , Retardo do Crescimento Fetal/patologia , Glicoproteínas/biossíntese , Glicosilação , Humanos , Lactente , Recém-Nascido , Pneumopatias/genética , Pneumopatias/patologia , Masculino , Transtornos Psicomotores/genética , Transtornos Psicomotores/patologia , Fosfatase Ácida Resistente a Tartarato/deficiênciaRESUMO
INTRODUCTION: TMEM16A is a calcium-activated chloride channel expressed in various secretory epithelia. Two siblings presented in early infancy with reduced intestinal peristalsis and recurrent episodes of haemorrhagic diarrhoea. In one of them, the episodes were characterised by hepatic pneumatosis with gas bubbles in the portal vein similar to necrotising enterocolitis of the newborn. METHODS: Exome sequencing identified a homozygous truncating pathogenic variant in ANO1. Expression analysis was performed using reverse transcription PCR, western blot and immunohistochemistry. Electrophysiological and cell biological studies were employed to characterise the effects on ion transport both in patient respiratory epithelial cells and in transfected HEK293 cells. RESULTS: The identified variant led to TMEM16A dysfunction, which resulted in abolished calcium-activated Cl- currents. Secondarily, CFTR function is affected due to the close interplay between both channels without inducing cystic fibrosis (CF). CONCLUSION: TMEM16A deficiency is a potentially fatal disorder caused by abolished calcium-activated Cl- currents in secretory epithelia. Secondary impairment of CFTR function did not cause a CF phenotyp, which may have implications for CF treatment.
Assuntos
Anoctamina-1/genética , Canais de Cloreto/genética , Predisposição Genética para Doença , Doenças do Recém-Nascido/genética , Proteínas de Neoplasias/genética , Anoctamina-1/deficiência , Transporte Biológico/genética , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Fibrose Cística/genética , Fibrose Cística/patologia , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/epidemiologia , Doenças do Recém-Nascido/patologia , Masculino , Proteínas de Neoplasias/deficiênciaRESUMO
DNA damage repair is a pivotal mechanism in life. The nucleotide excision repair pathway protects the cells against DNA damage and involves XPD, an ATP dependent helicase that is part of the multisubunit protein complex TFIIH. XPD is encoded by the excision repair cross-complementation group 2 gene (ERCC2). Only three patients with cerebro-oculo-facio-skeletal syndrome (COFS), caused by mutations in ERCC2, have been published so far. This report describes a boy with the homozygous amino acid change p.Gly47Arg in XPD. He presented with profound microcephaly, psychomotor retardation, failure to thrive, cutaneous photosensitivity, a bilateral hearing deficit and optic atrophy, thrombocytopenia, and recurrent episodes of pneumonia. We report the first homozygous occurrence of the pathogenic variant Gly47Arg in the ERCC2 gene. Occurring homozygous, this variant was associated with COFS syndrome, leading to early death of the patient at the age of 21 months.
Assuntos
Anormalidades Múltiplas/genética , Síndrome de Cockayne/genética , Mutação de Sentido Incorreto , Mutação Puntual , Proteína Grupo D do Xeroderma Pigmentoso/genética , Substituição de Aminoácidos , Consanguinidade , Criptorquidismo/genética , Infecções por Citomegalovirus/complicações , Deficiências do Desenvolvimento/genética , Suscetibilidade a Doenças , Evolução Fatal , Homozigoto , Humanos , Recém-Nascido , Cirrose Hepática/genética , Masculino , Transtornos Psicomotores/genética , Síndrome , Proteína Grupo D do Xeroderma Pigmentoso/deficiênciaRESUMO
3-Hydroxyisobutyric acid (3HiB) is an intermediate in the degradation of the branched-chain amino acid valine. Disorders in valine degradation can lead to 3HiB accumulation and its excretion in the urine. This article describes the first two patients with a new metabolic disorder, 3-hydroxyisobutyrate dehydrogenase (HIBADH) deficiency, its phenotype and its treatment with a low-valine diet. The detected mutation in the HIBADH gene leads to nonsense-mediated mRNA decay of the mutant allele and to a complete loss-of-function of the enzyme. Under strict adherence to a low-valine diet a rapid decrease of 3HiB excretion in the urine was observed. Due to limited patient numbers and intrafamilial differences in phenotype with one affected and one unaffected individual, the clinical phenotype of HIBADH deficiency needs further evaluation.
