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1.
J Biol Chem ; 292(28): 11980-11991, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28572511

RESUMO

Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Lisossomos/metabolismo , Erros Inatos do Metabolismo/genética , Modelos Moleculares , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Substituição de Aminoácidos , Domínio Catalítico , Linhagem Celular Transformada , Células Cultivadas , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/enzimologia , Lisossomos/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Simulação de Acoplamento Molecular , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/deficiência , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Vitamina B 12/metabolismo
2.
J Inherit Metab Dis ; 40(2): 297-306, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27743313

RESUMO

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.


Assuntos
Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Espasticidade Muscular/genética , Mutação de Sentido Incorreto/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Domínio Catalítico/genética , Fibroblastos/metabolismo , Genótipo , Humanos , Hiper-Homocisteinemia/genética , Cinética , Proteínas Mutantes/genética , NADP/genética , Polimorfismo de Nucleotídeo Único/genética , Transtornos Psicóticos/genética , Tetra-Hidrofolatos/genética
3.
Nucleic Acids Res ; 43(9): 4627-39, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25878036

RESUMO

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.


Assuntos
Anemia Megaloblástica/genética , Éxons , Ferredoxina-NADP Redutase/genética , Homocistinúria/genética , Mutação , Oligonucleotídeos , Splicing de RNA , Sequências Reguladoras de Ácido Ribonucleico , Anemia Megaloblástica/enzimologia , Linhagem Celular , Células Cultivadas , Ferredoxina-NADP Redutase/metabolismo , Células HEK293 , Homocistinúria/enzimologia , Humanos , Sítios de Splice de RNA
4.
Hum Mutat ; 37(5): 427-38, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26872964

RESUMO

Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations.


Assuntos
Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/genética , Mutação , Idade de Início , Animais , Domínio Catalítico , Bases de Dados Genéticas , Modelos Animais de Doenças , Humanos , Recém-Nascido , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Triagem Neonatal , Transtornos Psicóticos/genética
5.
Am J Hum Genet ; 93(3): 506-14, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-24011988

RESUMO

Derivatives of vitamin B12 (cobalamin) are essential cofactors for enzymes required in intermediary metabolism. Defects in cobalamin metabolism lead to disorders characterized by the accumulation of methylmalonic acid and/or homocysteine in blood and urine. The most common inborn error of cobalamin metabolism, combined methylmalonic acidemia and hyperhomocysteinemia, cblC type, is caused by mutations in MMACHC. However, several individuals with presumed cblC based on cellular and biochemical analysis do not have mutations in MMACHC. We used exome sequencing to identify the genetic basis of an X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the index case. Additional male subjects were ascertained through two international diagnostic laboratories, and 13/17 had one of five distinct missense mutations affecting three highly conserved amino acids within the HCFC1 kelch domain. A common phenotype of severe neurological symptoms including intractable epilepsy and profound neurocognitive impairment, along with variable biochemical manifestations, was observed in all affected subjects compared to individuals with early-onset cblC. The severe reduction in MMACHC mRNA and protein within subject fibroblast lines suggested a role for HCFC1 in transcriptional regulation of MMACHC, which was further supported by the identification of consensus HCFC1 binding sites in MMACHC. Furthermore, siRNA-mediated knockdown of HCFC1 expression resulted in the coordinate downregulation of MMACHC mRNA. This X-linked disorder demonstrates a distinct disease mechanism by which transcriptional dysregulation leads to an inborn error of metabolism with a complex clinical phenotype.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Genes Ligados ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Fator C1 de Célula Hospedeira/genética , Hiper-Homocisteinemia/genética , Mutação/genética , Vitamina B 12/genética , Idade de Início , Sequência de Aminoácidos , Sítios de Ligação , Análise Mutacional de DNA , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Células HEK293 , Fator C1 de Célula Hospedeira/química , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo
6.
J Inherit Metab Dis ; 39(1): 115-24, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26025547

RESUMO

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.


