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OBJECTIVE: Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. METHODS: Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. RESULTS: We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. INTERPRETATION: Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.
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CCDC186 protein is involved in the maturation of dense-core vesicles (DCVs) in the trans-Golgi network in neurons and endocrine cells. Mutations in genes involved in DCV regulation, other than CCDC186, have been described in patients with neurodevelopmental disorders. To date, only one patient, within a large sequencing study of 1000 cases, and a single case report with variants in CCDC186, had previously been described. However, no functional studies in any of these two cases had been performed. We identified three patients from two gypsy families, unrelated to each other, with mutations in the CCDC186 gene. Clinically, all patients presented with seizures, frontotemporal atrophy, hypomyelination, recurrent infections, and endocrine disturbances such as severe non-ketotic hypoglycemia. Low levels of cortisol, insulin, or growth hormone could only be verified in one patient. All of them had a neonatal onset and died between 7 months and 4 years of age. Whole exome sequencing identified a homozygous variant in the CCDC186 gene (c.2215C>T, p.Arg739Ter) in the index patients of both families. Protein expression studies demonstrated that CCDC186 was almost undetectable in fibroblasts and muscle tissue. These observations correlated with the transcriptomic analysis performed in fibroblasts in one of the patients, which showed a significant reduction of CCDC186 mRNA levels. Our study provides functional evidence that mutations in this gene have a pathogenic effect on the protein and reinforces CCDC186 as a new disease-associated gene. In addition, mutations in CCDC186 could explain the combined endocrine and neurologic alterations detected in our patients.
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Doenças do Sistema Endócrino , Transtornos do Neurodesenvolvimento , Recém-Nascido , Humanos , Sistema Nervoso Central , Transtornos do Neurodesenvolvimento/genética , Mutação , Rede trans-GolgiRESUMO
Isolated complex I (CI) deficiency is the most common cause of oxidative phosphorylation (OXPHOS) dysfunction. Whole-exome sequencing identified biallelic mutations in NDUFA8 (c.[293G > T]; [293G > T], encoding for an accessory subunit of CI, in two siblings with a favorable clinical evolution. The individuals reported here are practically asymptomatic, with the exception of slight failure to thrive and some language difficulties at the age of 6 and 9 years, respectively. These observations are remarkable since the vast majority of patients with CI deficiency, including the only NDUFA8 patient reported so far, showed an extremely poor clinical outcome. Western blot studies demonstrated that NDUFA8 protein was strongly reduced in the patients' fibroblasts and muscle extracts. In addition, there was a marked and specific decrease in the steady-state levels of CI subunits. BN-PAGE demonstrated an isolated defect in the assembly and the activity of CI with impaired supercomplexes formation and abnormal accumulation of CI subassemblies. Confocal microscopy analysis in fibroblasts showed rounder mitochondria and diminished branching degree of the mitochondrial network. Functional complementation studies demonstrated disease-causality for the identified mutation as lentiviral transduction with wild-type NDUFA8 cDNA restored the steady-state levels of CI subunits and completely recovered the deficient enzymatic activity in immortalized mutant fibroblasts. In summary, we provide additional evidence of the involvement of NDUFA8 as a mitochondrial disease-causing gene associated with altered mitochondrial morphology, CI deficiency, impaired supercomplexes formation, and very mild progression of the disease.
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Predisposição Genética para Doença , Doenças Mitocondriais/genética , NADH Desidrogenase/genética , Fosforilação Oxidativa , Criança , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Irmãos , Sequenciamento do ExomaRESUMO
3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.
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Proteínas de Membrana Transportadoras/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Biomarcadores , Transporte de Elétrons , Metabolismo Energético , Fibroblastos/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Fenótipo , Transporte Proteico , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética , Sequenciamento do ExomaRESUMO
Mucopolysaccharidosis type VII (MPS VII) is an inherited disease characterized by the cellular accumulation of undegraded GAGs due to the deficiency of the lysosomal enzyme ß-glucuronidase. We describe a case of a 2-year-old female affected by a moderate form of MPS VII and submitted twice to HSCT with the aim of stabilizing skeletal problems and preventing neurocognitive alterations. The child underwent a second transplantation due to the rejection of the graft after a reduced-intensity conditioning in the first transplant. A myeloablative regimen allowed to achieve a stable full donor engraftment and normal enzyme levels during the 6 years of follow-up. Clinically, we observed stabilization of skeletal deformities and normal neurocognitive development. This is one of the few reports of mucopolysaccharidosis type VII treated with allogeneic HSCT.
