RESUMEN
We describe two patients with mitochondrial DNA mutations in the gene encoding cytochrome b (m.15579A>G, p.Tyr278Cys and m.15045G>A p.Arg100Gln), which presented as a pure myopathic form (exercise intolerance), with an onset in childhood. Diagnosis was delayed, because acylcarnitine profile showed an increase in medium and long-chain acylcarnitines, suggestive of multiple acyl-CoA dehydrogenase deficiency, riboflavin transporter deficiency or FAD metabolism disorder. Implication of cytochrome b in fatty acid oxidation, and physiopathology of the mutations are discussed.
Asunto(s)
Citocromos b/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnóstico , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Mutación Missense , Adulto , Anciano , ADN Mitocondrial/genética , Diagnóstico Diferencial , Tolerancia al Ejercicio/genética , Humanos , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genéticaRESUMEN
WFS1 mutations are responsible for Wolfram syndrome (WS) characterized by juvenile-onset diabetes mellitus and optic atrophy, and for low-frequency sensorineural hearing loss (LFSNHL). Our aim was to analyze the French cohort of 96 patients with WFS1-related disorders in order (i) to update clinical and molecular data with 37 novel affected individuals, (ii) to describe uncommon phenotypes and, (iii) to precise the frequency of large-scale rearrangements in WFS1. We performed quantitative polymerase chain reaction (PCR) in 13 patients, carrying only one heterozygous variant, to identify large-scale rearrangements in WFS1. Among the 37 novel patients, 15 carried 15 novel deleterious putative mutations, including one large deletion of 17,444 base pairs. The analysis of the cohort revealed unexpected phenotypes including (i) late-onset symptoms in 13.8% of patients with a probable autosomal recessive transmission; (ii) two siblings with recessive optic atrophy without diabetes mellitus and, (iii) six patients from four families with dominantly-inherited deafness and optic atrophy. We highlight the expanding spectrum of WFS1-related disorders and we show that, even if large deletions are rare events, they have to be searched in patients with classical WS carrying only one WFS1 mutation after sequencing.
Asunto(s)
Estudios de Asociación Genética , Proteínas de la Membrana/genética , Mutación , Fenotipo , Síndrome de Wolfram/diagnóstico , Síndrome de Wolfram/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Niño , Estudios de Cohortes , Familia , Femenino , Francia , Genes Dominantes , Genes Recesivos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Succinate-CoA ligase deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Mutations in SUCLA2, the gene encoding a ß subunit of succinate-CoA ligase, have been reported in 17 patients until now. Mutations in SUCLG1, encoding the α subunit of the enzyme, have been described in two pedigrees only. METHODS AND FINDINGS: In this study, two unrelated patients harbouring three novel pathogenic mutations in SUCLG1 were reported. The first patient had a severe disease at birth. He was compound heterozygous for a missense mutation (p.Pro170Arg) and a c.97+3G>C mutation, which leads to the complete skipping of exon 1 in a minigene expression system. The involvement of SUCLG1 was confirmed by western blot analysis, which showed absence of SUCLG1 protein in fibroblasts. The second patient has a milder phenotype, similar to that of patients with SUCLA2 mutations, and is still alive at 12 years of age. Western blot analysis showed some residual SUCLG1 protein in patient's fibroblasts. CONCLUSIONS: Our results suggest that SUCLG1 mutations that lead to complete absence of SUCLG1 protein are responsible for a very severe disorder with antenatal manifestations, whereas a SUCLA2-like phenotype is found in patients with residual SUCLG1 protein. Furthermore, it is shown that in the absence of SUCLG1 protein, no SUCLA2 protein is found in fibroblasts by western blot analysis. This result is consistent with a degradation of SUCLA2 when its heterodimer partner, SUCLG1, is absent.
Asunto(s)
Ácido Metilmalónico/orina , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/fisiopatología , Mutación , Índice de Severidad de la Enfermedad , Succinato-CoA Ligasas/genética , Secuencia de Aminoácidos , Niño , Resultado Fatal , Humanos , Lactante , Masculino , Ácido Metilmalónico/sangre , Encefalomiopatías Mitocondriales/mortalidad , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/deficiencia , Succinato-CoA Ligasas/metabolismoRESUMEN
AIMS: Mutations of the WFS1 gene have been implicated in autosomal dominant diseases, such as low-frequency sensorineural hearing impairment (LFSNHI) and/or diabetes mellitus and/or optic atrophy. The aim was to investigate WFS1 gene sequences in a family with diabetes mellitus and hearing impairment. METHODS: Three members of a family with a maternally inherited combination of diabetes mellitus and hearing impairment, but no specific mutations in its mitochondrial genome, were investigated for mutations in the WFS1 gene. RESULTS: This pedigree, in which the proband had non-insulin-dependent diabetes mellitus and congenital hearing impairment and his mother a triple combination of diabetes mellitus, hearing impairment and optic atrophy, was found to be associated with autosomal dominant transmission of the E864K mutation of the WFS1 gene. CONCLUSIONS: In the light of this confirmatory study, we recommend the systematic analysis of WFS1 gene sequences in patients with parentally inherited diabetes mellitus and deafness (+/- optic atrophy), in particular when diabetogenic mtDNA mutations have been excluded.