Assuntos
Homocistinúria/enzimologia , Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Espasticidade Muscular/enzimologia , Espasticidade Muscular/genética , Ataxia/genética , Betaína/uso terapêutico , Criança , Feminino , Ácido Fólico/uso terapêutico , Estudos de Associação Genética/métodos , Homocistinúria/tratamento farmacológico , Humanos , Deficiência Intelectual/genética , Masculino , Metionina/uso terapêutico , Espasticidade Muscular/tratamento farmacológico , Mutação/genética , Fenótipo , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/enzimologia , Transtornos Psicóticos/genética , Estudos Retrospectivos , Doenças da Medula Espinal/genética , Vitamina B 12/uso terapêutico
7.
Hum Mutat ; 36(6): 611-21, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25736335

RESUMO

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.


Assuntos
Estudos de Associação Genética , Homocistinúria/diagnóstico , Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/genética , Alelos , Processamento Alternativo , Ativação Enzimática , Éxons , Fibroblastos/metabolismo , Homocistinúria/metabolismo , Humanos , Íntrons , Cinética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Espasticidade Muscular/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Estabilidade Proteica , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/genética , Transtornos Psicóticos/metabolismo
8.
Hum Mutat ; 35(12): 1449-58, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25125334

RESUMO

Methylmalonyl-CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild-type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in-depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations, and supports the development of alternative, chaperone-based therapy for patients not responding to current treatment.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Metilmalonil-CoA Mutase/genética , Mutação de Sentido Incorreto , Sequência de Bases , Western Blotting , Primers do DNA , Humanos
9.
Hum Mol Genet ; 21(6): 1410-8, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22156578

RESUMO

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patient's fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Homocistinúria/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação/genética , Deficiência de Vitamina B 12/metabolismo , Vitamina B 12/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Western Blotting , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/metabolismo , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/patologia
10.
J Inherit Metab Dis ; 37(5): 841-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24722857

RESUMO

In humans vitamin B12 (cobalamin, Cbl) must be converted into two coenzyme forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), in order to maintain intracellular homeostasis of homocysteine and methylmalonic acid, respectively. Previously we have shown that in cblD patients three types of MMADHC mutations exist: 1) null mutations N-terminal to Met116 cause isolated methylmalonic aciduria (cblD-MMA) due to AdoCbl deficiency; 2) null mutations across the C-terminus (p.Y140-R250) cause combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC) due to AdoCbl and MeCbl deficiency; 3) missense mutations in a conserved C-terminal region (p.D246-L259) cause isolated homocystinuria (cblD-HC) due to MeCbl deficiency. To better understand the domain boundaries related to MeCbl formation, we made selected point mutations and C-terminal truncations in MMADHC and tested rescue of MeCbl and AdoCbl synthesis in immortalized cblD-MMA/HC patient fibroblasts. Testing 20 mutations (15 missense and five C-terminal truncations) across p.P154-S287 revealed the presence of a region (p.R197-D226) responsible for MeCbl synthesis, which gave a similar cellular phenotype as cblD-HC. Further, mutation of the polypeptide stretch between the new and patient defined regions (p.D226-D246) and directly C-terminal to the patient region (p.L259-R266), gave cellular phenotypes intermediate to those of cblD-HC and cblD-MMA/HC. Finally, C-terminal truncation of more than 20 amino acids resulted in a cblD-MMA/HC like cellular phenotype, while truncation of between ten and 20 amino acids resulted in a cblD-HC like cellular phenotype. These data suggest that specific regions of MMADHC are involved in differential regulation of AdoCbl and MeCbl synthesis and help better define the boundaries of these regions.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Clonagem Molecular , Cobamidas/metabolismo , Coenzimas/metabolismo , Homocistinúria/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ácido Metilmalônico/urina , Dados de Sequência Molecular , Mutação/genética , Mutação de Sentido Incorreto/genética , Vitamina B 12/metabolismo
11.
Mol Genet Metab ; 105(4): 602-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22264772

RESUMO

Isolated 3-Methylcrotonyl-CoA carboxylase deficiency (MCC deficiency) is an organic aciduria presenting with a highly variable phenotype and has been part of newborn screening programs in various countries, in particular in the US. Here we present enzymatic and genetic characterisation of 22 individuals with increased 3-hydroxyisovalerylcarnitine and/or 3-methylcrotonylglycine suggesting MCC deficiency, but only partially reduced 3-methylcrotonyl-CoA carboxylase activity. Among these, 21 carried a single mutant allele in either MCCC1 (n=20) or MCCC2 (n=1). Our results suggest that heterozygosity for such a single deleterious mutation may lead to misdiagnosis of MCC deficiency.


