RESUMEN

Mitochondrial diseases are a group of heterogeneous disorders caused by dysfunctional mitochondria. Interestingly, a large proportion of mitochondrial diseases are caused by defects in genes associated with tRNA metabolism. We recently discovered that partial loss-of-function mutations in tRNA Nucleotidyl Transferase 1 (TRNT1), the nuclear gene encoding the CCA-adding enzyme essential for modifying both nuclear and mitochondrial tRNAs, causes a multisystemic and clinically heterogenous disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay; SIFD). However, it is not clear how mutations in a general and essential protein like TRNT1 cause disease with such clinically broad but unique symptomatology and tissue involvement. Using biochemical, cell, and mass spectrometry approaches, we demonstrate that TRNT1 deficiency is associated with sensitivity to oxidative stress, which is due to exacerbated, angiogenin-dependent cleavage of tRNAs. Furthermore, reduced levels of TRNT1 lead to phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha (eIF2α), increased reactive oxygen species (ROS) production, and changes in the abundance of distinct proteins. Our data suggest that the observed variable SIFD phenotypes are likely due to dysregulation of tRNA maturation and abundance, which in turn negatively affects the translation of distinct proteins.

Asunto(s)

Enfermedades Mitocondriales , Nucleotidiltransferasas , Humanos , Nucleotidiltransferasas/genética , Mitocondrias/genética , Mutación , Enfermedades Mitocondriales/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo

RESUMEN

The TRNT1 gene encodes tRNA nucleotidyltransferase 1, which catalyzes the addition of cytosine-cytosine-adenosine (CCA) to the ends of cytoplasmic and mitochondrial tRNAs. The most common clinical phenotype associated with TRNT1 is autosomal recessive sideroblastic anemia with B-cell immunodeficiency, periodic fever, and developmental delay (SIFD). Muscle involvement has rarely been reported in TRNT1-related disorders. Here we report a Chinese patient with incomplete SIFD and hyperCKemia, and explored the skeletal muscle pathological changes. The patient was a 3-year-old boy with sensorineural hearing loss, sideroblastic anemia, and developmental delay since infancy. At the age of 11 months, significantly increased levels of creatine kinase were noted, accompanied by mild muscle weakness. Whole-exome sequencing revealed compound heterozygous variants of the TRNT1 gene, c.443C > T (p.Ala148Val) and c.692C > G (p.Ala231Gly), in the patient. Western blot showed a decreased expression of TRNT1 and cytochrome c oxidase subunit IV (COX IV) in the skeletal muscle of the patient. Electron microscopy observation of skeletal muscle pathology revealed abnormal mitochondria of various sizes and shapes, supporting a diagnosis of mitochondrial myopathy. The present case indicates that in addition to the classic SIFD phenotype, TRNT1 mutations can cause mitochondrial myopathy, a rare clinical phenotype of TRNT1-related disorders.

RESUMEN

Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD) is a serious autosomal recessive syndrome caused by biallelic mutations in cytosine-cytosine-adenosine tRNA nucleotidyltransferase 1 (TRNT1). The main clinical features of SIFD are periodic fevers, developmental delay, sideroblastic or microcytic anemia, and immunodeficiency. Herein, we report three cases of SIFD with compound heterozygous variants of TRNT1. Patients 1 and 2 were siblings; they presented with periodic fevers, arthritis, low immunoglobulin A, bilateral cataracts, anemia, and neurodevelopmental and developmental delay. Patient 3 had severed clinical features with recurrent fever and infections. She was treated with infliximab and symptomatic treatments but without therapeutic effect. She received a stem cell transplantation of umbilical cord blood but died of posttransplant infection and posttransplant graft-vs.-host disease 17 days after transplantation. Finally, a literature review revealed that TRNT1 variants differed among SIFD patients. Our cases and literature review further expand existing knowledge on the phenotype and TRNT1 variations of SIFD and suggest that the early genomic diagnosis of TRNT1 is valuable to promptly assess bone marrow transplantation and tumor necrosis factor inhibitor treatments, which might be effective for the immunodeficiency and inflammation caused by SIFD.

