RESUMEN
Objective: To investigate the clinicopathological features of fumarate hydratase (FH) deficiency uterine leiomyoma. Methods: The data of 38 patients with FH deficiency uterine leiomyoma were screened and analyzed. The expressions of FH, S-(2-succino)-cysteine (2SC), desmin, p16, p53, CD10 and cell proliferation associated nuclear antigen (Ki-67) proteins were detected by immunohistochemistry, and their clinicopathological features were analyzed retrospectively. Results: (1) Clinical features: the median age of the patients was (42.5±7.4) years old. Twenty-one cases (55%) of them were myomas found in physical examination, and the median maximum diameter of the tumor was 6.0 cm (range: 5.0-7.5 cm); myomectomy was performed in 23 cases (61%), total hysterectomy with or without bilateral appendages in 15 cases (39%); laparoscopic surgery in 27 cases (71%), open surgery in 11 cases (29%); none of the patients had renal cell carcinoma. (2) Histological features: atypical nuclear cells were distributed locally or diffusely, eosinophilic nucleoli and intranuclear inclusion bodies could be seen, glass like globules could be seen in the cytoplasm, nuclear division was 0-4/10 high power field (HPF), and antler like blood vessels and pulmonary edema-like changes could be seen in the stroma. Among 38 patients with FH deficiency uterine leiomyoma, FH was negative in 37 cases (97%), and positive in 1 case (3%); 2SC, desmin, p16, p53, CD10 and Ki-67 showed focal positive expression in 38 cases (100%), including 35 cases (92%) with Ki-67 index<10% and 3 cases (8%) with Ki-67 index ≥10%. (3) Follow-up: 4 cases (11%) recurred, and there was no death. There were significant differences in age, family history, distribution of atypical nuclei and mitosis number between recurrent group and non-recurrent group (all P<0.05). Conclusions: FH deficiency uterine leiomyoma is a rare tumor, which needs pathological examination,immunohistochemical examination and clinical history. Patients younger than 43 years old, with family history, histologically atypical diffuse nuclear distribution and mitotic number ≥3/10 HPF should be alert to the risk of recurrence.
Asunto(s)
Fumarato Hidratasa , Leiomioma , Neoplasias Uterinas , Adulto , Desmina/metabolismo , Femenino , Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Leiomioma/enzimología , Leiomioma/patología , Leiomioma/cirugía , Errores Innatos del Metabolismo/enzimología , Persona de Mediana Edad , Hipotonía Muscular/enzimología , Trastornos Psicomotores/enzimología , Estudios Retrospectivos , Proteína p53 Supresora de Tumor , Neoplasias Uterinas/diagnósticoRESUMEN
BACKGROUND: Trimethylaminuria (TMAU) is a rare genetic disease characterized by the accumulation of trimethylamine (TMA) and its subsequent excretion trough main body fluids, determining the characteristic fish odour in affected patients. We realized an experimental study to investigate the role of several coding variants in the causative gene FMO3, that were only considered as polymorphic or benign, even if the available literature on them did not functionally explain their ineffectiveness on the encoded enzyme. METHODS: Mutational analysis of 26 TMAU patients was realized by Sanger sequencing. Detected variants were, subsequently, deeply statistically and in silico characterized to determine their possible effects on the enzyme activity. To achieve this goal, a docking prediction for TMA/FMO3 and an unbinding pathway study were performed. Finally, a TMAO/TMA urine quantification by 1H-NMR spectroscopy was performed to support modelling results. RESULTS: The FMO3 screening of all patients highlighted the presence of 17 variants distributed in 26 different haplotypes. Both non-sense and missense considered variants might impair the enzymatic kinetics of FMO3, probably reducing the interaction time between the protein catalytic site and TMA, or losing the wild-type binding site. CONCLUSIONS: Even if further functional assays will confirm our predictive results, considering the possible role of FMO3 variants with still uncertain effects, might be a relevant step towards the detection of novel scenarios in TMAU etiopathogenesis.