Assuntos
Oxirredutases do Álcool/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/dietoterapia , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Hidroxibutiratos/urina , Oxirredutases do Álcool/metabolismo , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Lactente , Masculino , Valina/metabolismoRESUMO
SLC25A36 is a pyrimidine nucleotide carrier playing an important role in maintaining mitochondrial biogenesis. Deficiencies in SLC25A36 in mouse embryonic stem cells have been associated with mtDNA depletion as well as mitochondrial dysfunction. In human beings, diseases triggered by SLC25A36 mutations have not been described yet. We report the first known case of SLC25A36 deficiency in a 12-year-old patient with hypothyroidism, hyperinsulinism, hyperammonemia, chronical obstipation, short stature, along with language and general developmental delay. Whole exome analysis identified the homozygous mutation c.803dupT, p.Ser269llefs*35 in the SLC25A36 gene. Functional analysis of mutant SLC25A36 protein in proteoliposomes showed a virtually abolished transport activity. Immunoblotting results suggest that the mutant SLC25A36 protein in the patient undergoes fast degradation. Supplementation with oral uridine led to an improvement of thyroid function and obstipation, increase of growth and developmental progress. Our findings suggest an important role of SLC25A36 in hormonal regulations and oral uridine as a safe and effective treatment.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/deficiência , Uridina/uso terapêutico , Criança , Pré-Escolar , Feminino , Crescimento e Desenvolvimento/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mutantes/metabolismo , Transporte Proteico/efeitos dos fármacos , Tireotropina/metabolismo , Uridina/farmacologiaRESUMO
Congenital disorders of glycosylation (CDG) are a growing group of inborn metabolic disorders with multiorgan presentation. SLC39A8-CDG is a severe subtype caused by biallelic mutations in the manganese transporter SLC39A8, reducing levels of this essential cofactor for many enzymes including glycosyltransferases. The current diagnostic standard for disorders of N-glycosylation is the analysis of serum transferrin. Exome and Sanger sequencing were performed in two patients with severe neurodevelopmental phenotypes suggestive of CDG. Transferrin glycosylation was analyzed by high-performance liquid chromatography (HPLC) and isoelectric focusing in addition to comprehensive N-glycome analysis using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). Atomic absorption spectroscopy was used to quantify whole blood manganese levels. Both patients presented with a severe, multisystem disorder, and a complex neurological phenotype. Magnetic resonance imaging (MRI) revealed a Leigh-like syndrome with bilateral T2 hyperintensities of the basal ganglia. In patient 1, exome sequencing identified the previously undescribed homozygous variant c.608T>C [p.F203S] in SLC39A8. Patient 2 was found to be homozygous for c.112G>C [p.G38R]. Both individuals showed a reduction of whole blood manganese, though transferrin glycosylation was normal. N-glycome using MALDI-TOF MS identified an increase of the asialo-agalactosylated precursor N-glycan A2G1S1 and a decrease in bisected structures. In addition, analysis of heterozygous CDG-allele carriers identified similar but less severe glycosylation changes. Despite its reliance as a clinical gold standard, analysis of transferrin glycosylation cannot be categorically used to rule out SLC39A8-CDG. These results emphasize that SLC39A8-CDG presents as a spectrum of dysregulated glycosylation, and MS is an important tool for identifying deficiencies not detected by conventional methods.
Assuntos
Gânglios da Base/fisiopatologia , Proteínas de Transporte de Cátions/genética , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/fisiopatologia , Adolescente , Proteínas de Transporte de Cátions/deficiência , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Manganês/metabolismo , Espectrometria de Massas , Fenótipo , Transferrina/análise , Sequenciamento do Exoma , Adulto JovemRESUMO
Superoxide dismutase 1 (SOD1) is the principal cytoplasmic superoxide dismutase in humans and plays a major role in redox potential regulation. It catalyses the transformation of the superoxide anion (O2â¢-) into hydrogen peroxide. Heterozygous variants in SOD1 are a common cause of familial amyotrophic lateral sclerosis. In this study we describe the homozygous truncating variant c.335dupG (p.C112Wfs*11) in SOD1 that leads to total absence of enzyme activity. The resulting phenotype is severe and marked by progressive loss of motor abilities, tetraspasticity with predominance in the lower extremities, mild cerebellar atrophy, and hyperekplexia-like symptoms. Heterozygous carriers have a markedly reduced enzyme activity when compared to wild-type controls but show no overt neurologic phenotype. These results are in contrast with the previously proposed theory that a loss of function is the underlying mechanism in SOD1-related motor neuron disease and should be considered before application of previously proposed SOD1 silencing as a treatment option for amyotrophic lateral sclerosis.
Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/genética , Superóxido Dismutase-1/deficiência , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica , Criança , Pré-Escolar , Mutação da Fase de Leitura , Humanos , Masculino , Linhagem , SíndromeRESUMO
Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.
Assuntos
Complexo de Golgi/genética , Homeostase , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Exoma , Feminino , Fibroblastos/citologia , Glicosilação , Complexo de Golgi/metabolismo , Células HeLa , Heterozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Accurate glycosylation of proteins is essential for their function and their intracellular transport. Numerous diseases have been described, where either glycosylation or intracellular transport of proteins is impaired. Coat protein I (COPI) is involved in anterograde and retrograde transport of proteins between endoplasmic reticulum and Golgi, where glycosylation takes place, but no association of defective COPI proteins and glycosylation defects has been described so far. We identified a patient whose phenotype at a first glance was reminiscent of PGM1 deficiency, a disease that also affects N-glycosylation of proteins. More detailed analyses revealed a different disease with a glycosylation deficiency that was only detectable during episodes of acute illness of the patient. Trio-exome analysis revealed a de novo loss-of-function mutation in ARCN1, coding for the delta-COP subunit of COPI. We hypothesize that the capacity of flow through Golgi is reduced by this defect and at high protein synthesis rates, this bottleneck also manifests as transient glycosylation deficiency.
Assuntos
Complexo I de Proteína do Envoltório/genética , Mutação com Perda de Função , Glicosilação , Humanos , Lactente , MasculinoRESUMO
RATIONALE: Familial sinus node and atrioventricular conduction dysfunction is a rare disorder that leads to paroxysmal dizziness, fatigue, and syncope because of a temporarily or permanently reduced heart rate. To date, only a few genes for familial sinus and atrioventricular conduction dysfunction are known, and the majority of cases remain pathogenically unresolved. OBJECTIVE: We aim to identify the disease gene in a large 3-generation family (n=25) with autosomal dominant sinus node dysfunction (SND) and atrioventricular block (AVB) and to characterize the mutation-related pathomechanisms in familial SND+AVB. METHODS AND RESULTS: Genome-wide linkage analysis mapped the SND+AVB disease locus to chromosome 7q21.1-q31.1 (2-point logarithm of the odds score: 4.64; θ=0); in this region, targeted exome sequencing identified a novel heterozygous mutation (p.Arg52Leu) in the GNB2 gene that strictly cosegregated with the SND+AVB phenotype. GNB2 encodes the ß2 subunit (Gß2) of the heterotrimeric G-protein complex that is being released from G-protein-coupled receptors on vagal stimulation. In 2 heterologous expression systems (HEK-293T cells and Xenopus laevis oocytes), an enhanced activation of the G-protein-activated K+ channel (GIRK; Kir3.1/Kir3.4) was shown when mutant Gß2 was coexpressed with Gγ2; this was in contrast to coexpression of mutant Gß2-Gγ2 with other cardiac ion channels (HCN4, HCN2, and Cav1.2). Molecular dynamics simulations suggested a reduced binding property of mutant Gß2 to cardiac GIRK channels when compared with native Gß2. CONCLUSIONS: A GNB2 gene mutation is associated with familial SND+AVB and leads to a sustained activation of cardiac GIRK channels, which is likely to hyperpolarize the myocellular membrane potential and thus reduces their spontaneous activity. Our findings describe for the first time a role of a mutant G-protein in the nonsyndromic pacemaker disease because of GIRK channel activation.
Assuntos
Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/fisiopatologia , Proteínas de Ligação ao GTP/genética , Mutação/genética , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/fisiopatologia , Adulto , Sequência de Aminoácidos , Bloqueio Atrioventricular/diagnóstico , Feminino , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , Células HEK293 , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome do Nó Sinusal/diagnóstico , Nó Sinoatrial/fisiologia , Adulto JovemRESUMO
SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably ß-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.