RESUMEN

PURPOSE: Sideroblastic anemia, immunodeficiency, periodic fevers, and developmental delay (SIFD) is an autosomal recessive syndrome caused by biallelic loss-of-function variant of tRNA nucleotidyl transferase 1 (TRNT1). Efficacious methods to treat SIFD are lacking. We identified two novel mutations in TRNT1 and an efficacious and novel therapy for SIFD. METHODS: We retrospectively summarized the clinical records of two patients with SIFD from different families and reviewed all published cases of SIFD. RESULTS: Both patients had periodic fever, developmental delay, rash, microcytic anemia, and B cell lymphopenia with infections. Whole-exome sequencing of patient 1 identified a previously unreported homozygous mutation of TRNT1 (c.706G > A/p.Glu236Lys). He received intravenous immunoglobulin (IVIG) replacement and antibiotics, but died at 1 year of age. Gene testing in patient 2 revealed compound heterozygous mutations (c.907C > G/p.Gln303Glu and c.88A > G/p.Met30Val) in TRNT1, the former of which is a novel mutation. Periodic fever was controlled in the first month after adalimumab therapy and IVIG replacement, but recurred in the second month. Adalimumab was discontinued and replaced with thalidomide, which controlled the periodic fever and normalized inflammatory markers effectively. A retrospective analysis of reported cases revealed 69 patients with SIFD carrying 46 mutations. The male: female ratio was 1: 1, and the mean age of onset was 3.0 months. The most common clinical manifestations in patients with SIFD were microcytic anemia (82.6%), hypogammaglobulinemia/B cell lymphopenia (75.4%), periodic fever (66.7%), and developmental delay (60.0%). In addition to the typical tetralogy, SIFD features several heterogeneous symptoms involving multiple systems. Corticosteroids, immunosuppressants, and anakinra have low efficacy, whereas etanercept suppressed fever and improved anemia in reports. Bone-marrow transplantation can be used to treat severe SIFD, but carries a high risk. In total, 28.2% (20/71) of reported patients died, mainly because of multi-organ failure. Biallelic mutations located in exon1-intron5 lead to more severe phenotypes and higher mortality. Furthermore, 15.5% (11/71) patients survived to adulthood. The symptoms could be resolved spontaneously in five patients. CONCLUSIONS: Thalidomide can control the inflammation of SIFD and represents a new treatment for SIFD.

Asunto(s)

Anemia Sideroblástica , Síndromes de Inmunodeficiencia , Linfopenia , Masculino , Humanos , Femenino , Talidomida , Estudios Retrospectivos , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/tratamiento farmacológico , Anemia Sideroblástica/genética , Adalimumab , Inmunoglobulinas Intravenosas/uso terapéutico , Síndromes de Inmunodeficiencia/genética , Fiebre/etiología , Fiebre/genética , Mutación/genética , Nucleotidiltransferasas/genética

RESUMEN

Introduction: Sideroblastic anemia with B cell immunodeficiency, periodic fevers, and developmental delay (SIFD) syndrome is caused by biallelic TRNT1 mutations. TRNT1 gene encodes a CCA-adding tRNA nucleotidyl transferase enzyme. Mutant TRNT1 results in immunodeficiency and anemia in various degrees, accompanied by several organ involvement. Case Presentation: We present here a 15-month old male, demonstrated brittle hair, growth hormone deficiency, recurrent fever, arthritis, recurrent infections, mild anemia, and hypogammaglobulinemia. The patient did not respond to colchicine treatment, and after establishing SIFD diagnosis with the presence of homozygote c.948-949delAAinsGG (p.Lys317Glu) mutation in TRNT1 gene, we commenced monthly intravenous immunoglobulin replacement and weekly subcutaneous etanercept. A rapid resolution of fever episodes and infections occurred after initiation of this treatment regimen. Afterward, both anemia and growth parameters have improved during follow-up. Conclusion: SIFD syndrome should be considered in patients with recurrent fever, arthritis, and growth retardation even in the absence of severe anemia and prominent hypogammaglobulinemia.

Asunto(s)

Agammaglobulinemia , Amiloidosis , Anemia Sideroblástica , Artritis , Síndromes de Inmunodeficiencia , Agammaglobulinemia/complicaciones , Agammaglobulinemia/tratamiento farmacológico , Agammaglobulinemia/genética , Amiloidosis/complicaciones , Anemia Sideroblástica/complicaciones , Anemia Sideroblástica/tratamiento farmacológico , Anemia Sideroblástica/genética , Artritis/complicaciones , Niño , Colchicina , Etanercept/uso terapéutico , Fiebre/complicaciones , Fiebre/tratamiento farmacológico , Hormona del Crecimiento , Humanos , Inmunoglobulinas Intravenosas , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/genética , Lactante , Masculino , Nucleotidiltransferasas/genética , ARN de Transferencia