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Transplante de Células-Tronco Hematopoéticas , Mucopolissacaridose VII/terapia , Pré-Escolar , Feminino , HumanosRESUMO
OBJECTIVE: To describe the clinical, biochemical, and genetic features of patients with congenital disorders of glycosylation (CDG) identified in Spain during the last 20 years. STUDY DESIGN: Patients were selected among those presenting with multisystem disease of unknown etiology. The isoforms of transferrin and of ApoC3 and dolichols were analyzed in serum; phosphomannomutase and mannosephosphate isomerase activities were measured in fibroblasts. Conventional or massive parallel sequencing (customized panel or Illumina Clinical-Exome Sequencing TruSight One Gene Panel) was used to identify genes and mutations. RESULTS: Ninety-seven patients were diagnosed with 18 different CDG. Eighty-nine patients had a type 1 transferrin profile; 8 patients had a type 2 transferrin profile, with 6 of them showing an alteration in the ApoC3 isoform profile. A total of 75% of the patients had PMM2-CDG presenting with a heterogeneous mutational spectrum. The remaining patients showed mutations in any of the following genes: MPI, PGM1, GFPT1, SRD5A3, DOLK, DPGAT1, ALG1, ALG6, RFT1, SSR4, B4GALT1, DPM1, COG6, COG7, COG8, ATP6V0A2, and CCDC115. CONCLUSION: Based on literature and on this population-based study of CDG, a comprehensive scheme including reported clinical signs of CDG is offered, which will hopefully reduce the timeframe from clinical suspicion to genetic confirmation. The different defects of CDG identified in Spain have contributed to expand the knowledge of CDG worldwide. A predominance of PMM2 deficiency was detected, with 5 novel PMM2 mutations being described.
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Acetiltransferases/metabolismo , Apolipoproteínas C/metabolismo , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/epidemiologia , Acetiltransferases/genética , Apolipoproteínas C/genética , Estudos de Coortes , Bases de Dados Factuais , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Incidência , Recém-Nascido , Masculino , Mutação , Estudos Retrospectivos , Medição de Risco , Espanha/epidemiologiaRESUMO
Most inborn errors of metabolism (IEM) remain without effective treatment mainly due to the incapacity of conventional therapeutic approaches to target the neurological symptomatology and to ameliorate the multisystemic involvement frequently observed in these patients. However, in recent years, the therapeutic use of small molecules has emerged as a promising approach for treating this heterogeneous group of disorders. In this review, we focus on the use of therapeutically active small molecules to treat IEM, including readthrough agents, pharmacological chaperones, proteostasis regulators, substrate inhibitors, and autophagy inducers. The small molecules reviewed herein act at different cellular levels, and this knowledge provides new tools to set up innovative treatment approaches for particular IEM. We review the molecular mechanism underlying therapeutic properties of small molecules, methodologies used to screen for these compounds, and their applicability in preclinical and clinical practice.
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Erros Inatos do Metabolismo/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Humanos , Recém-Nascido , Triagem Neonatal/métodosRESUMO
Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.
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Doença de Leigh/dietoterapia , Doença de Leigh/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Tiamina/metabolismo , Tiamina/uso terapêutico , Adolescente , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Doença de Leigh/sangue , Doença de Leigh/líquido cefalorraquidiano , Doença de Leigh/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Complexo Piruvato Desidrogenase/metabolismo , Tiamina/sangue , Tiamina/líquido cefalorraquidiano , Tiamina Pirofosfato/metabolismoRESUMO
Niemann-Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease-causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus-specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.