Asunto(s)
Errores Innatos del Metabolismo , Metilaminas/orina , Modelos Moleculares , Mutación , Oxigenasas , Adulto , Femenino , Humanos , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/orina , Resonancia Magnética Nuclear Biomolecular , Oxigenasas/química , Oxigenasas/genética , Oxigenasas/metabolismoRESUMEN
OBJECTIVES: Aromatase deficiency is a rare autosomal recessive disease that results in the absence of aromatase. In females it presents with ambiguous genitalia and lack of secondary sexual characteristics during puberty. Aromatase deficiency is not attributed to any specific population, but it is more commonly seen in consanguineous parents. Herein, we report the first Old Order Mennonite family with that diagnosis. CASE PRESENTATION: Our proband is an Old Order Mennonite female born with ambiguous genitalia who was identified to carry novel homozygous variant in the CYP19A1 gene c.1304G>A (p. Arg435His). Her older brother was later confirmed with the same genetic diagnosis. CONCLUSIONS: Recognizing the cultural sensitivity, unrecognized affected cases, and late presentation of males affected with aromatase deficiency, this condition may be more prevalent than believed in that population.
Asunto(s)
Trastornos del Desarrollo Sexual 46, XX/diagnóstico , Aromatasa/deficiencia , Ginecomastia/diagnóstico , Infertilidad Masculina/diagnóstico , Errores Innatos del Metabolismo/diagnóstico , Mutación , Trastornos del Desarrollo Sexual 46, XX/enzimología , Trastornos del Desarrollo Sexual 46, XX/genética , Adulto , Aromatasa/genética , Femenino , Ginecomastia/enzimología , Ginecomastia/genética , Homocigoto , Humanos , Recién Nacido , Infertilidad Masculina/enzimología , Infertilidad Masculina/genética , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , VirilismoRESUMEN
Fumarate hydratase (FH), encoded by the FH gene, is an enzyme which catalyses the conversion of fumarate to L-malate as part of the tricarboxylic acid cycle. Biallelic germline mutations in FH result in fumaric aciduria, a metabolic disorder resulting in severe neurological and developmental abnormalities. Heterozygous germline mutations in FH result in hereditary leiomyomatosis and renal cell carcinoma, a cancer predisposition syndrome. FH deficiency has multiple oncogenic mechanisms including through promotion of aerobic glycolysis, induction of pseudohypoxia, post-translational protein modification and impairment of DNA damage repair by homologous recombination. FH-deficient neoplasms can present with characteristic morphological features that raise suspicion for FH alterations and also frequently demonstrate loss of FH immunoreactivity and intracellular accumulation of 2-succinocysteine, also detected by immunohistochemistry.
Asunto(s)
Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/metabolismo , Errores Innatos del Metabolismo/enzimología , Hipotonía Muscular/enzimología , Neoplasias/enzimología , Trastornos Psicomotores/enzimología , Animales , Fumarato Hidratasa/genética , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Leiomiomatosis/enzimología , Leiomiomatosis/genética , Leiomiomatosis/patología , Errores Innatos del Metabolismo/genética , Hipotonía Muscular/genética , Mutación , Neoplasias/genética , Neoplasias/patología , Síndromes Neoplásicos Hereditarios/enzimología , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/patología , Fenotipo , Trastornos Psicomotores/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Uterinas/enzimología , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologíaRESUMEN
Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.
