RESUMEN
Genomic copy number variations (CNVs) can be detected by chromosomal microarray testing. However, upon final diagnosis, other methods may be recommended for a validation method to confirm CNVs. Trio analyses or carrier detection in family members are also frequently required. Previously, fluorescence in situ hybridization and/or quantitative PCR have been used; however, these methods present limitations. The purpose of this study was to establish a simple and rapid method to detect genomic copy numbers. We utilized droplet digital PCR (dPCR) with an intercalation method. Thirteen patients, who were diagnosed with MECP2 duplications via chromosomal microarray testing, were enrolled in this study. Four of their female relatives, who were verified as carriers of MECP2 duplications, were also included. Genomic copy numbers of MECP2 and IRAK1 were analyzed in comparison with reference genes: XIST and AR on the X-chromosome, and RPP30 and RPPH1 on the autosomal chromosomes. As a result, genomic copy numbers of MECP2 were rapidly and precisely detected by the dPCR system established in this study. This method can be widely applied as a diagnostic method to confirm CNVs on other chromosomal regions.
Asunto(s)
Duplicación de Gen/genética , Quinasas Asociadas a Receptores de Interleucina-1/genética , Proteína 2 de Unión a Metil-CpG/genética , Reacción en Cadena de la Polimerasa/métodos , Autoantígenos/genética , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , Femenino , Genoma/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Proteína 2 de Unión a Metil-CpG/aislamiento & purificación , Análisis por Micromatrices , Linaje , ARN Largo no Codificante/genética , Receptores Androgénicos/genética , Ribonucleasa P/genéticaRESUMEN
Methyl CpG binding protein 2 (MeCP2) is a canonical intrinsically disordered protein (IDP), that is, it lacks stable secondary structure throughout its entire polypeptide chain. Because IDPs often have the propensity to become locally ordered, we tested whether full-length MeCP2 and its constituent domains would gain secondary structure in 2,2,2-trifluoroethanol (TFE), a cosolvent that stabilizes intramolecular hydrogen bonding in proteins. The α-helix, ß-strand/turn, and unstructured content were determined as a function of TFE concentration by deconvolution of circular dichroism data. Results indicate that approximately two-thirds of the unstructured residues present in full-length MeCP2 were converted to α-helix in 70% TFE without a change in ß-strand/turn. Thus, much of the MeCP2 polypeptide chain undergoes coil-to-helix transitions under conditions that favor intrachain hydrogen bond formation. The unstructured residues of the N-terminal (NTD) and C-terminal (CTD) domains were partially converted to α-helix in 70% TFE. In contrast, the central transcription regulation domain (TRD) became almost completely α-helical in 70% TFE. Unlike the NTD, CTD, and TRD, the unstructured content of the methyl DNA binding domain and the intervening domain did not change with increasing TFE concentration. These results indicate that the coil-to-helix transitions that occur in full-length MeCP2 are localized to the NTD, CTD, and TRD, with the TRD showing the greatest tendency for helix formation. The potential relationships between intrinsic disorder, coil-to-helix transitions, and MeCP2 structure and function are discussed.
Asunto(s)
Proteínas de Unión al ADN/química , Proteína 2 de Unión a Metil-CpG/química , Pliegue de Proteína , Trifluoroetanol/química , Animales , Pollos , Proteínas de Unión al ADN/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Histonas/química , Humanos , Enlace de Hidrógeno , Proteína 2 de Unión a Metil-CpG/aislamiento & purificación , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solventes/química , Relación Estructura-Actividad , Trifluoroetanol/farmacologíaRESUMEN
Myelination is critical for normal functioning of mammalian central nervous system. Central nervous system myelin is created and maintained by oligodendrocytes. Protein expression patterns change as the oligodendrocyte progenitors differentiate into myelinating oligodendrocytes. Several proteins, including the cell surface proteoglycan NG2, proteolipid protein, myelin basic protein, and myelin-associated glycoprotein are critical for normal myelination. The molecular regulation of myelination is for the most part unknown, although several transcription factors have been identified as regulating myelin protein expression. We have identified a known transcriptional regulator, methyl-CpG-binding protein 2, as regulating myelin specific gene expression in a transgenic mouse. Our findings show a potential role for myelin in the pathophysiology of methyl-CpG-binding protein 2 mutation-associated disorders.
Asunto(s)
Discapacidades del Desarrollo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteína 2 de Unión a Metil-CpG/genética , Vaina de Mielina/genética , Animales , Animales Recién Nacidos , Química Encefálica/genética , Células Cultivadas , Discapacidades del Desarrollo/metabolismo , Femenino , Proteína 2 de Unión a Metil-CpG/biosíntesis , Proteína 2 de Unión a Metil-CpG/deficiencia , Proteína 2 de Unión a Metil-CpG/aislamiento & purificación , Ratones , Ratones Noqueados , Ratones Transgénicos , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the wild type or mutant MECP2 in each of their cells. To test the hypothesis that MECP2 mutations result in genome-wide transcriptional deregulation and identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we performed gene expression profiling of pure populations of untransformed T-lymphocytes that express either a mutant or a wild-type allele. METHODS: Single T lymphocytes from a patient with a c.473C>T (p.T158M) mutation and one with a c.1308-1309delTC mutation were subcloned and subjected to short term culture. Gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. RESULTS: Expression profiling yielded 44 upregulated genes and 77 downregulated genes. We compared this gene list with expression profiles of independent microarray experiments in cells and tissues of RTT patients and mouse models with Mecp2 mutations. These comparisons identified a candidate MeCP2 target gene, SPOCK1, downregulated in two independent microarray experiments, but its expression was not altered by quantitative RT-PCR analysis on brain tissues from a RTT mouse model. CONCLUSION: Initial expression profiling from T-cell clones of RTT patients identified a list of potential MeCP2 target genes. Further detailed analysis and comparison to independent microarray experiments did not confirm significantly altered expression of most candidate genes. These results are consistent with other reported data.
Asunto(s)
Perfilación de la Expresión Génica , Linfocitos/metabolismo , Síndrome de Rett/genética , Animales , Células Cultivadas , Células Clonales/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/aislamiento & purificación , Ratones , Análisis por Micromatrices , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteoglicanos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Síndrome de Rett/inmunologíaRESUMEN
DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15).