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1.
Int J Mol Sci ; 24(12)2023 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-37373084

RESUMEN

Congenital dyserythropoietic anemia type II (CDA II) is an inherited autosomal recessive blood disorder which belongs to the wide group of ineffective erythropoiesis conditions. It is characterized by mild to severe normocytic anemia, jaundice, and splenomegaly owing to the hemolytic component. This often leads to liver iron overload and gallstones. CDA II is caused by biallelic mutations in the SEC23B gene. In this study, we report 9 new CDA II cases and identify 16 pathogenic variants, 6 of which are novel. The newly reported variants in SEC23B include three missenses (p.Thr445Arg, p.Tyr579Cys, and p.Arg701His), one frameshift (p.Asp693GlyfsTer2), and two splicing variants (c.1512-2A>G, and the complex intronic variant c.1512-3delinsTT linked to c.1512-16_1512-7delACTCTGGAAT in the same allele). Computational analyses of the missense variants indicated a loss of key residue interactions within the beta sheet and the helical and gelsolin domains, respectively. Analysis of SEC23B protein levels done in patient-derived lymphoblastoid cell lines (LCLs) showed a significant decrease in SEC23B protein expression, in the absence of SEC23A compensation. Reduced SEC23B mRNA expression was only detected in two probands carrying nonsense and frameshift variants; the remaining patients showed either higher gene expression levels or no expression changes at all. The skipping of exons 13 and 14 in the newly reported complex variant c.1512-3delinsTT/c.1512-16_1512-7delACTCTGGAAT results in a shorter protein isoform, as assessed by RT-PCR followed by Sanger sequencing. In this work, we summarize a comprehensive spectrum of SEC23B variants, describe nine new CDA II cases accounting for six previously unreported variants, and discuss innovative therapeutic approaches for CDA II.


Asunto(s)
Anemia Diseritropoyética Congénita , Humanos , Anemia Diseritropoyética Congénita/genética , Anemia Diseritropoyética Congénita/metabolismo , Mutación , Mutación Missense , Exones , Alelos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
2.
Int J Mol Sci ; 23(8)2022 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-35457224

RESUMEN

Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron.


Asunto(s)
Anemia Hipocrómica , Anemia , Sobrecarga de Hierro , Anemia/genética , Anemia Hipocrómica/genética , Animales , Humanos , Hierro/metabolismo , Sobrecarga de Hierro/metabolismo , Mamíferos/metabolismo , Mutación
3.
Int J Mol Sci ; 22(11)2021 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-34064225

RESUMEN

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a rare disease characterized by high serum ferritin levels, congenital bilateral cataracts, and the absence of tissue iron overload. This disorder is produced by mutations in the iron responsive element (IRE) located in the 5' untranslated regions (UTR) of the light ferritin (FTL) gene. A canonical IRE is a mRNA structure that interacts with the iron regulatory proteins (IRP1 and IRP2) to post-transcriptionally regulate the expression of proteins related to iron metabolism. Ferritin L and H are the proteins responsible for iron storage and intracellular distribution. Mutations in the FTL IRE abrogate the interaction of FTL mRNA with the IRPs, and de-repress the expression of FTL protein. Subsequently, there is an overproduction of ferritin that accumulates in serum (hyperferritinemia) and excess ferritin precipitates in the lens, producing cataracts. To illustrate this disease, we report two new families affected with hereditary hyperferritinemia-cataract syndrome with previous known mutations. In the diagnosis of congenital bilateral cataracts, HHCS should be taken into consideration and, therefore, it is important to test serum ferritin levels in patients with cataracts.


Asunto(s)
Catarata/congénito , Ferritinas/genética , Trastornos del Metabolismo del Hierro/congénito , Adulto , Catarata/genética , Niño , Femenino , Humanos , Hierro/metabolismo , Trastornos del Metabolismo del Hierro/genética , Proteínas Reguladoras del Hierro/genética , Mutación/genética
4.
Mol Genet Metab ; 131(3): 349-357, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33153867

RESUMEN

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.


