RESUMO
Limb-girdle muscular dystrophy (LGMD) is a group of myopathies that lead to progressive muscle weakness, predominantly involving the shoulder and pelvic girdles; it has a heterogeneous genetic etiology, with variation in the prevalence of subtypes according to the ethnic backgrounds and geographic origins of the populations. The aim of the present study was to analyze a series of patients with autosomal recessive LGMD (LGMD-R) to contribute to a better characterization of the disease and to find the relative proportion of the different subtypes in a Southern Brazil cohort. The sample population consisted of 36 patients with LGMD-R. A 9-gene targeted next-generation sequencing panel revealed variants in 23 patients with LGMD (64%), and it identified calpainopathy (LGMD-R1) in 26%, dysferlinopathy (LGMD-R2) in 26%, sarcoglycanopathies (LGMD-R3-R5) in 13%, telethoninopathy (LGMD-R7) in 18%, dystroglicanopathy (LGMD-R9) in 13%, and anoctaminopathy (LGMD-R12) in 4% of the patients. In these 23 patients with LGMD, there were 27 different disease-related variants in the ANO5, CAPN3, DYSF, FKRP, SGCA, SGCB, SGCG, and TCAP genes. There were different causal variants in different exons of these genes, except for the TCAP gene, for which all patients carried the p.Gln53* variant, and the FKRP gene, which showed recurrence of the p.Leu276Ile variant. We analyzed the phenotypic, genotypic and muscle immunohistochemical features of this Southern Brazilian cohort.
A distrofia muscular de cinturas (DMC) é um grupo de miopatias que leva à fraqueza muscular progressiva, e envolvendo predominante as cinturas escapular e pélvica. A DMCtem uma etiologia genética heterogênea, com variação na prevalência de subtipos de acordo com as origens étnicas e geográficas das populações. O objetivo deste estudo foi analisar uma série de pacientes com DMC do tipo autossômico recessivo (DMC-R) para contribuir para uma melhor caracterização da doença e encontrar a proporção relativa dos diferentes subtipos em uma coorte do Sul do Brasil. A população amostral foi composta por 36 pacientes com DMC-R. O painel de sequenciamento de nova geração com 9 genes revelou variantes em 23 pacientes com DMC (64%), e identificou calpainopatia (DMC-R1) em 26%, disferlinopatia (DMC-R2) em 26%, sarcoglicanopatias (DMC-R3R5) em 13%, teletoninopatia (D-MCR7) em 18%, distroglicanopatia (D-MCR9) em 13%, e anoctaminopatia (DMC-R12) em 4% dos pacientes. Nesses 23 pacientes com DMC, havia 27 variantes diferentes nos genes ANO5, CAPN3, DYSF, FKRP, SGCA, SGCB, SGCG e TCAP. Foram encontradas diferentes variantes em diferentes éxons desses genes, com exceção do gene TCAP, para o qual todos os pacientes eram portadores da variante p.Gln53*, e do gene FKRP, que apresentou recorrência da variante p.Leu276Ile. As características fenotípicas, genotípicas e imuno-histoquímicas musculares desta coorte do Sul do Brasil foram analisadas.
Assuntos
Distrofia Muscular do Cíngulo dos Membros , Humanos , Anoctaminas/genética , Brasil , Debilidade Muscular , Distrofia Muscular do Cíngulo dos Membros/genética , Pentosiltransferases/genéticaRESUMO
The complete mutational spectrum of dystrophinopathies and limb-girdle muscular dystrophy (LGMD) remains unknown in Mexican population. Seventy-two unrelated Mexican male patients (73% of pediatric age) with clinical suspicion of muscular dystrophy and no evidence of DMD gene deletion on multiplex polymerase chain reaction (mPCR) analysis were analyzed by multiplex ligation-dependent probe amplification (MLPA). Those with a normal result were subjected to Sanger sequencing or to next-generation sequencing for DMD plus 10 selected LGMD-related genes. We achieved a diagnostic genotype in 80.5% (n = 58/72) of patients with predominance of dystrophinopathy-linked genotypes (68%, n = 49/72), followed by autosomal recessive LGMD-related genotypes (types 2A-R1, 2C-R5, 2E-R4, 2D-R3 and 2I-R9; 12.5%, n = 9/72). MLPA showed 4.2% of false-negatives for DMD deletions assessed by mPCR. Among the small DMD variants, 96.5% (n = 28/29) corresponded to null-alleles, most of which (72%) were inherited through a carrier mother. The FKRP p.[Leu276Ile]; [Asn463Asp] genotype is reported for the first time in Mexican patients as being associated with dilated cardiomyopathy. Absence of dysferlinopathies could be related to the small sample size and/or the predominantly pediatric age of patients. The employed strategy seems to be an affordable diagnosis approach for Mexican muscular dystrophy male patients and their families.
