Findings from Stanford University, Medical Department advance knowledge in quantitative PCR assays
2007 OCT 22 -- "' MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand movements and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures," scientists in the United States report. "Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy. To identify the pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out in cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes. Of several hundred genes with altered expression levels in the mutants, twice as many were increased than decreased, and only 27 were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with the exon 3 deletion (Mecp2(tm1.1Jae)) than in mice with the larger deletion (Mecp2(tm1.1Bird)). Between the mutants, few genes overlapped at each time point. Real- time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase; Reln, encoding an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2. Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2- deficient mice and individuals with Rett syndrome," wrote C. Jordan and colleagues, Stanford University, Medical Department. The researchers concluded: "Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on." Jordan and colleagues published their study in BMC Medical Genetics (Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets. BMC Medical Genetics, 2007;8():36). For additional information, contact U. Francke, Stanford University, School Medical, Dept. of Genetics, Stanford, CA 94305, USA. The publisher's contact information for the journal BMC Medical Genetics is: Biomedical Central Ltd., Middlesex House, 34-42 Cleveland St., London W1T 4LB, England. Keywords: United States, Stanford, Quantitative PCR Assays, DNA, Diagnostics, Genetics, Medical Device, Quantitative PCR Assay, Rett Syndrome, Stanford University, Medical Department. This article was prepared by Proteomics Weekly editors from staff and other reports. Copyright 2007, Proteomics Weekly via NewsRx.com.
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