Asunto(s)
Predisposición Genética a la Enfermedad , Enfermedades Mitocondriales/genética , NADH Deshidrogenasa/genética , Fosforilación Oxidativa , Niño , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patología , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/patología , Hermanos , Secuenciación del Exoma
5.
Hum Mutat ; 40(10): 1700-1712, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31058414

RESUMEN

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.


Asunto(s)
Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Biomarcadores , Transporte de Electrón , Metabolismo Energético , Fibroblastos/metabolismo , Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Fenotipo , Transporte de Proteínas , Espasmos Infantiles/diagnóstico , Espasmos Infantiles/genética , Secuenciación del Exoma
8.
Hum Mol Genet ; 23(7): 1907-15, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24256811

RESUMEN

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.


Asunto(s)
Aciltransferasas/genética , Lipoilación/genética , Oxo-Ácido-Liasas/genética , Acidosis Láctica/genética , Acidosis Láctica/mortalidad , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Metabolismo Energético/genética , Femenino , Humanos , Recién Nacido , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación Missense , Complejo Piruvato Deshidrogenasa/genética , Ácido Tióctico/metabolismo
10.
J Inherit Metab Dis ; 39(6): 781-793, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27586888

RESUMEN

Lipoic acid (LA) is an essential cofactor required for the activity of five multienzymatic complexes that play a central role in the mitochondrial energy metabolism: four 2-oxoacid dehydrogenase complexes [pyruvate dehydrogenase (PDH), branched-chain ketoacid dehydrogenase (BCKDH), 2-ketoglutarate dehydrogenase (2-KGDH), and 2-oxoadipate dehydrogenase (2-OADH)] and the glycine cleavage system (GCS). LA is synthesized in a complex multistep process that requires appropriate function of the mitochondrial fatty acid synthesis (mtFASII) and the biogenesis of iron-sulphur (Fe-S) clusters. Defects in the biosynthesis of LA have been reported to be associated with multiple and severe defects of the mitochondrial energy metabolism. In recent years, disease-causing mutations in genes encoding for proteins involved in LA metabolism have been reported: NFU1, BOLA3, IBA57, LIAS, GLRX5, LIPT1, ISCA2, and LIPT2. These studies represented important progress in understanding the pathophysiology and molecular bases underlying these disorders. Here we review current knowledge regarding involvement of LA synthesis defects in human diseases with special emphasis on the diagnostic strategies for these disorders. The clinical and biochemical characteristics of patients with LA synthesis defects are discussed and a workup for the differential diagnosis proposed.


Asunto(s)
Metabolismo Energético/genética , Ácido Tióctico/biosíntesis , Ácido Tióctico/genética , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Aminoácido Oxidorreductasas/genética , Animales , Proteínas Portadoras/genética , Diagnóstico Diferencial , Humanos , Cetona Oxidorreductasas/genética , Mitocondrias/genética , Complejos Multienzimáticos/genética , Transferasas/genética
11.
Mol Genet Metab ; 110(1-2): 73-7, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23707711

RESUMEN

3-Methylglutaconic aciduria (3-MGA-uria) is a heterogeneous group of syndromes characterized by an increased excretion of 3-methylglutaconic and 3-methylglutaric acids. Five types of 3-MGA-uria (I to V) with different clinical presentations have been described. Causative mutations in TAZ, OPA3, DNAJC19, ATP12, ATP5E, and TMEM70 have been identified. After excluding the known genetic causes of 3-MGA-uria we used exome sequencing to investigate a patient with Leigh syndrome and 3-MGA-uria. We identified a homozygous variant in SERAC1 (c.202C>T; p.Arg68*), that generates a premature stop codon at position 68 of SERAC1 protein. Western blot analysis in patient's fibroblasts showed a complete absence of SERAC1 that was consistent with the prediction of a truncated protein and supports the pathogenic role of the mutation. During the course of this project a parallel study identified mutations in SERAC1 as the genetic cause of the disease in 15 patients with MEGDEL syndrome, which was compatible with the clinical and biochemical phenotypes of the patient described here. In addition, our patient developed microcephaly and optic atrophy, two features not previously reported in MEGDEL syndrome. We highlight the usefulness of exome sequencing to reveal the genetic bases of human rare diseases even if only one affected individual is available.


Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Exoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Errores Innatos del Metabolismo/genética , Adolescente , Adulto , Niño , Femenino , Humanos , Lactante , Masculino , Errores Innatos del Metabolismo/patología , Mutación
12.
J Inherit Metab Dis ; 36(5): 841-7, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23179554

RESUMEN

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1 were recently identified in patients with fatal encephalopathy displaying a biochemical phenotype consistent with defects in lipoic acid-dependent enzymatic activities and respiratory chain complexes. This discovery highlighted the molecular function of NFU1 as an iron-sulfur(Fe-S) cluster protein necessary for lipoic acid biosynthesis and respiratory chain complexes activities. To understand the pathophysiological mechanisms underlying this disease we have characterized the protein expression profiles of patients carrying NFU1 mutations. Fibroblasts from patients with the p.Gly208Cys mutation showed complete absence of protein-bound lipoic acid and decreased SDHA and SDHB subunits of complex II. In contrast, subunits of other respiratory chain complexes were normal. Protein lipoylation was also decreased in muscle and liver but not in other tissues available (brain, kidney, lung) from NFU1 patients. Although levels of the respiratory chain subunits were unaltered in tissues, BN-PAGE showed an assembly defect for complex II in muscle, consistent with the low enzymatic activity of this complex. This study provides new insights into the molecular bases of NFU1 disease as well as into the regulation of NFU1 protein in human tissues. We demonstrate a ubiquitous expression of NFU1 protein and further suggest that defects in lipoic acid biosynthesis and complex II are the main molecular signature of this disease, particularly in patients carrying the p.Gly208Cys mutation.


Asunto(s)
Proteínas Portadoras/genética , Enfermedades Mitocondriales/genética , Mutación Missense , Biosíntesis de Proteínas/genética , Proteínas/genética , Transcriptoma/genética , Transporte de Electrón/genética , Fibroblastos/metabolismo , Genotipo , Homocigoto , Humanos , Lactante , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Proteínas/metabolismo , Ácido Tióctico/genética , Ácido Tióctico/metabolismo
13.
Genes (Basel) ; 12(12)2021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34946929

RESUMEN

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A-p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D-p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.


Asunto(s)
Proteínas Ligadas a GPI/genética , Proteína de la Hemocromatosis/genética , Hemocromatosis/genética , Receptores de Transferrina/genética , Adulto , Codón sin Sentido/genética , Femenino , Proteínas Ligadas a GPI/metabolismo , Hemocromatosis/fisiopatología , Proteína de la Hemocromatosis/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Hierro/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Linaje , Receptores de Transferrina/metabolismo
14.
Mitochondrion ; 26: 72-80, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26688339

RESUMEN

Mutations in NFU1 were recently identified in patients with fatal encephalopathy. NFU1 is an iron-sulfur cluster protein necessary for the activity of the mitochondrial respiratory chain complexes I-II and the synthesis of lipoic acid. We report two NFU1 compound heterozygous individuals with normal complex I and lipoic acid-dependent enzymatic activities and low, but detectable, levels of lipoylated proteins. We demonstrated a leaky splicing regulation due to a splice site mutation (c.545+5G>A) that produces small amounts of wild type NFU1 mRNA that might result in enough protein to partially lipoylate and restore the activity of lipoic acid-dependent enzymes and the assembly and activity of complex I. These results allowed us to gain insights into the molecular basis underlying this disease and should be considered for the diagnosis of NFU1 patients.


Asunto(s)
Encefalopatías Metabólicas/diagnóstico por imagen , Encefalopatías Metabólicas/genética , Proteínas Portadoras/genética , Mutación , Sitios de Empalme de ARN , Empalme del ARN , Encefalopatías Metabólicas/metabolismo , Proteínas Portadoras/metabolismo , Femenino , Humanos , Lactante , Lipoilación/genética , Masculino , Radiografía
15.
Neurotherapeutics ; 12(4): 874-86, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26169295

RESUMEN

Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthrough-promoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.


Asunto(s)
Codón sin Sentido/genética , Fibroblastos/metabolismo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Esfingomielina Fosfodiesterasa/genética , Acetilglucosaminidasa/genética , Acetilglucosaminidasa/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Gentamicinas/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Mutación/genética , Oxadiazoles/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo
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