Asunto(s)
Ácidos Grasos/metabolismo , Fibroblastos/enzimología , Errores Innatos del Metabolismo Lipídico/enzimología , Lipidómica , Metaboloma , Piel/enzimología , Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Estudios de Casos y Controles , Células Cultivadas , Ceramidas/metabolismo , Femenino , Humanos , Errores Innatos del Metabolismo Lipídico/genética , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/genética , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Oxidación-Reducción , Esfingolípidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Glycine conjugation is an important phase II reaction and represents a central detoxification pathway which is essential for the recycling of free coenzyme A. Only few sequence variants have been reported in the human GLYAT gene and only two studies have overexpressed the human protein in bacterial systems and partially characterized it. This has prompted us to study the wild-type enzyme and two sequence variants not only in the E. coli strain Origami 2(DE3), but also to overexpress GLYAT in HEK293 cells, a human-derived cell line. Following purification of the recombinant proteins from E. coli the wild-type GLYAT protein and sequence variants, p.(Gln61Leu) yielded decreased specific activity than the wild-type enzyme, while specific activity of p.(Asn156Ser) activity of the latter variant was somewhat increased. KM values were similar for the three forms of GLYAT overexpressed in bacteria and for the wild-type enzyme overexpressed in HEK293 cells. Localization studies demonstrated intramitochondrial localization of human wild-type GLYAT, conjugated with eGFP, in the HEK293 cells. As p.(Gln61Leu) does not only impair GLYAT activity in vitro, but is of high prevalence in a Caucasian Afrikaner cohort in South Africa, potential pharmacogenetic implications, warrant further studies of GLYAT.
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Aciltransferasas , Errores Innatos del Metabolismo , Mutación Missense , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Sustitución de Aminoácidos , Células HEK293 , Humanos , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genéticaRESUMEN
Phaeochromocytomas and paragangliomas are rare neuroendocrine tumours. So far, over 20 causative genes have been identified, of which the most frequent and strongest indicator for malignancies are mutations in succinate dehydrogenase subunit B. No curative therapy is available for patients with metastases resulting in poor prognosis. Therapy development has been hindered by lack of suitable model systems. The succinate dehydrogenase complex is located in the inner membrane of the mitochondria and plays a crucial role in the oxidative phosphorylation chain and the tricarboxylic acid-cycle. Succinate dehydrogenase deficiency results in accumulation of the oncometabolite succinate inducing hypoxia inducible factor stabilization, deoxyribonucleic acid and histone methylation inhibition, and impaired production of adenosine triphosphate. It remains unknown which combination of pathways and/or triggers are decisive for metastases development. In this review, the role of mitochondria in malignant succinate dehydrogenase subunit B-associated phaeochromocytomas and paragangliomas and implications for mitochondria as therapeutic target are discussed.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/enzimología , Complejo II de Transporte de Electrones/deficiencia , Errores Innatos del Metabolismo/enzimología , Mitocondrias/enzimología , Enfermedades Mitocondriales/enzimología , Paraganglioma/enzimología , Feocromocitoma/enzimología , Succinato Deshidrogenasa/metabolismo , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Neoplasias de las Glándulas Suprarrenales/patología , Animales , Complejo II de Transporte de Electrones/genética , Complejo II de Transporte de Electrones/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Mutación , Paraganglioma/genética , Paraganglioma/metabolismo , Paraganglioma/patología , Feocromocitoma/genética , Feocromocitoma/metabolismo , Feocromocitoma/patología , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/genéticaRESUMEN
Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (~50% and ~10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.