Asunto(s)
Diabetes Mellitus/genética , Pérdida Auditiva/genética , Proteínas de la Membrana/genética , Mutación Missense/genética , Adulto , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).
Asunto(s)
ADN Mitocondrial/genética , Endonucleasas , Pruebas Genéticas/métodos , Enfermedades Neuromusculares/genética , Adolescente , Adulto , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/genética , LinajeRESUMEN
The 2001 International Classification of Constitutional Disorders of Bone has included in the group of multicentric hands and feet osteolysis syndromes three autosomal recessive inherited disorders: Winchester, Torg and nodulosis-arthropathy-osteolysis (NAO) syndromes. Nosographic delineations of these rare syndromes are difficult to define, and there is no consensus. In 2001, two mutations in the matrix metalloproteinase 2 gene (MMP2) have been identified in two families with a NAO phenotype. In a recent study, a homozygous MMP2 mutation has also been identified in a patient presenting with Winchester syndrome. We report the clinical evolution of two sisters with a Winchester phenotype. Clinical review over 23 years provides information on the general evolution of osteolysis and points to an intrafamilial variation with clinical and radiological changes during the patients' life. In both sisters, we identified a new homozygous mutation in the catalytic domain of the MMP2 gene. Our study results are consistent with the involvement of MMP2 in Winchester syndrome and with the hypothesis that Winchester and NAO syndromes are allelic disorders that form a continuous clinical spectrum. At last, our observation emphasizes the interest of molecular analysis in genetic counselling of this consanguineous family.
Asunto(s)
Metaloproteinasa 2 de la Matriz/genética , Mutación , Osteólisis Esencial/enzimología , Osteólisis Esencial/genética , Adulto , Secuencia de Bases , Dominio Catalítico/genética , ADN/genética , Femenino , Homocigoto , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/deficiencia , Osteólisis Esencial/patología , Fenotipo , Eliminación de Secuencia , SíndromeRESUMEN
TRBP1 and TRBP2 are isoforms of a double-stranded RNA-binding protein that differ in their N-terminal end and were each identified by binding to human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive RNA. TRBP1 and TRBP2 also bind and modulate the function of the double-stranded RNA-activated protein kinase, protein kinase R. Both proteins increase long terminal repeat expression in human and murine cells, and their gene has been mapped to human chromosome 12. We have isolated and characterized the complete tarbp2 gene (5493 bp) coding for the two TRBP proteins. Two adjacent promoters initiate transcription of alternative first exons for TRBP1 and TRBP2 mRNAs that are spliced onto common downstream exons. TRBP2 transcription and translation start sites are localized within the first intron of TRBP1. TRBP promoters are TATA-less but have CCAAT boxes, a CpG island, and several potential binding sites for transcriptional factors. Promoter deletion analysis identified two regions from position -1397 to -330 for TRBP1 and from position -330 to +38 for TRBP2 that are important for promoter function. TRBP2 promoter activity was expressed at a higher level compared with TRBP1 promoter. In addition, a specific down-regulation of TRBP1 and TRBP2 promoter activity was identified in human astrocytic cell line U251MG compared with HeLa cells. This minimal TRBP promoter activity may account for minimal HIV-1 replication in astrocytes.
Asunto(s)
Empalme Alternativo/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/fisiología , Proteínas de Unión al ARN/genética , Astrocitos/fisiología , Secuencia de Bases , Línea Celular , Clonación Molecular , Exones/genética , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.
Asunto(s)
Duplicado del Terminal Largo de VIH/genética , Proteínas de Unión al ARN/química , eIF-2 Quinasa/metabolismo , Aminoácidos/química , Sitios de Unión , Dimerización , Eliminación de Gen , Genes Reporteros , Células HeLa , Humanos , Luciferasas/metabolismo , Modelos Genéticos , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
In vitro, LSLCL is expressed by numerous myeloid, promyelocytic, and T or B lymphoblastoid cell lines. In vivo, LSLCL is strongly expressed in bone marrow and only faintly in lymphoid organs. We show here that, in bone marrow, LSLCL is detected: (i) concentrated in the cytoplasm of immature neutrophils but not in myeloblasts nor in mature neutrophils, (ii) in extracellular bone marrow fluid. Besides, numerous cDNAs, similar to LSLCL (identity of 93-99%), are found in 'expressed sequence tags' databases from various origins, mostly fetal and undifferentiated tumour tissues. Since LSLCL and various closely related cDNAs are expressed at definite stages of cellular maturation processes, we hypothesize that this class of proteins could play an important role in the control of cellular differentiation.
