RESUMO
To estimate the error rate of the gene expression machinery and its possible age-related increase, we compared the occurrence of polymerase errors during replication and transcription in (A)/(T) runs, in DNA and RNA of young and old individuals and of early- and late-passage cultured fibroblasts. We analyzed three human genes: TPRD, TGFBR2, and ATRX containing stretches of (A)8, (A)10, and (T)13, respectively. The error rate was determined by sequencing 100 cloned PCR or RT-PCR fragments from each DNA and RNA sample. The error rates in replication and transcription increased with the stretch length. The pooled error rates for genomic DNA were: TPRD (A)8, TGFBR2 (A)10, and ATRX (T)13: 1%+/-0.41, 15.8%+/-1.3, and 31.3%+/-2.9, while those for RNA were: 3.8%+/-0.5, 19.3%+/-2.1, and 54.3%+/-1.8, respectively. The deletions of one nucleotide were the most frequent errors. In the replication analysis, a significant difference was found in old versus young individuals for the ATRX (T)13. In the transcription analysis, significantly higher error rates were obtained in old versus young individuals for the TPRD (A)8 and TGFBR2 (A)10. For these genes, the error rate in RNA isolated from fibroblasts was significantly higher than that in blood. The data show a trend of age-related increase in replication/transcription errors; however further studies are necessary to confirm this hypothesis, since the sample size is small. This imperfect fidelity of the gene expression process may explain the evolutionary disadvantage of nucleotide repeats within coding sequences, and that these repeats are targets for mutations in human diseases.
Assuntos
Envelhecimento/genética , DNA Helicases , Replicação do DNA/genética , Proteínas Nucleares , Transcrição Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Linhagem Celular , DNA/genética , Dano ao DNA , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Genoma Humano , Humanos , Mutação , Proteínas Serina-Treonina Quinases , Proteínas/genética , RNA/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/genética , Proteína Nuclear Ligada ao XRESUMO
Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.
Assuntos
DNA Satélite/genética , Surdez/congênito , Surdez/enzimologia , Genes Recessivos/genética , Proteínas de Membrana , Mutagênese Insercional/genética , Proteínas de Neoplasias , Serina Endopeptidases/genética , Adulto , Idade de Início , Sequência de Bases , Criança , Consanguinidade , Mapeamento de Sequências Contíguas , Análise Mutacional de DNA , Surdez/epidemiologia , Surdez/genética , Éxons/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Hibridização in Situ Fluorescente , Israel , Masculino , Dados de Sequência Molecular , Paquistão , Linhagem , Sítios de Splice de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Serina Endopeptidases/metabolismoRESUMO
Trisomy 21, or Down syndrome (DS), is the most common genetic cause of mental retardation. Changes in the neuropathology, neurochemistry, neurophysiology, and neuropharmacology of DS patients' brains indicate that there is probably abnormal development and maintenance of central nervous system structure and function. The segmental trisomy mouse (Ts65Dn) is a model of DS that shows analogous neurobehavioral defects. We have studied the global gene expression profiles of normal and Ts65Dn male and normal female mice brains (P30) using the serial analysis of gene expression (SAGE) technique. From the combined sample we collected a total of 152,791 RNA tags and observed 45,856 unique tags in the mouse brain transcriptome. There are 14 ribosomal protein genes (nine under expressed) among the 330 statistically significant differences between normal male and Ts65Dn male brains, which possibly implies abnormal ribosomal biogenesis in the development and maintenance of DS phenotypes. This study contributes to the establishment of a mouse brain transcriptome and provides the first overall analysis of the differences in gene expression in aneuploid versus normal mammalian brain cells.
Assuntos
Química Encefálica/genética , Síndrome de Down/genética , Perfilação da Expressão Gênica/métodos , Transcrição Gênica , Animais , Northern Blotting , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Biblioteca Gênica , Marcadores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Sitios de Sequências Rotuladas , Trissomia/genéticaRESUMO
Autoimmune polyendocrinopathy syndrome type 1 (APS-1; MIM# 240300) is a rare autosomal recessively inherited disease characterised by destructive autoimmune diseases of endocrine glands. The gene responsible for APS-1, known as AIRE (for autoimmune regulator), was recently identified and contains motifs suggestive of a transcription regulator. To date, nine APS-1-associated mutations have been identified in the AIRE gene, including two common mutations R257X and 1094-1106del. In addition to these two mutations, we report seven novel mutations in 16 APS-1 patients from North America. We found that 1094-1106del and R257X were the most common mutations in this population of mixed geoethnic origin, accounting for 17/32 and 4/32 alleles, respectively. Haplotype analyses suggest that both are recurrent mutations, occurring on several different haplotypes with closely linked markers. All the novel mutations appear to be rare, occurring in only single APS-1 families. After examining all coding sequences and exon/intron boundaries of the AIRE gene, the other APS-1 allele remained unidentified in three patients. Genotype-phenotype correlations for APS-1 remain difficult, suggesting that other genetic or environmental factors, or both, influence the clinical presentation and disease progression in individual APS-1 patients.
Assuntos
Deleção de Genes , Poliendocrinopatias Autoimunes/genética , Fatores de Transcrição/genética , Análise Mutacional de DNA , Feminino , Genótipo , Haploidia , Humanos , Masculino , América do Norte/etnologia , Fenótipo , Poliendocrinopatias Autoimunes/etnologia , Deleção de Sequência , Proteína AIRERESUMO
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.