Related Experiment Video
Updated: Aug 7, 2026
Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations
Published on: January 19, 2017
Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients
Ivan J Delgado1, Dong Sun Kim, Karen N Thatcher
1Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA. idelgado@identigene.com
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.
Insights
Rett syndrome (RTT) research identified potential methyl-CpG-binding protein 2 (MECP2) target genes in T-cells. Further analysis did not confirm widespread gene expression changes in RTT patients.
Area of Science:
- Genetics
- Molecular Biology
- Neuroscience
Background:
- Rett syndrome (RTT) is linked to mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, crucial for transcriptional regulation.
- X-chromosome inactivation (XCI) in females leads to functional mosaicism, complicating the study of MECP2's role.
Purpose of the Study:
- To investigate genome-wide transcriptional deregulation caused by MECP2 mutations.
- To identify MECP2 target genes using a system that bypasses XCI-induced mosaicism.
Main Methods:
- Gene expression profiling was performed on pure T-lymphocyte clones from RTT patients with specific MECP2 mutations.
- Microarray analysis compared gene expression between wild-type and mutant MECP2 T-cell clones.
Main Results:
- Expression profiling identified 44 upregulated and 77 downregulated genes.
- A candidate MECP2 target gene, SPOCK1, was downregulated in RTT samples but not confirmed by RT-PCR in mouse brain tissue.
- Most candidate genes identified did not show significantly altered expression in further analyses.
Conclusions:
- Initial T-cell gene expression profiling identified potential MECP2 target genes in Rett syndrome.
- Subsequent detailed analysis and comparison with independent datasets failed to confirm most candidate genes, consistent with existing literature.