Assuntos
Proteínas de Transporte de Cátions/genética , Defeitos Congênitos da Glicosilação/genética , Nanismo/genética , Manganês/sangue , Espasmos Infantis/genética , Sequência de Aminoácidos , Sequência de Carboidratos , Proteínas de Transporte de Cátions/deficiência , Cátions Bivalentes , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/complicações , Defeitos Congênitos da Glicosilação/dietoterapia , Nanismo/sangue , Nanismo/complicações , Nanismo/dietoterapia , Feminino , Galactose/uso terapêutico , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Transporte de Íons , Manganês/deficiência , Dados de Sequência Molecular , Mutação , Linhagem , Alinhamento de Sequência , Espasmos Infantis/sangue , Espasmos Infantis/complicações , Espasmos Infantis/dietoterapiaRESUMO
PurposeSLC39A8 deficiency is a severe inborn error of metabolism that is caused by impaired function of manganese metabolism in humans. Mutations in SLC39A8 lead to impaired function of the manganese transporter ZIP8 and thus manganese deficiency. Due to the important role of Mn2+ as a cofactor for a variety of enzymes, the resulting phenotype is complex and severe. The manganese-dependence of ß-1,4-galactosyltransferases leads to secondary hypoglycosylation, making SLC39A8 deficiency both a disorder of trace element metabolism and a congenital disorder of glycosylation. Some hypoglycosylation disorders have previously been treated with galactose administration. The development of an effective treatment of the disorder by high-dose manganese substitution aims at correcting biochemical, and hopefully, clinical abnormalities.MethodsTwo SCL39A8 deficient patients were treated with 15 and 20 mg MnSO4/kg bodyweight per day. Glycosylation and blood manganese were monitored closely. In addition, magnetic resonance imaging was performed to detect potential toxic effects of manganese.ResultsAll measured enzyme dysfunctions resolved completely and considerable clinical improvement regarding motor abilities, hearing, and other neurological manifestations was observed.ConclusionHigh-dose manganese substitution was effective in two patients with SLC39A8 deficiency. Close therapy monitoring by glycosylation assays and blood manganese measurements is necessary to prevent manganese toxicity.
Assuntos
Proteínas de Transporte de Cátions/deficiência , Estudos de Associação Genética , Predisposição Genética para Doença , Alelos , Biomarcadores , Suplementos Nutricionais , Eletroencefalografia , Feminino , Estudos de Associação Genética/métodos , Glicosilação/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Manganês/administração & dosagem , Manganês/efeitos adversos , Manganês/uso terapêutico , Mutação , Fenótipo , Resultado do TratamentoRESUMO
BACKGROUND: Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS: Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS: Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS: Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).
Assuntos
Glucofosfatos/genética , Doença de Depósito de Glicogênio/genética , Fenótipo , Fosfoglucomutase/genética , Galactose/uso terapêutico , Genes Recessivos , Glucose/metabolismo , Glucofosfatos/metabolismo , Doença de Depósito de Glicogênio/dietoterapia , Doença de Depósito de Glicogênio/metabolismo , Glicoproteínas/biossíntese , Glicosilação , Humanos , Masculino , Mutação , Fosfoglucomutase/metabolismo , RNA Mensageiro/análiseRESUMO
Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.
Assuntos
Encéfalo/metabolismo , Ciclinas/genética , Deficiência Intelectual/genética , Magnésio/metabolismo , Mutação de Sentido Incorreto/genética , Convulsões/genética , Adolescente , Animais , Proteínas de Transporte de Cátions , Linhagem Celular , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/metabolismo , Rim/metabolismo , Masculino , Fenótipo , Convulsões/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.
Assuntos
Hipercalcemia/genética , Doenças do Recém-Nascido/genética , Erros Inatos do Metabolismo/genética , Mutação , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Animais , Genes Recessivos , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos KnockoutRESUMO
AIMS: Elevated Carbohydrate-deficient transferrin (CDT) levels are used as a biomarker in order to screen for chronic alcohol abuse. Transferrin (Tf) variants can impair methods to measure elevated CDT levels such as high-performance liquid chromatography (HPLC). We present a Tf variant affecting the second glycosylation site of Tf and the complications it causes in diagnosing alcoholism. METHODS: A blood sample from a patient with suspected alcohol abuse was analyzed with HPLC, isoelectric focusing, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), immunoprecipitation and SDS-Page. Sanger sequencing of Tf was performed to detect Tf mutations. RESULTS: HPLC, SDS-Page and IEF showed a distinctly increased disialo-Tf fraction while the tetrasialo-Tf fraction was decreased, ESI-TOF-MS confirmed these results. Sanger sequencing revealed the Tf mutation c.1889 A>C, deleting a Tf glycosylations site and thereby causing elevated disialo-Tf levels. CONCLUSIONS: Transferrin mutations can severely impair the diagnostics of chronic alcohol abuse by causing false positive results. This has to be considered when CDT screening is used to detect alcoholism.