Asunto(s)
Membrana Celular/enzimología , Metilaminas/metabolismo , Oxígeno/metabolismo , Oxigenasas/genética , Polimorfismo de Nucleótido Simple/genética , Argentina , Niño , Femenino , Humanos , Errores Innatos del Metabolismo/enzimología , Metilaminas/orina , Oxigenasas/metabolismoRESUMEN
BACKGROUND: Dihidropyrimidinase (DHP) deficiency is an inherited inborn error of pyrimidine metabolism with a variable clinical presentation and even asymptomatic subjects. Dihydropyrimidinase is encoded by the DPYS gene, thus pathogenic mutations in this gene can cause DHP deficiency. To date, several variations in the DPYS gene have been reported but only 23 of them have been confirmed to be pathogenic. Therefore, the biochemical, clinical and genetic aspects of this disease are still unclear. CASE PRESENTATION: Here, we report a 22-year-old woman with DHP deficiency. To identify the genetic cause of DHP deficiency in this patient, Whole Exome Sequencing (WES) was performed, which revealed a novel homozygote stop gain mutation (NM_001385: Exon 9, c.1501 A > T, p.K501X) in the DPYS gene. Sanger sequencing was carried out on proband and other family members in order to confirm the identified mutation. According to the homozygote genotype of the patient and heterozygote genotype of her parents, the autosomal recessive pattern of inheritance was confirmed. In addition, bioinformatics analysis of the identified variant using Mutation Taster and T-Coffee Multiple Sequence Alignment showed the pathogenicity of mutation. Moreover, mRNA expression level of DPYS gene in the proband's liver biopsy showed about 6-fold reduction compared to control, which strongly suggested the pathogenicity of the identified mutation. CONCLUSIONS: This study identified a novel pathogenic stop gain mutation in DPYS gene in a DHP deficient patient. Our findings can improve the knowledge about the genetic basis of the disease and also provide information for accurate genetic counseling for the families at risk of these types of disorders.
Asunto(s)
Amidohidrolasas/genética , Codón sin Sentido/genética , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Mutación/genética , Amidohidrolasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Linaje , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
Carnitine palmitoyltransferase (CPT) catalyzes the transfer of long- and medium-chain fatty acids from cytoplasm into mitochondria, where oxidation of fatty acids takes place. Deficiency of CPT enzyme is associated with rare diseases of fatty acid metabolism. CPT is present in two subforms: CPT I at the outer mitochondrial membrane and carnitine palmitoyltransferase II (CPT II) inside the mitochondria. Deficiency of CPT II results in the most common inherited disorder of long-chain fatty acid oxidation affecting skeletal muscle. There is a lethal neonatal form, a severe infantile hepato-cardio-muscular form, and a rather mild myopathic form characterized by exercise-induced myalgia, weakness, and myoglobinuria. Total CPT activity (CPT I + CPT II) in muscles of CPT II-deficient patients is generally normal. Nevertheless, in some patients, not detectable to reduced total activities are also reported. CPT II protein is also shown in normal concentration in patients with normal CPT enzymatic activity. However, residual CPT II shows abnormal inhibition sensitivity towards malonyl-CoA, Triton X-100 and fatty acid metabolites in patients. Genetic studies have identified a common p.Ser113Leu mutation in the muscle form along with around 100 different rare mutations. The biochemical consequences of these mutations have been controversial. Hypotheses include lack of enzymatically active protein, partial enzyme deficiency and abnormally regulated enzyme. The recombinant enzyme experiments that we recently conducted have shown that CPT II enzyme is extremely thermoliable and is abnormally inhibited by different emulsifiers and detergents such as malonyl-CoA, palmitoyl-CoA, palmitoylcarnitine, Tween 20 and Triton X-100. Here, we present a conceptual overview on CPT II deficiency based on our own findings and on results from other studies addressing clinical, biochemical, histological, immunohistological and genetic aspects, as well as recent advancements in diagnosis and therapeutic strategies in this disorder.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Errores Innatos del Metabolismo/enzimología , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Carnitina/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Malonil Coenzima A/metabolismo , Malonil Coenzima A/farmacología , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/fisiopatología , Mitocondrias/patología , Músculo Esquelético/citología , Músculo Esquelético/patología , Oxidación-ReducciónRESUMEN
BACKGROUND: Primary neonatal hypocholinesterase is rare; its genetic pattern and mutation still need to be further studied. METHODS: The patient and his parents are studied using next-generation sequencing technology. RESULTS: A boy one day after birth is admitted to the Neonatal Intensive Care Unit at our hospital after experiencing intermittent vomiting for 12 hours. The patient's serum cholinesterase level (113 - 283 U/L) is lower than normal value (4,000 - 12,600 U/L). Many factors of low serum cholinesterase are excluded. We highly suspect that it may be related to congenital factors. Molecular genetic test results show that the patient carried the BCHE gene (NM_000055.2) and has homozygous frameshift mutations at exon 2 c.401dupA (p.Asn134fs) of chromosome 3q26. It is a pathogenicity mutation. This locus mutation belongs to a novel pathogenic mutation. As a result of this mutation, the 134th amino acid Asn began to frameshift and the translation is terminated early. It can cause the Encoding of protein to truncate and lose its normal function. His parents' serum cholinesterase levels (father: 5,135 U/L; mother: 4,367 U/L) are in the normal value range, but his parents carried a heterozygous BCHE gene. CONCLUSIONS: This study suggests that gene sequence detection should be carried out early in hypocholinesterase of nknown cause in neonates. This study can not only improve understanding of the etiology and pathological mechanism of hypocholinesterase, but also it can enlarge the hypocholinesterase gene mutation spectrum.