Asunto(s)
Regulación de la Expresión Génica , Factores de Crecimiento de Célula Hematopoyética , Lectinas Tipo C , Lectinas/genética , Neutrófilos/fisiología , Animales , Northern Blotting , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Pollos , Bases de Datos de Ácidos Nucleicos , Etiquetas de Secuencia Expresada , Femenino , Enfermedades Hematológicas/sangre , Enfermedades Hematológicas/genética , Humanos , Lectinas/sangre , Leucemia/sangre , Leucemia/genética , Neutrófilos/citologíaRESUMEN
TRBP1 and TRBP2 cDNAs have been isolated based on the ability of the protein that they encode to bind HIV-1 TAR RNA. The two cDNAs have different 5' end-termini resulting in 21 additional amino acids for TRBP2 protein compared to TRBP1. The corresponding gene is conserved in mammalian species. By PCR amplification of a human library, we have isolated an additional 22 nucleotides in the 5' end of TRBP2 cDNA. Based on the addition of these 22 new nucleotides, the first 87 nucleotides of TRBP2 mRNA can fold into a stable stem-loop structure that resembles TAR RNA. We have also isolated the DNA sequence that represents the TRBP processed pseudogene. The absence of full alignment between TRBP2 full-length cDNA and this sequence suggests that the stem-loop structure could have prevented a complete reverse transcription during pseudogene formation. Using different antibodies, three forms of TRBP can be identified in primate cells at 40, 43 and 50 kD, suggesting a differential expression from the cDNAs and post-translational modifications. Both TRBP1 and TRBP2 activate the basal and the Tat-activated level of the HIV-1 LTR in human and murine cells. Our data indicate that TRBP proteins act at a level prior to Tat function. TRBP could contribute to improved HIV expression in murine models.
Asunto(s)
Duplicado del Terminal Largo de VIH , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Células HeLa , Humanos , Células Jurkat , Ratones , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Seudogenes , Proteínas de Unión al ARN/química , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoAsunto(s)
Factores de Crecimiento de Célula Hematopoyética , Lectinas Tipo C , Lectinas/genética , Mucinas/genética , Mapeo Cromosómico , Cromosomas Humanos Par 19/genética , Clonación Molecular , ADN/química , ADN/genética , Exones/genética , Humanos , Hibridación Fluorescente in Situ , Linfocitos/metabolismo , Datos de Secuencia Molecular , Mucinas/metabolismo , Análisis de Secuencia de ADNRESUMEN
We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), (Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265-274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609-1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass approximately 47 and approximately 40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking alpha-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites for O-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.
Asunto(s)
Antígenos CD , Factores de Crecimiento de Célula Hematopoyética , Lectinas Tipo C , Lectinas/genética , Activación de Linfocitos , Linfocitos T/química , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor , Proteínas Sanguíneas/química , Clonación Molecular , Glicosilación , Infecciones por VIH/metabolismo , VIH-1 , Células HL-60 , Humanos , Células Jurkat , Lectinas/química , Leucina Zippers , Antígenos Comunes de Leucocito/química , Leucosialina , Datos de Secuencia Molecular , Peso Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Conejos , Receptores de IgE/química , Alineación de Secuencia , Sialoglicoproteínas/química , Linfocitos T/virologíaRESUMEN
In previous studies, we have demonstrated that O-glycans at the surface of HIV-1-infected cell lines were hyposialylated. Moreover, we and others have shown that HIV+ individuals produced autoantibodies that react with hyposialylated CD43, on T cell lines. Since the autoantigen responsible for this abnormal immune response was not easily found in the peripheral blood cells of corresponding patients, we searched for its possible presence in other sites. Using fluorescence staining of alveolar macrophages with various lectins, we show that the binding of the PNA lectin specific for asialo O-glycans is much more efficient on cells from HIV-1-infected individuals. Moreover, the degree of reactivity of PNA is correlated with the clinical stage of the illness.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1 , Macrófagos Alveolares/metabolismo , Polisacáridos/metabolismo , Adulto , Infecciones por VIH/patología , Humanos , Lectinas/metabolismo , Macrófagos Alveolares/inmunología , Persona de Mediana Edad , Ácido N-AcetilneuramínicoRESUMEN
Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.