RESUMEN

Sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD; MIM #616084) is an autosomal recessive disorder of mitochondrial and cytosolic tRNA processing caused by pathogenic, biallelic variants in TRNT1. Other features of this disorder include central nervous system, renal, cardiac, ophthalmological features, and sensorineural hearing impairment. SIFD was first described in 2013 and to date, it has been reported in 46 patients. Herein, we review the literature and describe two siblings with SIFD and note the novel phenotype of hypoglycemia in the context of growth hormone (GH) deficiency. GH deficiency without hypoglycemia has previously been reported in three patients with SIFD, but GH deficiency had not been firmly ascribed to SIFD. We propose to expand the phenotype to include GH deficiency, hypoglycemia, and previously unreported dysmorphic features. Furthermore, we highlight the intrafamilial variability of the disease by the discordance of our patients' clinical phenotypes and biochemical profiles measured by untargeted metabolomics analysis. Several metabolomic abnormalities were observed in both patients, and these may represent a potential biochemical signature for SIFD.

Asunto(s)

Anemia Sideroblástica , Anemia Sideroblástica/genética , Fiebre/complicaciones , Fiebre/genética , Humanos , Mutación , Nucleotidiltransferasas/genética , Fenotipo

RESUMEN

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Asunto(s)

Endorribonucleasas/genética , Interacciones Huésped-Patógeno/genética , Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN de Transferencia/genética , Theilovirus/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Virus de la Encefalomiocarditis/genética , Virus de la Encefalomiocarditis/crecimiento & desarrollo , Virus de la Encefalomiocarditis/metabolismo , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Nucleotidiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Theilovirus/crecimiento & desarrollo , Theilovirus/metabolismo , Carga Viral

RESUMEN

Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD) syndrome is a serious autosomal recessive genetic disease. So far, <40 cases have been reported worldwide, and only one case has been reported in China. The main clinical features of SIFD are sideroblastic or microcytic anemia, immune deficiency, and recurrent episodes of inflammation. Here, we describe two unrelated cases of SIFD from China with different clinical manifestations and mild symptoms. Patient 1 was hospitalized at the age of 3.5 years due to persistent joint swelling with imaging of multiple joint effusions. Patient 2 was hospitalized at the age of 12 years due to repeated rashes on both lower limbs and oral ulcers. SIFD was detected using gene testing, which revealed the following compound heterozygous variants in TRNT1 in cases 1 and 2, respectively: c.88A > G/c.363G > T and c.302 T > C/c.1234cC > T. Searches of the HGMD databases revealed that these variants were all novel. Molecular dynamics simulations revealed that the missense variants c.363G > T and c.302 T > C would cause changes in protein structure and thus affect protein function. Finally, through literature reviewing, we found that the mortality in cases of SIFD was approximately 44% (14/32), and about 79% of individuals who died carried the hot-spot mutation c.668 T > C. Moreover, variants in the non-coding region were significantly more common among patients who died than among survivors. Our cases further expand the existing knowledge of the phenotype and variation spectrums of SIFD and suggest that genomic diagnosis is valuable for the hierarchical clinical management of this disease.

Asunto(s)

Anemia Sideroblástica , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/genética , Linfocitos B , Niño , Preescolar , China , Fiebre/genética , Humanos , Mutación , Nucleotidiltransferasas/genética

RESUMEN

Since the first description of the syndrome of sideroblastic anemia with immunodeficiency, fevers and development delay (SIFD), clinical pictures lacking both neurological and hematological manifestations have been reported. Moreover, prominent skin involvement, such as with relapsing erythema nodosum, is not a common finding. Up to this moment, no genotype and phenotype correlation could be done, but mild phenotypes seem to be located in the N or C part. B-cell deficiency is a hallmark of SIFD syndrome, and multiple others immunological defects have been reported, but not high levels of double negative T cells. Here we report a Brazilian patient with a novel phenotype of SFID syndrome, carrying multiple immune defects and harboring a novel mutation on TRNT1 gene.

