RESUMO
The Na-Cl cotransporter (NCC) in the distal convoluted tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels >30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Membro 3 da Família 12 de Carreador de Soluto/urina , Idoso , Animais , Exossomos/química , Feminino , Síndrome de Gitelman/urina , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fosforilação , Pseudo-Hipoaldosteronismo/urina , Inibidores de Simportadores de Cloreto de SódioRESUMO
Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although â¼ 15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785,720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P = 5.3 × 10(-10)). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P = 1.2 × 10(-43), odds ratio = 190.8, 95% confidence interval = 71.7-507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.
Assuntos
Adenosina Trifosfatases/genética , Estudo de Associação Genômica Ampla , Doença de Moyamoya/genética , Polimorfismo de Nucleotídeo Único , Animais , Povo Asiático/genética , Linhagem Celular , Família , Predisposição Genética para Doença , Haplótipos , Humanos , Camundongos , Modelos Biológicos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia , Dedos de Zinco/genéticaRESUMO
Glycine encephalopathy (GE) is caused by an inherited deficiency of the glycine cleavage system (GCS) and characterized by accumulation of glycine in body fluids and various neurologic symptoms. Coma and convulsions develop in neonates in typical GE while psychomotor retardation and behavioral abnormalities in infancy and childhood are observed in mild GE. Recently, we have established a transgenic mouse line (low-GCS) with reduced GCS activity (29% of wild-type (WT) C57BL/6) and accumulation of glycine in the brain (Stroke, 2007; 38:2157). The purpose of the present study is to characterize behavioral features of the low-GCS mouse as a model of mild GE. Two other transgenic mouse lines were also analyzed: high-GCS mice with elevated GCS activity and low-GCS-2 mice with reduced GCS activity. As compared with controls, low-GCS mice manifested increased seizure susceptibility, aggressiveness and anxiety-like activity, which resembled abnormal behaviors reported in mild GE, whereas high-GCS mice were less sensitive to seizures, hypoactive and less anxious. Antagonists for the glycine-binding site of the N-methyl-D-aspartate receptor significantly ameliorated elevated locomotor activity and seizure susceptibility in the low-GCS mice. Our results suggest the usefulness of low-GCS mice as a mouse model for mild GE and a novel therapeutic strategy.
Assuntos
Aminoácido Oxirredutases/metabolismo , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/fisiopatologia , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Glicina/metabolismo , Complexos Multienzimáticos/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transferases/metabolismo , Agressão/efeitos dos fármacos , Agressão/fisiologia , Aminoácido Oxirredutases/genética , Animais , Ansiedade/tratamento farmacológico , Ansiedade/fisiopatologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encefalopatias Metabólicas/tratamento farmacológico , Proteínas de Transporte/genética , Maleato de Dizocilpina/farmacologia , Maleato de Dizocilpina/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Glicina/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Complexos Multienzimáticos/genética , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêutico , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/fisiopatologia , Transferases/genéticaRESUMO
BACKGROUND: Non-ketotic hyperglycinaemia (NKH) is an inborn error of metabolism characterised by accumulation of glycine in body fluids and various neurological symptoms. NKH is caused by deficiency of the glycine cleavage multienzyme system with three specific components encoded by GLDC, AMT and GCSH. Most patients are deficient of the enzymatic activity of glycine decarboxylase, which is encoded by GLDC. Our recent study has suggested that there are a considerable number of GLDC mutations which are not identified by the standard exon-sequencing method. METHODS: A screening system for GLDC deletions by multiplex ligation-dependent probe amplification (MLPA) has been developed. Two distinct cohorts of patients with typical NKH were screened by this METHOD: the first cohort consisted of 45 families with no identified AMT or GCSH mutations, and the second cohort was comprised of 20 patients from the UK who were not prescreened for AMT mutations. RESULTS: GLDC deletions were identified in 16 of 90 alleles (18%) in the first cohort and in 9 of 40 alleles (22.5%) in the second cohort. 14 different types of deletions of various lengths were identified, including one allele where all 25 exons were missing. Flanking sequences of interstitial deletions in five patients were determined, and Alu-mediated recombination was identified in three of five patients. CONCLUSIONS: GLDC deletions are a significant cause of NKH, and the MLPA analysis is a valuable first-line screening for NKH genetic testing.