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Bases de Dados Genéticas , Mutação , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo B/genética , RNA Mensageiro/genética , Esfingomielina Fosfodiesterase/genética , Éxons , Expressão Gênica , Genes Recessivos , Estudos de Associação Genética , Genótipo , Humanos , Íntrons , Doença de Niemann-Pick Tipo A/diagnóstico , Doença de Niemann-Pick Tipo A/patologia , Doença de Niemann-Pick Tipo B/diagnóstico , Doença de Niemann-Pick Tipo B/patologia , Fases de Leitura Aberta , Fenótipo , Splicing de RNARESUMO
Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-ß-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-ß-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-ß-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.
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1-Desoxinojirimicina/análogos & derivados , Adamantano/análogos & derivados , Doença de Gaucher/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , 1-Desoxinojirimicina/farmacologia , Adamantano/farmacologia , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Análise Mutacional de DNA , Neurônios Dopaminérgicos/enzimologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática/efeitos dos fármacos , Doença de Gaucher/patologia , Expressão Gênica , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fator 4 Semelhante a Kruppel , Lisossomos/enzimologia , Macrófagos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transporte Proteico , Bibliotecas de Moléculas Pequenas , TranscriptomaRESUMO
Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 µmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.
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Antioxidantes/uso terapêutico , Fibroblastos/efeitos dos fármacos , Mucopolissacaridose III/tratamento farmacológico , Ubiquinona/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Exocitose , Glicosaminoglicanos/metabolismo , Humanos , Lisossomos/metabolismo , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Ubiquinona/uso terapêuticoRESUMO
Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.
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Proteínas de Arabidopsis , Doença de Leigh , Humanos , Doença de Leigh/genética , Doença de Leigh/patologia , Mitocôndrias/patologia , Proteínas/genética , Mutação/genética , Fenótipo , Proteínas de Ligação a RNA , Proteínas de Arabidopsis/genéticaRESUMO
Mucopolysaccharidosis type IVA (Morquio A) is an inherited metabolic disease with autosomal recessive inheritance. The pathology is due to a deficient activity of N-acetylgalactosamine-6-sulfate-sulfatase, which is involved in the degradation of keratan sulfate and chondroitin-6-sulfate. To date more than 150 mutations have been described in the GALNS gene in different populations. The aim of this study was to analyze the mutations and polymorphisms in Spain in order to know the epidemiology of our population and also to offer genetic counseling to affected families. We found 30 mutant alleles in the 15 families analyzed completing all the genotypes. Most of the mutations that we found were missense mutations, six of which were novel: p.S74F, p.E121D, p.Y254C, p.E260K, p.T394P and p.N495Y; we also found a small deletion (c.1142delC) and a probable deep intronic mutation that causes the loss of exon 5 (c.423_566del) found in cDNA. Both mutations are described in this study for the first time. We also identified 20 polymorphisms previously reported and 2 novel ones: (c.633+222T/C and c.898+25C>G). In conclusion, we have identified the mutations responsible for Mucopolysaccharidosis IV A in Spain. We found great allelic heterogeneity, as occurs in other populations, which hinders the establishment of genotype-phenotype correlations in Spain. This study has been very useful for genetic counseling to the affected families.
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Condroitina Sulfatases/genética , Mucopolissacaridose IV/genética , Adolescente , Adulto , Idade de Início , Alelos , Sequência de Bases , Criança , Pré-Escolar , Família , Feminino , Ordem dos Genes , Haplótipos , Humanos , Lactente , Masculino , Mucopolissacaridose IV/epidemiologia , Taxa de Mutação , Polimorfismo de Nucleotídeo Único , Espanha/epidemiologia , Adulto JovemRESUMO
OBJECTIVE: Nonimmune hydrops fetalis (NIHF) is defined by the excessive fluid accumulation in more than one foetal compartments and body cavities because of nonimmune reasons. It has been described that 14 lysosomal diseases may be causative of NIHF. The aim of this study was to design a fast protocol to investigate the most frequent lysosomal diseases that are reported that may cause NIHF. METHOD: We analysed the glycosaminoglycans excretion in the amniotic fluid supernatant and four different lysosomal enzymatic activities in the amniotic cultured cells of the different NIHF amniotic fluids we received. RESULTS: We investigated 30 NIHF cases, using this fast protocol. We detected two cases of NIHF because of lysosomal diseases, which represent 6.6%. We diagnosed one case of mucopolysaccharidosis type VII and one case of Gaucher disease. CONCLUSION: The fast protocol we designed analyses seven of the most frequent lysosomal pathologies that have been described that may cause NIHF, with only five different determinations, which make the analysis of NIHF fast, cost-effective and without need of too much amniotic fluid. We believe this protocol may be useful for the analysis of lysosomal diseases in NIHF.