Asunto(s)
Butirilcolinesterasa/genética , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad/genética , Errores Innatos del Metabolismo/genética , Butirilcolinesterasa/sangre , Butirilcolinesterasa/deficiencia , Salud de la Familia , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Recién Nacido , Masculino , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/enzimologíaRESUMEN
Methylmalonic acidemia/aciduria (MMA) is a genetically heterogeneous group of inherited metabolic disorders biochemically characterized by the accumulation of methylmalonic acid. Isolated MMA is primarily caused by the deficiency of methylmalonyl-CoA mutase (MMA mut; EC 5.4.99.2). A systematic literature review and a meta-analysis were undertaken to assess and compile published epidemiological data on MMA with a focus on the MMA mut subtype (OMIM #251000). Of the 1114 identified records, 227 papers were assessed for eligibility in full text, 48 articles reported on disease epidemiology, and 39 articles were included into the quantitative synthesis. Implementation of newborn screening in various countries has allowed for the estimation of birth prevalence of MMA and its isolated form. Meta-analysis pooled point estimates of MMA (all types) detection rates were 0.79, 1.12, 1.22 and 6.04 per 100,000 newborns in Asia-Pacific, Europe, North America and the Middle East and North Africa (MENA) regions, respectively. The detection rate of isolated MMA was < 1 per 100,000 newborns in all regions with the exception of MENA where it approached 6 per 100,000 newborns. Few studies published data on the epidemiology of MMA mut, therefore no meta-analysis could have been performed on this subtype. Most of the identified papers reported birth prevalence estimates below 1 per 100,000 newborns for MMA mut. The systematic literature review clearly demonstrates that MMA and its subtypes are ultra-rare disorders.
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Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Metilmalonil-CoA Mutasa/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Femenino , Humanos , Recién Nacido , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/epidemiología , Errores Innatos del Metabolismo/genética , Metilmalonil-CoA Mutasa/deficiencia , Tamizaje NeonatalRESUMEN
Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.
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Cardiomiopatía Dilatada/enzimología , Cardiomiopatía Dilatada/genética , Catarata/enzimología , Catarata/genética , Hipogonadismo/enzimología , Hipogonadismo/genética , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Pirofosfatasas/deficiencia , Animales , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Homocigoto , Humanos , Inosina/metabolismo , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/enzimología , Mutación , Linaje , Pirofosfatasas/genética , ARN/genética , ARN/metabolismo , Secuenciación del ExomaRESUMEN
Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.