Assuntos
Glicina Desidrogenase (Descarboxilante)/genética , Hiperglicinemia não Cetótica/genética , Proteínas Mutantes/genética , Deleção de Sequência , Idade de Início , Alelos , Sequência de Bases , Estudos de Coortes , Análise Mutacional de DNA/métodos , Sondas de DNA , Éxons/genética , Frequência do Gene , Testes Genéticos , Humanos , Hiperglicinemia não Cetótica/diagnóstico , Hiperglicinemia não Cetótica/enzimologia , Recém-Nascido , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Mutação Puntual , Reação em Cadeia da PolimeraseRESUMO
Mental retardation (MR) is a common trait, affecting approximately 2-3% of individuals in the general population. Although the etiology of MR remains largely unknown, genetics apparently play a major role. Recent molecular studies of X-linked form of MR in European and North American countries have revealed 24 nonsyndromic X-linked mental retardation (NS-XLMR) genes including FTSJ1, a human homolog of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ gene. Here we identified a novel FTSJ1 mutation in an XLMR family through mutation screening of a cohort of 73 unrelated Japanese male probands with MR. Sequence analysis of the proband and his mother revealed a G > A substitution at the consensus for the donor splicing site in intron 8 (c.571 + 1G > A) of FTSJ1. This mutation prevented the removal intron 8 from the pre-mRNA, thereby leading to a frameshift in the mutant FTSJ1 mRNA and resulting in a premature termination in exon 9. Quantitative RT-PCR showed a significant reduction of mutant FTSJ1 mRNA in the patient's lymphoblast cells, which was restored by treatment with cycloheximide, a potent inhibitor of nonsense-mediated mRNA decay (NMD). Therefore, mRNAs carrying this mutation are likely subject to degradation by NMD. Together, loss-of-function of FTSJ1 may be a mechanism for the cognitive dysfunction observed in this family. Our study also suggested that the FTSJ1 mutation probably accounts for XLMR in Japanese at a similar frequency (1-2%) as in Europeans.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Metiltransferases/genética , Mutação , Proteínas Nucleares/genética , Adolescente , Povo Asiático , Análise Mutacional de DNA , Humanos , Masculino , Metiltransferases/deficiência , Núcleo Familiar , Proteínas Nucleares/deficiência , Sítios de Splice de RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/sangue , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Bronchial asthma is a chronic airway disorder characterized by bronchial inflammation. Oxidative stress is a key component of inflammation. Glutathione S-transferase P1 (GSTP1), the abundant isoform of glutathione S-transferases (GSTs) in lung epithelium, plays a key role in cellular protection against oxidative stress. Several studies have shown that the GSTP1 geneis involved in the pathogenesis of asthma and a gene-gene interaction may occur within the GST gene superfamily. METHODS: We screened single-nucleotide polymorphisms (SNPs) at the GSTP1 locus and performed an association study in the Japanese population using two independent case-control groups (group 1: 391 pediatric patients with asthma, 462 adult patients with asthma, and 639 controls, and group 2: 115 pediatric patients with asthma and 184 controls). The effect of GSTM1 null/present genotype on the association between GSTP1 Ile105Val and asthma was also investigated. RESULTS: We identified 20 SNPs at this locus and found this region consisted of one linkage disequilibrium block represented by four SNPs (tag SNPs). The association between the Ile105Val polymorphism in the GSTP1 gene and childhood asthma was significant in both groups (p = 0.047 in group 1, and p = 0.021 in group 2). This association was only significant in patients with GSTM1-positive genotype in both groups (group 1: GSTM1 present p = 0.013 and GSTM1 null p = 0.925, and group 2: GSTM1 present p = 0.015 and GSTM1 null p = 0.362). CONCLUSIONS: These findings suggest that the GSTP1 gene is a childhood asthma susceptible gene, and the GSTM1 gene is a modifier gene of GSTP1 for the risk of childhood asthma in the Japanese population.
Assuntos
Asma/genética , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Adolescente , Adulto , Idoso , Asma/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Smith-Magenis syndrome is characterized by multiple congenital anomalies and mental retardation caused by the heterozygous deletion of chromosomal region 17p11.2. We present a long-term follow-up study of a girl with Smith-Magenis syndrome and West syndrome. West syndrome became apparent at 7 months of age. Since then, mental retardation, particularly in terms of language development, became increasingly more obvious. The patient's spasms and hypsarrhythmia disappeared after a course of adrenocorticotropic hormone therapy, but focal seizures reappeared at the age of 3 years and 3 months. Her craniofacial dysmorphia and mental retardation became increasingly evident compared to her condition at the onset of West syndrome. Chromosome analysis detected the characteristic 17p deletion, which was then confirmed via fluorescent in situ hybridization analysis. This is the second report of a patient with Smith-Magenis syndrome and West syndrome; taken together, these results suggest that Smith-Magenis syndrome may be a further cause of West syndrome.
