RESUMEN
X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase, ALAS2, which encodes the first enzyme in heme biosynthesis. A related sideroblastic anemia is due to mutations in SLC25A38, which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited studies on the pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knock-in mouse models that recapitulate the main features of XLSA and XLPP, and, using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP porphyria, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ ï¢-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.
RESUMEN
Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.
Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación de Línea Germinal , Síndromes Mielodisplásicos/genética , Neutropenia/congénito , Septinas/genética , Línea Celular , Células Cultivadas , Enfermedades Genéticas Ligadas al Cromosoma X/complicaciones , Humanos , Recién Nacido , Masculino , Síndromes Mielodisplásicos/complicaciones , Neutropenia/complicaciones , Neutropenia/genética , TetraploidíaAsunto(s)
5-Aminolevulinato Sintetasa , Anemia Sideroblástica , Enfermedades Genéticas Ligadas al Cromosoma X , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Anemia Sideroblástica/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Mutación , Empalme del ARN/genéticaRESUMEN
The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.
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Anemia Sideroblástica/congénito , Genotipo , Proteínas de Transporte de Membrana Mitocondrial/genética , Mutación , Fenotipo , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
BACKGROUND: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. METHODS: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. FINDINGS: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. CONCLUSIONS: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. FUNDING: Medical Research Council, UK.
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Deficiencias de Hierro , Trastornos del Metabolismo del Hierro , Animales , Hepcidinas/genética , Humanos , Inmunidad Humoral , Hierro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Porcinos , VacunaciónRESUMEN
The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.
Asunto(s)
Anemia Sideroblástica/genética , Chaperonas Moleculares/genética , Mutación , Adolescente , Anemia Sideroblástica/congénito , Anemia Sideroblástica/metabolismo , Animales , Niño , Análisis Mutacional de ADN , Femenino , Mutación del Sistema de Lectura , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Hierro-Azufre/deficiencia , Proteínas Hierro-Azufre/genética , Células K562 , Masculino , Ratones , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Linaje , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Adulto Joven , Pez CebraRESUMEN
SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.
Asunto(s)
Linfocitos B/inmunología , Senescencia Celular , Eliminación de Gen , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Neoplasias Experimentales/genética , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Receptores de Antígenos de Linfocitos B/inmunología , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Empalme Alternativo , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Senescencia Celular/efectos de los fármacos , Daño del ADN , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Fenotipo , Fosfoproteínas/metabolismo , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Factores de Empalme de ARN/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
YARS2 variants have previously been described in patients with myopathy, lactic acidosis and sideroblastic anemia 2 (MLASA2). YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase, which is responsible for conjugating tyrosine to its cognate mt-tRNA for mitochondrial protein synthesis. Here we describe 14 individuals from 11 families presenting with sideroblastic anemia and YARS2 variants that we identified using a sideroblastic anemia gene panel or exome sequencing. The phenotype of these patients ranged from MLASA to isolated congenital sideroblastic anemia. As in previous cases, inter- and intra-familial phenotypic variability was observed, however, this report includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of five novel missense variants showed that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency =0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, including severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype.
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Acidosis Láctica/genética , Anemia Sideroblástica/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación de Línea Germinal , Síndrome MELAS/genética , Proteínas Mitocondriales/genética , Tirosina-ARNt Ligasa/genética , Acidosis Láctica/enzimología , Adolescente , Anemia Sideroblástica/enzimología , Femenino , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/enzimología , Humanos , Lactante , Síndrome MELAS/enzimología , Masculino , Persona de Mediana Edad , Mutación Missense , Adulto JovenAsunto(s)
Anemia Sideroblástica/genética , ADN Mitocondrial/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Mutación Missense , Adolescente , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , Niño , Análisis Mutacional de ADN/métodos , Femenino , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Lactante , Masculino , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mutación Puntual , Recurrencia , SíndromeRESUMEN
Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.
Asunto(s)
Mutación con Pérdida de Función , Mielofibrosis Primaria/congénito , Receptores Inmunológicos/genética , Trombocitopenia/congénito , Adolescente , Adulto , Animales , Plaquetas/metabolismo , Plaquetas/patología , Niño , Preescolar , Femenino , Técnicas de Sustitución del Gen , Humanos , Lactante , Masculino , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Trombocitopenia/genética , Trombocitopenia/patología , Adulto JovenAsunto(s)
Anemia de Diamond-Blackfan/genética , Enfermedades de la Médula Ósea/genética , Pruebas Genéticas , Lipomatosis/etiología , Anemia de Diamond-Blackfan/complicaciones , Anemia de Diamond-Blackfan/diagnóstico , Enfermedades de la Médula Ósea/diagnóstico , Humanos , Lipomatosis/genética , Enfermedades PancreáticasRESUMEN
During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.
