Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

Abstract

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.

Figure 1

Experimental design of T-lymphocyte clone isolation. Two technical replicate hybridizations for the wild type MECP2 and mutant MECP2-expressing clonal cell-cultures (RT211 with p.T158M mutation and RT208 with c. 1308-1309delTC mutation) were performed, as well as a biological replicate hybridization with an independent clone of patient RT211. (PBLy = peripheral blood lymphocytes; Ly clone = T-lymphocyte clone; * indicates unused in array hybridization analysis because of insufficient RNA quality)

Figure 2

Quantitative RT-PCR (qPCR) analysis of Spock1 expression. Quantitative RT-PCR analysis of the expression of (A.) human SPOCK1 in the clonal lymphocyte RNAs (Mt Ly, Wt Ly) used for the microarray experiments and of mouse Spock1 in differentiating wild-type (Wt EB) and Mecp2^R308/Y mutant (Mt EB) male mouse embryoid bodies; (B.) cerebral cortex (Wt Cx, Mt Cx), cerebellum (Wt Cb, Mt Cb) and olfactory bulb (Wt OB, Mt OB) of adult (5 week-old) wild-type and Mecp2^R308/Y mutant male mice; and (C.) E16.5 embryo head (Wt head, Mt head) and body (Wt body, Mt body) (4 independent samples were tested for each tissue and genotype). The Y-axis shows the average fold change in expression +/- standard deviation) of mutant compared to wild-type, which is set as one-fold baseline, except for the Ly samples, where the one-fold baseline was set in the mutant for figure clarity (light bars = wild-type; dark bars = mutant), p-values are given under each respective graph.