Assuntos
Alcoolismo/diagnóstico , Alcoolismo/genética , Variação Genética/genética , Mutação/genética , Transferrina/genética , Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodos , Transferrina/análogos & derivados , Transferrina/análiseRESUMO
We compared the consequences of an ABCA1 mutation that produced an apparent lack of atherosclerosis (Tangier family 1, N935S) with an ABCA1 mutation with functional ABCA1 knockout that was associated with severe atherosclerosis (Tangier family 2, Leu(548):Leu(575)-End), using primary and telomerase-immortalized fibroblasts. Telomerase-immortalized Tangier fibroblasts of family 1 (TT1) showed 30% residual cholesterol efflux capacity in response to apolipoprotein A-I, whereas telomerase-immortalized Tangier fibroblasts of family 2 (TT2) showed only 20%. However, there were a number of secondary differences that were often stronger and may help to explain the more rapid development of atherosclerosis in family 2. First, the total cellular cholesterol content increase was 2-3-fold and 3-5-fold in TT1 and TT2 cells, respectively. The corresponding increase in esterified cholesterol concentration was 10- and 40-fold, respectively. Second, 24-, 25-, and 27-hydroxycholesterol concentrations were moderately increased in TT1 cells, but were increased as much as 200-fold in TT2 cells. Third, cholesterol biosynthesis was moderately decreased in TT1 cells, but was markedly decreased in TT2 cells. Fourth, potentially atheroprotective LXR-dependent SREBP1c signaling was normal in TT1, but was rather suppressed in TT2 cells. Cultivated primary Tangier fibroblasts were characterized by premature aging in culture and were associated with less obvious biochemical differences. In summary, these results may help to understand the differential atherosclerotic susceptibility in Tangier disease and further demonstrate the usefulness of telomerase-immortalized cells in studying this cellular phenotype. The data support the contention that side chain-oxidized oxysterols are strong suppressors of cholesterol biosynthesis under specific pathological conditions in humans.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/biossíntese , Fibroblastos/metabolismo , Doença de Tangier/metabolismo , Telomerase/biossíntese , Transportador 1 de Cassete de Ligação de ATP/genética , Adulto , Substituição de Aminoácidos , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Linhagem Celular Transformada , Senescência Celular/genética , Colesterol/genética , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Mutação de Sentido Incorreto , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Doença de Tangier/genética , Doença de Tangier/patologia , Telomerase/genéticaRESUMO
BACKGROUND: The serotonin transporter (5-HTT) and the 5-HTTLPR/rs25531 polymorphisms in its gene (SLC6A4) have been associated with depression, increased stress-response, and brain structural alterations such as reduced hippocampal volumes. Recently, epigenetic processes including SLC6A4 promoter methylation were shown to be affected by stress, trauma, or maltreatment and are regarded to be involved in the etiology of affective disorders. However, neurobiological correlates of SLC6A4 promoter methylation have never been studied or compared to genotype effects by means of human neuroimaging hitherto METHODS: Healthy subjects were recruited in two independent samples (N = 94, N = 95) to obtain structural gray matter images processed by voxel-based morphometry (VBM8), focusing on hippocampal, amygdala, and anterior cingulate gyrus gray matter structure. SLC6A4 promoter methylation within an AluJb element and 5-HTTLPR/rs25531 genotypes were analyzed in view of a possible impact on local gray matter volume RESULTS: Strong associations of AluJb methylation and hippocampal gray matter volumes emerged within each sample separately, which in the combined sample withstood most conservative alpha-corrections for the entire brain. The amygdala, insula, and caudate nucleus showed similar associations. The 5-HTTLPR/rs25531 showed no main effect on gray matter, and the effect of methylation rates on hippocampal structure was comparable among the genotype groups CONCLUSIONS: Methylation within the AluJb appears to have strong effects on hippocampal gray matter volumes, indicating that epigenetic processes can alter brain structures crucially involved in stress-related disorders. Different ways of regulating SLC6A4 expression might involve exonization or transcription factor binding as potentially underlying mechanisms, which, however, is speculative and warrants further investigation.