Assuntos
Carbono-Carbono Ligases/genética , Mutação/genética , Triagem Neonatal , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/genética , Acil Coenzima A/metabolismo , Carbono-Carbono Ligases/deficiência , Carnitina/análogos & derivados , Carnitina/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Glicina/análogos & derivados , Glicina/metabolismo , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Pele/citologia , Pele/enzimologia
12.
N Engl J Med ; 358(14): 1454-64, 2008 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-18385497

RESUMO

BACKGROUND: Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS: We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS: The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS: Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.


Assuntos
Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/genética , Ácido Metilmalônico/urina , Proteínas Mitocondriais/genética , Vitamina B 12/metabolismo , Adolescente , Sequência de Aminoácidos , Criança , Cromossomos Humanos Par 2 , Análise Mutacional de DNA , Feminino , Homocistinúria/genética , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Transporte da Membrana Mitocondrial , Dados de Sequência Molecular , Análise de Sequência de Proteína
13.
J Inherit Metab Dis ; 34(1): 121-6, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20446115

RESUMO

To date, only very few genetic disorders due to defects in lysosomal membrane transport are known. This paper reviews the identification of the underlying molecular defect causing an intriguing inborn error of vitamin B12 metabolism, namely, defective lysosomal release of vitamin B12 (cblF defect). Using microcell-mediated chromosome transfer of wild-type human chromosomes into immortalized fibroblasts from a cblF patient and genome-wide homozygosity mapping in 12 unrelated cblF patients, we identified LMBRD1 as a positional candidate gene on chromosome 6q13. Five different frameshift mutations leading to loss of function of both LMBRD1 alleles were detected in the affected patients. Transfection of the LMBRD1 wild-type construct into fibroblasts derived from cblF patients restored cobalamin coenzyme synthesis and function. LMBRD1 encodes a novel lysosomal membrane protein with significant homology to lipocalin membrane receptors. These studies give further insight into the intracellular transport of vitamins, challenge the views on lipocalin receptors, and add to our understanding of lysosomal diseases.


Assuntos
Proteínas de Transporte Nucleocitoplasmático/fisiologia , Deficiência de Vitamina B 12/genética , Vitamina B 12/metabolismo , Animais , Humanos , Modelos Biológicos , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Vitamina B 12/sangue , Deficiência de Vitamina B 12/metabolismo
14.
J Biol Chem ; 284(42): 28953-7, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19706617

RESUMO

3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha (MCCA) and smaller beta (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G-->A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G-->A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G-->A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G-->A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Carbono-Carbono Ligases/deficiência , Carbono-Carbono Ligases/genética , Processamento Alternativo , Consanguinidade , Análise Mutacional de DNA , DNA Complementar/metabolismo , Éxons , Insuficiência de Crescimento/genética , Fibroblastos/metabolismo , Humanos , Modelos Genéticos , Mutação , Mutação de Sentido Incorreto , Análise de Sequência de DNA , Pele/metabolismo
15.
J Inherit Metab Dis ; 33(1): 17-24, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20127417

RESUMO

In the cblF defect of vitamin B(12) (cobalamin) metabolism, cobalamin is trapped in lysosomes. Consequently, cobalamin coenzyme synthesis is blocked, and cofactors for methionine synthase and methylmalonyl-coenzyme A (CoA) mutase are deficient. We recently identified LMBRD1 as the causative gene located on chromosome 6q13 and showed that 18 out of 24 alleles in unrelated patients carried the deletion c.1056delG (p.L352fsX18) (Rutsch et al. (Nat Genet 41:234-239, 2009). LMBRD1 encodes the lysosomal membrane protein LMBD1, which presumably facilitates lysosomal cobalamin export. Our patient is the second child of consanguineous Turkish parents. He presented on the second day of life with cerebral seizures due to intraventricular hemorrhage. Plasma homocysteine and urinary methylmalonic acid levels were elevated, and serum cobalamin level was decreased. Synthesis of both cobalamin coenzymes was deficient in cultured skin fibroblasts. The cblF defect was confirmed by somatic complementation analysis. Sequencing of LMBRD1 revealed the novel deletion c.1405delG (p.D469fsX38) on both alleles. Real-time polymerase chain reaction (PCR) revealed reduced messenger RNA (mRNA) levels in patient fibroblasts compared with controls. Transfection of patient fibroblasts with the LMBD1 wild-type complement DNA (cDNA) rescued coenzyme synthesis and function, confirming this new deletion as an additional cause of the cblF defect. This case adds to the spectrum of clinical presentations and mutations of this rare disorder of lysosomal transport.