Asunto(s)
Genes Recesivos/genética , Lisina/análogos & derivados , Mutación , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Alelos , Secuencia de Aminoácidos , Niño , Preescolar , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Femenino , Haplotipos , Humanos , Lisina/biosíntesis , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Linaje , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Convulsiones/enzimología , Convulsiones/genética , Adulto Joven , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder. Its clinical phenotypes are classified into the muscle, severe infantile, and lethal neonatal forms. Among Caucasians, the muscle form predominates, and the c.338C > T (p.S113L) variant is detected in most cases, whereas among the Japanese, c.1148T > A (p.F383Y) is the variant allele occurring with the highest frequency and can apparently cause symptoms of the severe infantile form. Newborn screening (NBS) for this potentially fatal disease has not been established. We encountered an infantile case of CPT II deficiency not detected in NBS using C16 and C18:1 concentrations as indices, and therefore we adopted the (C16 + C18:1)/C2 ratio as an alternative primary index. As a result, the disease was diagnosed in nine of 31 NBS-positive subjects. The values for (C16 + C18:1)/C2 in the affected newborns partly overlapped with those in unaffected ones. Among several other indices proposed previously, C14/C3 has emerged as a more promising index. Based on these findings, nationwide NBS for CPT II deficiency using both (C16 + C18:1)/C2 and C14/C3 as indices was officially approved and started in April 2018. We diagnosed the disease in four young children presenting with symptoms of the muscle form, whose values for the new indices were not elevated. Although it is still difficult to detect all cases of the muscle form of CPT II deficiency in NBS, our system is expected to save many affected children in Japan with the severe infantile form predominating.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Errores Innatos del Metabolismo/diagnóstico , Tamizaje Neonatal/métodos , Humanos , Recién Nacido , Errores Innatos del Metabolismo/enzimología , PronósticoRESUMEN
1. Flavin-containing monooxygenase 3 (FMO3) in humans is polymorphic in several ethnic groups, including Caucasians, Africans and Asians. Some FMO3 variants are associated with a disorder trimethylaminuria. 2. In the current study, we used the results from urinary phenotyping assays to identify 63 subjects with <85% FMO3 metabolic capacity with respect to trimethylamine N-oxidation among 787 Japanese volunteers with self-reported trimethylaminuria. The 63 subjects with reduced FMO3 activity were screened and investigated in detail to identify novel FMO3 variants. 3. Homozygous or heterozygous individuals for new single nucleotide substitution variants/haplotypes p.(Pro282Leu), p.[(Glu158Lys; Glu308Gly; Thr329Ala)], p.[(Glu158Lys; Glu308Gly; Asp429Gly)], p.[(Val257Met; Leu473Pro)], p.[(Glu158Lys; Glu308Gly; Ile441Thr)], and p.[(Arg205Cys; Gly503Arg)] were identified in six proband subjects and their family members after pedigree analyses. 4. These variant FMO3 proteins recombinantly expressed in Escherichia coli membranes exhibited decreased N-oxygenation activities toward trimethylamine (Vmax/Km < 40% that of the wild-type). 5. Although the allele frequencies of the six new variants and/or haplotypes were low, the present results indicated that individuals homozygous or heterozygous for any of these novel missense FMO3 variants or known nonsense mutations such as p.(Cys197Ter) or p.(Arg205Cys) highly found in this self-reported Japanese trimethylaminuria cohort may have reduced FMO3 activity with respect to the N-oxygenation of trimethylamine.
Asunto(s)
Alelos , Codón sin Sentido , Frecuencia de los Genes , Haplotipos , Errores Innatos del Metabolismo/genética , Metilaminas/orina , Mutación Missense , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Pueblo Asiatico , Preescolar , Femenino , Humanos , Lactante , Japón , Masculino , Errores Innatos del Metabolismo/enzimología , Metilaminas/metabolismo , Persona de Mediana Edad , OxigenasasRESUMEN
OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.
Asunto(s)
Tolerancia al Ejercicio/fisiología , Enfermedades Genéticas Ligadas al Cromosoma X/enzimología , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/fisiopatología , Fosfoglicerato Quinasa/deficiencia , Fosfoglicerato Quinasa/metabolismo , Ergometría , Prueba de Esfuerzo , Enfermedades Genéticas Ligadas al Cromosoma X/complicaciones , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Humanos , Discapacidad Intelectual/sangre , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/fisiopatología , Ácido Láctico/sangre , Masculino , Errores Innatos del Metabolismo/complicaciones , Errores Innatos del Metabolismo/diagnóstico , Músculo Esquelético/metabolismo , Enfermedades Musculares/sangre , Enfermedades Musculares/complicaciones , Enfermedades Musculares/enzimología , Enfermedades Musculares/fisiopatología , Fenotipo , Fosfoglicerato Quinasa/sangreRESUMEN
The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in â¼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.