Assuntos
Distrofina/genética , Testes Genéticos/normas , Distrofia Muscular de Duchenne/genética , Mutação , Adolescente , Adulto , Criança , Pré-Escolar , Reações Falso-Negativas , Testes Genéticos/métodos , Genótipo , Humanos , Masculino , México , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Distrofia Muscular de Duchenne/patologia , Pentosiltransferases/genéticaRESUMO
Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8-10) across a broad range of temperatures (30-70 °C) and ionic strengths (0-500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2'-fluoro-2'-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2'-fluoro-2'-deoxyguanosine, ara G, and ara H by a 2'-deoxyribosyltransferase is reported.
Assuntos
Leishmania mexicana/enzimologia , Nucleosídeos/biossíntese , Pentosiltransferases/metabolismo , Purinas/química , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Biologia Computacional , Enzimas Imobilizadas , Concentração de Íons de Hidrogênio , Leishmania mexicana/genética , Pentosiltransferases/genética , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5Î-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.
Assuntos
Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Pentosiltransferases/genética , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Pentosiltransferases/metabolismo , Uracila/metabolismo , Uracila/farmacologia , VirulênciaAssuntos
Anormalidades Craniofaciais/genética , Nanismo/genética , Homozigoto , Instabilidade Articular/genética , Mutação de Sentido Incorreto , Ossificação Heterotópica/genética , Pentosiltransferases/genética , Polidactilia/genética , Criança , Pré-Escolar , Consanguinidade , Anormalidades Craniofaciais/patologia , Nanismo/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Instabilidade Articular/patologia , Nucleotidases/genética , Ossificação Heterotópica/patologia , Polidactilia/patologia , UDP Xilose-Proteína XilosiltransferaseRESUMO
Ethambutol inhibits arabinogalactan and lipoarabinomannan biosynthesis in mycobacteria. The occurrence of mutations in embB codon 306 in ethambutol-susceptible isolates and their absence in resistant isolates has raised questions regarding the utility of this codon as a potential marker for resistance against ethambutol. The characterization of mutations on embB 306 will contribute to a better understanding of the mechanisms of resistance to this drug; therefore, the purpose of this study was to investigate the association between embB 306 mutations and first-line drug resistance profiles in tuberculosis isolates. We sequenced the region surrounding the embB 306 codon in 175 tuberculosis clinical isolates, divided according to drug sensitivity, in three groups: 110 were resistant to at least one first-line drug, of which 61 were resistant to ethambutol (EMB(r)), 49 were sensitive to ethambutol (EMB(s)) but were resistant to another drug, and 65 were pansensitive isolates (P(s)). The associations between embB 306 mutations and phenotypic resistance to all first-line drugs were determined, and their validity and safety as a diagnostic marker were assessed. One of the P(s) isolates (1/65), one of the EMB(s) isolates (1/49), and 20 of the EMB(r) isolates (20/61) presented with an embB 306 mutation. Four different single-nucleotide polymorphisms (SNPs) at embB 306 were associated with simultaneous resistance to ethambutol, isoniazid, and rifampin (odds ratio [OR], 17.7; confidence interval [CI], 5.6 to 56.1) and showed a positive predictive value of 82%, with a specificity of 97% for diagnosing multidrug resistance associated with ethambutol, indicating its potential as a molecular marker for several drugs.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Pentosiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Mutação/genética , Mycobacterium tuberculosis/genética , Pentosiltransferases/genética , Tuberculose Resistente a Múltiplos Medicamentos/genéticaRESUMO
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.
Assuntos
Antituberculosos/farmacologia , Resistência Microbiana a Medicamentos/genética , Etambutol/farmacologia , Mycobacterium tuberculosis/genética , Pentosiltransferases/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Substituição de Aminoácidos , Códon/genética , Cuba/epidemiologia , Análise Mutacional de DNA , DNA Bacteriano/genética , República Dominicana/epidemiologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Pentosiltransferases/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologiaRESUMO
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Mycobacterium tuberculosis/genética , Pentosiltransferases/genética , Resistência Microbiana a Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Análise Mutacional de DNA , Cuba/epidemiologia , Códon/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Pentosiltransferases/fisiologia , Relação Dose-Resposta a Droga , Reprodutibilidade dos Testes , República Dominicana/epidemiologia , Sensibilidade e Especificidade , Substituição de Aminoácidos , Testes de Sensibilidade Microbiana , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologiaRESUMO
Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
Assuntos
Mycobacterium tuberculosis/enzimologia , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Reação em Cadeia da Polimerase , Análise de Sequência , Especificidade por SubstratoRESUMO
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.