Asunto(s)

Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/etiología , Discapacidades del Desarrollo/diagnóstico , Susceptibilidad a Enfermedades , Fiebre , Síndromes de Inmunodeficiencia/diagnóstico , Fenotipo , Alelos , Biopsia , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Mutación

RESUMEN

Mutations in the gene encoding tRNA nucleotidyltransferase 1 (TRNT1) are associated with heterogeneous phenotypes and multisystem involvement of variable severity and progression. Immunodeficiency and inflammation are recurrent-associated features. The use of cytokine inhibitors in suppressing the inflammatory phenotype has been recently reported, with a 3-year follow-up for patients treated with Etanercept. We report on two unrelated patients sharing the same clinical condition, who had been referred to our Pediatric Rheumatology Unit because of recurrent fever associated with cutaneous lesions and increased levels of inflammatory markers since their first months of life. Whole exome sequencing allowed to identify compound heterozygosity for functionally relevant variants in TRNT1 as the only molecular event shared by the two patients. Both patients have been treated with Etanercept during 11 years, documenting normalization of inflammatory indexes and resolution of recurrent fever and associated symptoms. This is the longest follow-up assessment of Etanercept treatment in patients with TRNT1 mutations. Our findings confirm efficacy and safety of the treatment. Key Points • Mutations in TRNT1 have been associated with phenotypic heterogeneity. • We report on two patients with early-onset autoinflammatory syndrome. • Whole exome sequencing led to reveal compound heterozygosity for two variants in TRNT1 in both patients. • The patients were successfully treated with Etanercept for more than 10 years, the longest follow-up described in literature.

Asunto(s)

Síndromes de Inmunodeficiencia , Nucleotidiltransferasas , Etanercept/uso terapéutico , Humanos , Mutación , Nucleotidiltransferasas/genética , Fenotipo

RESUMEN

Sideroblastic anaemia, B-cell immunodeficiency, periodic fever and developmental delay (SIFD) is caused by mutations of TRNT1, an enzyme essential for mitochondrial protein synthesis, and has been reported in 23 cases. A 6-month-old girl was evaluated with recurrent fever, failure to thrive, skin lesions and anaemia. She received blood transfusions and empirical antibiotics. Skin lesions, previously interpreted as insect bites, consisted of numerous firm asymptomatic erythematous papules and nodules, distributed over trunk and limbs. Skin histopathology revealed an intense dermal neutrophilic infiltrate extending to the subcutaneous, with numerous atypical myeloid cells, requiring the diagnosis of leukaemia cutis, to be ruled out. Over the follow-up, she developed herpetic stomatitis, tonsillitis, lobar pneumonia and Metapneumovirus tracheitis, and also deeper skin lesions, resembling panniculitis. Hypogammaglobulinaemia was diagnosed. An autoinflammatory disease was confirmed by whole exome sequencing: heterozygous mutations for TRNT1 NM_182916 c.495_498del, p.F167Tfs * 9 and TRNT1 NM_182916 c.1246A>G, p.K416E. The patient has been treated with subcutaneous immunoglobulin and etanercept. She presented with developmental delay and short stature for age. The fever, anaemia, skin neutrophilic infiltration and the inflammatory parameters improved. We describe a novel mutation in SIFD and the first to present skin manifestations, namely neutrophilic dermal and hypodermal infiltration.

Asunto(s)

Anemia Sideroblástica/diagnóstico , Discapacidades del Desarrollo/complicaciones , Síndromes de Inmunodeficiencia/diagnóstico , Neutrófilos/metabolismo , Enfermedades de la Piel/etiología , Anemia Sideroblástica/genética , Dermis/metabolismo , Discapacidades del Desarrollo/genética , Femenino , Fiebre/etiología , Humanos , Síndromes de Inmunodeficiencia/congénito , Síndromes de Inmunodeficiencia/genética , Lactante , Mutación , Nucleotidiltransferasas/genética , Secuenciación del Exoma

RESUMEN

tRNA nucleotidyl transferase 1 (TRNT1) is an essential enzyme catalyzing the addition of terminal cytosine-cytosine-adenosine (CCA) trinucleotides to all mature tRNAs, which is necessary for aminoacylation. It was recently discovered that partial loss-of-function mutations in TRNT1 are associated with various, seemingly unrelated human diseases including sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD), retinitis pigmentosa with erythrocyte microcytosis, and progressive B-cell immunodeficiency. In addition, even within the same disease, the severity and range of the symptoms vary greatly, suggesting a broad, pleiotropic impact of imparting TRNT1 function on diverse cellular systems. Here, we describe the current state of knowledge of the TRNT1 function and the phenotypes associated with mutations in TRNT1.