Asunto(s)
Células Eritroides/citología , Eritropoyesis/fisiología , Proteínas Ribosómicas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Anemia/genética , Anemia Hipocrómica/genética , Animales , Eritrocitos/citología , Eritrocitos/enzimología , Células Eritroides/enzimología , Eritropoyesis/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica , Reticulocitos/citología , Reticulocitos/enzimología , Ribosomas/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Symptomatic ß-thalassemia is one of the globally most common inherited disorders. The initial clinical presentation is variable. Although common hematological analyses are typically sufficient to diagnose the disease, sometimes the diagnosis can be more challenging. We describe a series of patients with ß-thalassemia whose diagnosis was delayed, required bone marrow examination in one affected member of each family, and revealed ringed sideroblasts, highlighting the association of this morphological finding with these disorders. Thus, in the absence of characteristic congenital sideroblastic mutations or causes of acquired sideroblastic anemia, the presence of ringed sideroblasts should raise the suspicion of ß-thalassemia.
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Anemia Sideroblástica/patología , Células de la Médula Ósea/patología , Eritroblastos/patología , Talasemia beta/patología , Adolescente , Adulto , Anemia Sideroblástica/diagnóstico , Células de la Médula Ósea/citología , Examen de la Médula Ósea , Niño , Eritroblastos/citología , Eritrocitos Anormales , Femenino , Enfermedades Hematológicas/complicaciones , Humanos , Lactante , Masculino , Talasemia beta/diagnósticoRESUMEN
More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.
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Eritropoyesis/fisiología , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Empalmosomas/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Eritropoyesis/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Transgénicos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Fosfoproteínas/deficiencia , Fosfoproteínas/metabolismo , Mutación Puntual , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Empalme del ARN , Factores de Empalme de ARN/deficiencia , Factores de Empalme de ARN/metabolismoRESUMEN
The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.
Asunto(s)
Anemia Sideroblástica/genética , Secuencia de Bases , Cromosomas Humanos X/genética , Complejo I de Transporte de Electrón/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Eliminación de Secuencia , Adolescente , Adulto , Anciano , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , Niño , Preescolar , Cromosomas Humanos X/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Humanos , Células K562 , Masculino , Persona de Mediana EdadRESUMEN
Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.
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Hidroliasas/deficiencia , Síndrome MELAS/patología , Músculos/patología , Músculos/fisiología , Animales , Modelos Animales de Enfermedad , Histocitoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de TransmisiónRESUMEN
Pathogenic variants in mitochondrial aminoacyl-tRNA synthetases result in a broad range of mitochondrial respiratory chain disorders despite their shared role in mitochondrial protein synthesis. LARS2 encodes the mitochondrial leucyl-tRNA synthetase, which attaches leucine to its cognate tRNA. Sequence variants in LARS2 have previously been associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss (OMIM #615300). In this study, we report variants in LARS2 that are associated with a severe multisystem metabolic disorder. The proband was born prematurely with severe lactic acidosis, hydrops, and sideroblastic anemia. She had multisystem complications with hyaline membrane disease, impaired cardiac function, a coagulopathy, pulmonary hypertension, and progressive renal disease and succumbed at 5 days of age. Whole exome sequencing of patient DNA revealed compound heterozygous variants in LARS2 (c.1289C>T; p.Ala430Val and c.1565C>A; p.Thr522Asn). The c.1565C>A (p.Thr522Asn) LARS2 variant has previously been associated with Perrault syndrome and both identified variants are predicted to be damaging (SIFT, PolyPhen). Muscle and liver samples from the proband did not display marked mitochondrial respiratory chain enzyme deficiency. Immunoblotting of patient muscle and liver showed LARS2 levels were reduced in liver and complex I protein levels were reduced in patient muscle and liver. Aminoacylation assays revealed p.Ala430Val LARS2 had an 18-fold loss of catalytic efficiency and p.Thr522Asn a 9-fold loss compared to wild-type LARS2. We suggest that the identified LARS2 variants are responsible for the severe multisystem clinical phenotype seen in this baby and that mutations in LARS2 can result in variable phenotypes.
RESUMEN
The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.
Asunto(s)
Anemia Sideroblástica/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Mitocondriales/genética , Adulto , Anciano , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.