Assuntos
Anormalidades Múltiplas , Deficiência Intelectual , Espasmos Infantis , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/fisiopatologia , Hormônio Adrenocorticotrópico/uso terapêutico , Encéfalo/patologia , Encéfalo/fisiopatologia , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 17 , Eletroencefalografia , Feminino , Seguimentos , Hormônios/uso terapêutico , Humanos , Hibridização in Situ Fluorescente , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Deficiência Intelectual/fisiopatologia , Imageamento por Ressonância Magnética , Análise de Sequência de DNA , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/patologia , Espasmos Infantis/fisiopatologia , SíndromeRESUMO
Pyridoxine dependent seizure (PDS) is a disorder of neonates or infants with autosomal recessive inheritance characterized by seizures, which responds to pharmacological dose of pyridoxine. Recently, mutations have been identified in the ALDH7A1 gene in Caucasian families with PDS. To elucidate further the genetic background of PDS, we screened for ALDH7A1 mutations in five PDS families (patients 1-5) that included four Orientals. Diagnosis as having PDS was confirmed by pyridoxine-withdrawal test. Exon sequencing analysis of patients 1-4 revealed eight ALDH7A1 mutations in compound heterozygous forms: five missense mutations, one nonsense mutation, one point mutation at the splicing donor site in intron 1, and a 1937-bp genomic deletion. The deletion included the entire exon 17, which was flanked by two Alu elements in introns 16 and 17. None of the mutations was found in 100 control chromosomes. In patient 5, no mutation was found by the exon sequencing analysis. Furthermore, expression level or nucleotide sequences of ALDH7A1 mRNA in lymphoblasts were normal. Plasma pipecolic acid concentration was not elevated in patient 5. These observations suggest that ALDH7A1 mutation is unlikely to be responsible for patient 5. Abnormal metabolism of GABA/glutamate in brain has long been suggested as the underlying pathophysiology of PDS. CSF glutamate concentration was elevated during the off-pyridoxine period in patient 3, but not in patient 2 or 5. These results suggest allelic and non-allelic heterogeneities of PDS, and that the CSF glutamate elevation does not directly correlate with the presence of ALDH7A1 mutations.
Assuntos
Aldeído Desidrogenase/genética , Análise Mutacional de DNA , Polimorfismo de Nucleotídeo Único , Piridoxina/toxicidade , Convulsões/enzimologia , Convulsões/etiologia , Genes Recessivos , Ácido Glutâmico/líquido cefalorraquidiano , Humanos , Recém-Nascido , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/genéticaRESUMO
OBJECTIVE: It is currently problematic to confirm the clinical diagnosis of glycine encephalopathy, requiring either invasive liver biopsy for enzymatic analysis of the glycine cleavage system or exhaustive mutation analysis. Because the glycine cleavage system breaks down glycine generating carbon dioxide, we suppose that the glycine cleavage system activity could be evaluated in vivo by measuring exhaled (13)CO(2) after administration of [1-(13)C]glycine. METHODS: The [1-(13)C]glycine breath test was performed in 10 control subjects and 5 glycine encephalopathy patients with GLDC mutation, including 1 patient with mild glycine encephalopathy. RESULTS: All the patients showed lower (13)CO(2) excretion than any control subject. INTERPRETATION: Not only typical GE but also atypical GE can be reliably diagnosed by the (13)C-glycine breath test. Because it is rapid, non-invasive, and requires little expertise, the breath test could be useful as a standard test for diagnosing GE.
Assuntos
Dano Encefálico Crônico/diagnóstico , Glicina/análise , Glicina/fisiologia , Hiperglicemia/diagnóstico , Adolescente , Dano Encefálico Crônico/genética , Testes Respiratórios , Dióxido de Carbono/metabolismo , Isótopos de Carbono , Criança , Pré-Escolar , Feminino , Glicina/metabolismo , Humanos , Hiperglicemia/genética , Lactente , Masculino , Oxirredução , Reprodutibilidade dos TestesRESUMO
Autosomal-dominant, nonsyndromic hearing impairment is clinically and genetically heterogeneous. We encountered a large Japanese pedigree in which nonsyndromic hearing loss was inherited in an autosomal-dominant fashion. A genome-wide linkage study indicated linkage to the DFNA2 locus on chromosome 1p34. Mutational analysis of KCNQ4 encoding a potassium channel revealed a novel one-base deletion in exon 1, c.211delC, which generated a profoundly truncated protein without transmembrane domains (p.Q71fsX138). Previously, six missense mutations and one 13-base deletion, c.211_223del, had been reported in KCNQ4. Patients with the KCNQ4 missense mutations had younger-onset and more profound hearing loss than patients with the 211_223del mutation. In our current study, 12 individuals with the c.211delC mutation manifested late-onset and pure high-frequency hearing loss. Our results support the genotype-phenotype correlation that the KCNQ4 deletions are associated with later-onset and milder hearing impairment than the missense mutations. The phenotypic difference may be caused by the difference in pathogenic mechanisms: haploinsufficiency in deletions and dominant-negative effect in missense mutations.