Asunto(s)

Adenosina/metabolismo , Citosina/metabolismo , Enfermedad/genética , ARN de Transferencia/metabolismo , Animales , Humanos , Mitocondrias/metabolismo , Nucleotidiltransferasas/metabolismo

RESUMEN

Mutations in the gene encoding transfer RNA (tRNA) nucleotidyltransferase, CCA-adding 1 (TRNT1), an enzyme essential for the synthesis of the 3'-terminal CCA sequence in tRNA molecules, are associated with a rare syndrome of congenital sideroblastic anemia, B cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Clinical manifestations and immunological phenotypes were assessed in a Chinese patient with novel compound heterozygous mutations in TRNT1. The patient required multiple hospitalizations starting at the age of 2 years for recurrent fevers without an infective cause. During the febrile episode, the patient was found to have microcytic hypochromic anemia, B cell lymphopenia, and hypogammaglobulinemia. Targeted gene sequencing identified novel compound heterozygous mutations in the TRNT1 gene (c.525delT, p.Leu176X; c.938T>C, p.Leu313Ser). Immunophenotyping revealed increased CD8+ T cells, CD4+ terminally differentiated effector memory helper T lymphocytes (CD4 TEMRA), and CD4+ effector memory lymphocytes (CD4 EM). Analysis of CD4+ T subsets identified decreased T follicular helper cells (Tfh) with a biased phenotype to Th2-like cells. The patient also showed a lower percentage of switched memory B (smB) cells. Additionally, defects in the cytotoxicity of the patient's NK and γδT cells were shown by CD107alpha expression. In conclusion, T RNT1 mutations may lead to multiple immune abnormality especially humoral and cytotoxicity defects, which indicate that SIFD is not only suffered 'Predominantly antibody deficiencies' in IUIS classification system, and further studies are needed to understand the pathogenesis of immunodeficiency in these patients.

RESUMEN

Post-transcriptional non-template additions of nucleotides to 3'-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3'-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3'-ends of the sequence 5'-NCCA-3', which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen 'CCA-RNAs' that only contain CCA as non-genomic sequences, and eleven 'NCCA-RNAs' that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.

Asunto(s)

Mitocondrias/genética , Nucleotidiltransferasas/genética , ARN Mitocondrial/genética , ARN de Transferencia/genética , Emparejamiento Base , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Biología Computacional/métodos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Células Epiteliales , Genoma Mitocondrial , Células HEK293 , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células MCF-7 , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/metabolismo , ARN Mitocondrial/química , ARN Mitocondrial/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Rayos Ultravioleta

RESUMEN

The I326T mutation in the TRNT1 gene encoding human tRNA nucleotidyltransferase (tRNA-NT) is linked to a relatively mild form of SIFD. Previous work indicated that the I326T variant was unable to incorporate AMP into tRNAs in vitro, however, expression of the mutant allele from a strong heterologous promoter supported in vivo CCA addition to both cytosolic and mitochondrial tRNAs in a yeast strain lacking tRNA-NT. To address this discrepancy, we determined the biochemical and biophysical characteristics of the I326T variant enzyme and the related variant, I326A. Our in vitro analysis revealed that the I326T substitution decreases the thermal stability of the enzyme and causes a ten-fold reduction in enzyme activity. We propose that the structural changes in the I326T variant that lead to these altered parameters result from a rearrangement of helices within the body domain of the protein which can be probed by the inability of the monomeric enzyme to form a covalent dimer in vitro mediated by C373. In addition, we confirm that the effects of the I326T or I326A substitutions are relatively mild in vivo by demonstrating that the mutant alleles support both mitochondrial and cytosolic CCA-addition in yeast.

Asunto(s)

Sustitución de Aminoácidos , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Anemia Sideroblástica/genética , Dominio Catalítico , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Nucleotidiltransferasas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica

RESUMEN

Mutation in the gene encoding tRNA nucleotidyl transferase, CCA-adding 1 (TRNT1), an enzyme essential for the synthesis of the 3'-terminal CCA sequence in tRNA molecules, results in a disorder that features sideroblastic anemia, B-cell immunodeficiency, periodic fever, and developmental delay. Mutations in TRNT1 are also linked to phenotypes including retinitis pigmentosa, cataracts, and cardiomyopathy. To date, it has remained unclear how defective TRNT1 is linked to B-cell deficiency. Here we report the case of a 12-year-old boy without sideroblastic anemia who harbors novel compound heterozygous mutations in TRNT1. Immunophenotypic analysis revealed severely decreased levels of B cells and follicular helper T cells. In the bone marrow, B-cell maturation stopped at the CD19+CD10+CD20+/- pre-B-cell stage. Severe combined immunodeficiency mice transplanted with bone marrow hematopoietic stem cells from the patient showed largely normal B-cell engraftment and differentiation in the bone marrow and periphery at 24 weeks post-transplantation, comparable to those in mouse transplanted with healthy hematopoietic stem cells. Biochemical analysis revealed augmented endoplasmic reticulum (ER) stress response in activated T cells. Peripheral B-cell deficiency of TRNT1 deficiency may be associated with augmented ER stress in immature B cells in the bone marrow.