Assuntos
Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Alelos , Feminino , Fibroblastos/metabolismo , Homocisteína/sangue , Humanos , Lisossomos/metabolismo , Masculino , Ácido Metilmalônico/urina , Metilmalonil-CoA Mutase/genética , Turquia , Vitamina B 12/sangue
16.
Hum Mutat ; 30(7): 1072-81, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19370762

RESUMO

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B(12) (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype-phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late-onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late- or early-onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early-onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late-onset c.394C>T and c.482G>A mutations. The early-onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real-time RT-PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early-onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life.


Assuntos
Alelos , Proteínas de Transporte/genética , Regulação da Expressão Gênica , Mutação , Idade de Início , Canadá , Análise Mutacional de DNA , Feminino , Genótipo , Homocistinúria/genética , Humanos , Itália , Masculino , Oxirredutases , Fenótipo , RNA Mensageiro/análise , Deficiência de Vitamina B 12/genética
17.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1265-1272, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30682498

RESUMO

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of d-methylmalonyl-CoA and l-methylmalonyl-CoA in propionate catabolism. Autosomal recessive pathogenic variations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Šstructure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. We solved the structure of MCEE-Arg143Cys to 1.9 Šand found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Predisposição Genética para Doença/genética , Mutação , Racemases e Epimerases/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Códon sem Sentido , Cristalografia por Raios X , Homozigoto , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Racemases e Epimerases/química , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo
18.
Nat Commun ; 9(1): 2261, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891918

RESUMO

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Humanos , Cinética , Espectrometria de Massas , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , NADP/metabolismo , Fosforilação , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
19.
Hum Mutat ; 26(2): 164, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16010683

RESUMO

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder that appears to be the most frequent organic aciduria detected in tandem mass spectrometry (TMS)-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. We report mutation analysis in 28 MCC-deficient probands, 19 of whom were asymptomatic newborns detected by TMS newborn screening, and nine presented with clinical symptoms. Ten have mutations in MCCA, and 18 in MCCB. We identified 10 novel MCCA and 14 novel MCCB mutant alleles including missense, nonsense, frameshift and splice site mutations, and show that three of the missense mutations result in severely decreased MCC activity when expressed in MCC-deficient cell lines. Our data demonstrate no clear correlation between genotype and phenotype suggesting that factors other than the genotype at the MCC loci have a major influence on the phenotype of MCC deficiency.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Carbono-Carbono Ligases/deficiência , Carbono-Carbono Ligases/genética , Análise Mutacional de DNA , Técnicas de Diagnóstico Molecular/métodos , Triagem Neonatal/métodos , Alelos , Biotina/química , Genótipo , Humanos , Recém-Nascido , Mutação , Fases de Leitura Aberta , Fenótipo
20.
Hum Mutat ; 25(3): 239-47, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15714522

RESUMO

The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/deficiência , Ferredoxina-NADP Redutase/deficiência , Terapia Genética , Homocistinúria/genética , Substituição de Aminoácidos , Betaína/uso terapêutico , Encéfalo/patologia , Linhagem Celular Transformada/enzimologia , Linhagem Celular Transformada/patologia , Códon sem Sentido , Análise Mutacional de DNA , Ferredoxina-NADP Redutase/genética , Fibroblastos/enzimologia , Fibroblastos/patologia , Ácido Fólico/uso terapêutico , Genes Sintéticos , Teste de Complementação Genética , Haplótipos/genética , Homocisteína/sangue , Homocistinúria/sangue , Homocistinúria/classificação , Homocistinúria/tratamento farmacológico , Homocistinúria/enzimologia , Homocistinúria/patologia , Homocistinúria/terapia , Humanos , Hidroxocobalamina/uso terapêutico , Mutação de Sentido Incorreto , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Transfecção , População Branca/genética
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