Assuntos
Perda Auditiva de Alta Frequência/genética , Canais de Potássio KCNQ/genética , Deleção de Sequência , Povo Asiático , Sequência de Bases , Análise Mutacional de DNA , Genótipo , Perda Auditiva de Alta Frequência/diagnóstico , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , FenótipoRESUMO
Bronchial asthma (BA) is a common chronic inflammatory disease characterized by hyperresponsive airways, excess mucus production, eosinophil activation, and the production of IgE. The complement system plays an immunoregulatory role at the interface of innate and acquired immunities. Recent studies have provided evidence that C3, C3a receptor, and C5 are linked to airway hyperresponsiveness. To determine whether genetic variations in the genes of the complement system affect susceptibility to BA, we screened single nucleotide polymorphisms (SNPs) in C3, C5, the C3a receptor gene (C3AR1), and the C5a receptor gene (C5R1) and performed association studies in the Japanese population. The results of this SNP case-control study suggested an association between 4896C/T in the C3 gene and atopic childhood BA (P = 0.0078) as well as adult BA (P = 0.010). When patient data were stratified according to elevated total IgE levels, 4896C/T was more closely associated with adult BA (P = 0.0016). A patient-only association study suggested that severity of childhood BA was associated with 1526G/A of the C3AR1 gene (P = 0.0057). We identified a high-risk haplotype of the C3 gene for childhood (P = 0.0021) and adult BA (P = 0.0058) and a low-risk haplotype for adult BA (P = 0.00011). We also identified a haplotype of the C5 gene that was protective against childhood BA (P = 1.4 x 10(-6)) and adult BA (P = 0.00063). These results suggest that the C3 and C5 pathways of the complement system play important roles in the pathogenesis of BA and that polymorphisms of these genes affect susceptibility to BA.
Assuntos
Asma/genética , Complemento C3/genética , Complemento C5/genética , Variação Genética , Receptores de Complemento/genética , Adulto , Análise de Variância , Criança , Predisposição Genética para Doença/genética , Haplótipos/genética , Humanos , Japão , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
We devised a simple method using a TaqMan fluorogenic probe for detection of a prevalent G6PT1 mutation W118R among Japanese patients with glycogen storage disease type Ib. The W118R mutation was detected in three of six newly diagnosed Japanese patients. The W118R-negative alleles were screened for causative mutations by sequencing analysis, revealing five novel mutations. The genetic tests using the simple TaqMan method coupled with sequencing analysis would facilitate the early diagnosis of this disorder.
Assuntos
Antiporters/genética , Doença de Depósito de Glicogênio Tipo I/genética , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Técnicas de Amplificação de Ácido Nucleico/métodos , Adulto , Alelos , Sequência de Bases , Análise Mutacional de DNA , Feminino , Testes Genéticos , Humanos , Lactente , Japão , Masculino , Dados de Sequência Molecular , Taq Polimerase/metabolismoRESUMO
Several studies have shown linkage of chromosome region 12q13-24 to bronchial asthma and related phenotypes in ethnically diverse populations. In the Japanese population, a genome-wide study failed to show strong evidence of linkage of this region. Chromosome 12 genes that showed association with the disease in at least one report include: the signal transducer and activator of transcription 6 gene ( STAT6), the nitrogen oxide synthetase 1 gene ( NOS1), the interferon gamma gene ( IFNG), and the activation-induced cytidine deaminase gene ( AICDA). To evaluate the linkage between chromosome 12 and childhood asthma in the Japanese population, we performed sib-pair linkage analysis on childhood asthma families using 18 microsatellite markers on chromosome 12. To investigate association between chromosome 12 candidate genes and asthma, distributions of alleles and genotypes of repeat polymorphisms of STAT6, NOS1, and IFNG were compared between controls and patients. Single nucleotide polymorphism of AICDA was also investigated. Chromosome region 12q24.23-q24.33 showed suggestive linkage to asthma. The NOS1 intron 2 GT repeat and STAT6 exon 1 GT repeat were associated with asthma. Neither the IFNG intron 1 CA repeat nor 465C/T of AICDA showed any association with asthma. Our results suggest that NOS1 and STAT6 are asthma-susceptibility genes and that chromosome region 12q24.23-q24.33 contains other susceptibility gene(s).