Asunto(s)

Heterocigoto , Síndromes de Inmunodeficiencia , Nucleotidiltransferasas , Antígenos CD/sangre , Antígenos CD/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Síndromes de Inmunodeficiencia/sangre , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Lactante , Masculino , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/patología

RESUMEN

Mutations in the human TRNT1 gene encoding tRNA nucleotidyltransferase (tRNA-NT), an essential enzyme responsible for addition of the CCA (cytidine-cytidine-adenosine) sequence to the 3'-termini of tRNAs, have been linked to disease phenotypes including congenital sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD) or retinitis pigmentosa with erythrocyte microcytosis. The effects of these disease-linked mutations on the structure and function of tRNA-NT have not been explored. Here we use biochemical and biophysical approaches to study how five SIFD-linked amino acid substitutions (T154I, M158V, L166S, R190I and I223T), residing in the N-terminal head and neck domains of the enzyme, affect the structure and activity of human tRNA-NT in vitro. Our data suggest that the SIFD phenotype is linked to poor stability of the T154I and L166S variant proteins, and to a combination of reduced stability and altered catalytic efficiency in the M158 V, R190I and I223T variants.

Asunto(s)

Anemia Sideroblástica , Enfermedades Genéticas Ligadas al Cromosoma X , Calor , Mutación Missense , Nucleotidiltransferasas/química , Sustitución de Aminoácidos , Catálisis , Estabilidad de Enzimas , Humanos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Dominios Proteicos

RESUMEN

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Asunto(s)

Anemia Sideroblástica/genética , Antiinflamatorios/uso terapéutico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Síndromes de Inmunodeficiencia/genética , Mutación , Nucleotidiltransferasas/genética , ARN de Transferencia/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anemia Sideroblástica/sangre , Niño , Preescolar , Citocinas/sangre , Citocinas/genética , Discapacidades del Desarrollo/genética , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Humanos , Inmunofenotipificación , Masculino , Linaje , Fenotipo , Factor de Necrosis Tumoral alfa/análisis , Secuenciación del Exoma

Asunto(s)

Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Mutación , Nucleotidiltransferasas/genética , Anomalías Múltiples/inmunología , Agammaglobulinemia/diagnóstico , Anemia Sideroblástica/diagnóstico , Linfocitos B/inmunología , Linfocitos B/patología , Cardiomiopatía Hipertrófica/diagnóstico , Discapacidades del Desarrollo/diagnóstico , Homocigoto , Humanos , Lactante , Infecciones/diagnóstico , Masculino , Linaje , Recurrencia , Linfocitos T/inmunología , Linfocitos T/patología , Secuenciación del Exoma/métodos

RESUMEN

Retinitis pigmentosa (RP) is a heterogeneous group of monogenic disorders characterized by progressive death of the light-sensing photoreceptor cells of the outer neural retina. We recently identified novel hypomorphic mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1) gene that cause early-onset RP. To model this disease in vitro, we generated patient-specific iPSCs and iPSC-derived retinal organoids from dermal fibroblasts of patients with molecularly confirmed TRNT1-associated RP. Pluripotency was confirmed using rt-PCR, immunocytochemistry, and a TaqMan Scorecard Assay. Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived retinal organoids. Patient-specific iPSCs and iPSC-derived retinal organoids exhibited a deficit in autophagy, as evidenced by aberrant accumulation of LC3-II and elevated levels of oxidative stress. Autologous stem cell-based disease modeling will provide a platform for testing multiple avenues of treatment in patients suffering from TRNT1-associated RP.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Nucleotidiltransferasas/metabolismo , Retinitis Pigmentosa/patología , Retinitis Pigmentosa/fisiopatología , Autofagosomas/metabolismo , Autofagia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación/genética , Organoides/metabolismo , Estrés Oxidativo , Retina/metabolismo