Asunto(s)
Colorantes Fluorescentes/química , Ácido Hipocloroso/análisis , Neutrófilos/enzimología , Fagocitosis , Fagosomas/metabolismo , Rodaminas/química , Bioensayo , Colorantes Fluorescentes/metabolismo , Humanos , Ácido Hipocloroso/inmunología , Ácido Hipocloroso/metabolismo , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/inmunología , Errores Innatos del Metabolismo/patología , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Neutrófilos/inmunología , Neutrófilos/patología , Proteínas Opsoninas/química , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasa/inmunología , Fagosomas/inmunología , Fagosomas/ultraestructura , Cultivo Primario de Células , Rodaminas/metabolismo , Espectrometría de Fluorescencia , Staphylococcus aureus/inmunología , Zimosan/químicaRESUMEN
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mitochondrial HMG-CoA synthase deficiency or mHS deficiency, OMIM #605911) is an inborn error of metabolism that affects ketone body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycemia and dicarboxylic aciduria. The diagnosis is difficult due to the relatively unspecific clinical and biochemical presentation, and fewer than 30 patients have been described. This work describes three new patients with mHS deficiency and two missense mutations c.334C>T (p.R112W) and c.430G>T (p.V144L) previously not reported. We developed a new method to express and measure the activity of the enzyme and in this work the study is extended to ten new missense variants including those of our patients. Enzymatic assays showed that three of the mutant proteins retained some but seven completely lacked activity. The identification of a patient homozygous for a mutation that retains 70% of enzyme activity opens the door to a new interpretation of the disease by demonstrating that a modest impairment of enzyme function can actually produce symptoms. This is also the first study employing molecular dynamics modelling of the enzyme mutations. We show that the correct maintenance of the dimerization surface is crucial for retaining the structure of the active center and therefore the activity of the enzyme.
Asunto(s)
Hidroximetilglutaril-CoA Sintasa/deficiencia , Errores Innatos del Metabolismo , Proteínas Mitocondriales/deficiencia , Mutación Missense , Multimerización de Proteína , Sustitución de Aminoácidos , Preescolar , Femenino , Humanos , Lactante , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patologíaRESUMEN
Several inherited metabolic disorders are associated with an accumulation of reactive acyl-CoA metabolites that can non-enzymatically react with lysine residues to modify proteins. While the role of acetylation is well-studied, the pathophysiological relevance of more recently discovered acyl modifications, including those found in inherited metabolic disorders, warrants further investigation. We recently showed that sirtuin 4 (SIRT4) removes glutaryl, 3-hydroxy-3-methylglutaryl, 3-methylglutaryl, and 3-methylglutaconyl modifications from lysine residues. Thus, we used SIRT4 knockout mice, which can accumulate these novel post-translational modifications, as a model to investigate their physiological relevance. Since SIRT4 is localized to mitochondria and previous reports have shown SIRT4 influences metabolism, we thoroughly characterized glucose and lipid metabolism in male and female SIRT4KO mice across different genetic backgrounds. While only minor perturbations in overall lipid metabolism were observed, we found SIRT4KO mice consistently had elevated glucose- and leucine-stimulated insulin levels in vivo and developed accelerated age-induced insulin resistance. Importantly, elevated leucine-stimulated insulin levels in SIRT4KO mice were dependent upon genetic background since SIRT4KO mice on a C57BL/6NJ genetic background had elevated leucine-stimulated insulin levels but not SIRT4KO mice on the C57BL/6J background. Taken together, the data suggest that accumulation of acyl modifications on proteins in inherited metabolic disorders may contribute to the overall metabolic dysfunction seen in these patients.