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Hidropisia Fetal/diagnóstico , Hidropisia Fetal/etiologia , Doenças por Armazenamento dos Lisossomos/complicações , Doenças por Armazenamento dos Lisossomos/diagnóstico , Diagnóstico Pré-Natal/métodos , Adulto , Líquido Amniótico/química , Líquido Amniótico/citologia , Líquido Amniótico/metabolismo , Feminino , Doença de Gaucher/complicações , Doença de Gaucher/diagnóstico , Doença de Gaucher/diagnóstico por imagem , Humanos , Hidropisia Fetal/epidemiologia , Hidropisia Fetal/metabolismo , Doenças por Armazenamento dos Lisossomos/epidemiologia , Gravidez , Fatores de Tempo , UltrassonografiaRESUMO
The association between Parkinson's disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD.
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The objective of this study is to describe the alterations occurring during the neurodegenerative process in skin fibroblast cultures from C9orf72 patients. We characterized the oxidative stress, autophagy flux, small ubiquitin-related protein SUMO2/3 levels as well as the mitochondrial function in skin fibroblast cultures from C9orf72 patients. All metabolic and bioenergetic findings were further correlated with gene expression data obtained from RNA sequencing analysis. Fibroblasts from C9orf72 patients showed a 30% reduced expression of C9orf72, ~3-fold increased levels of oxidative stress and impaired mitochondrial function obtained by measuring the enzymatic activities of mitochondrial respiratory chain complexes, specifically of complex III activity. Furthermore, the results also reveal that C9orf72 patients showed an accumulation of p62 protein levels, suggesting the alteration of the autophagy process, and significantly higher protein levels of SUMO2/3 (p = 0.03). Our results provide new data reinforcing that C9orf72 cells suffer from elevated oxidative damage to biomolecules and organelles and from increased protein loads, leading to insufficient autophagy and an increase in SUMOylation processes.
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The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.
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In the field of laboratory medicine, proficiency testing is a vehicle used to improve the reliability of reported results. When proficiency tests are unavailable for a given analyte, an alternative approach is required to ensure adherence to the International Organization for Standardization (ISO) 15189:2012 standard. In this study, we report the results of a split-sample testing program performed as an alternative to a formal PT. This testing method was based on recommendations provided in the Clinical and Laboratory Standards Institute (CLSI) QMS24 guideline. Two different laboratories measured, in duplicate, the heparan sulfate concentration in five samples using ultra-performance liquid chromatography and tandem mass spectrometry. The data analysis to determine the criterion used for the comparability assessment between the two laboratories was based on Appendix E of the QMS24 guideline. Mean interlaboratory differences fell within the maximum allowable differences calculated from the application of the QMS24 guideline, indicating that the results obtained by the two laboratories were comparable across the concentrations tested. Application of the QMS24 split-sample testing procedure allows laboratories to objectively assess test results, thus providing the evidence needed to face an accreditation audit with confidence. However, due to the limitations of statistical analyses in small samples (participants and/or materials), laboratory specialists should assess whether the maximum allowable differences obtained are suitable for the intended use, and make adjustments if necessary.
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Laboratórios Clínicos/normas , Ensaio de Proficiência Laboratorial/métodos , Controle de Qualidade , Cromatografia Líquida/normas , Heparitina Sulfato/análise , Heparitina Sulfato/sangue , Humanos , Espectrometria de Massas em Tandem/normasRESUMO
Clinical exome sequencing is a powerful approach to overcome the wide clinical and genetic heterogeneity of mucopolysaccharidosis. These data could be useful for prenatal diagnosis of MPS VII, genetic counseling, and preimplantation genetic testing.