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1.  Altered DNA Methylation in Leukocytes with Trisomy 21 
PLoS Genetics  2010;6(11):e1001212.
The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2′deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.
Author Summary
Down syndrome (DS; trisomy 21) is caused by the gain of a single extra chromosome 21. However, the mechanisms by which this extra chromosome produces the medical abnormalities seen in DS, including not only mental retardation but also susceptibility to autoimmune diseases and recurrent infections, are still not understood. DNA methylation is a mechanism that might contribute to these abnormalities. To test this possibility, we profiled DNA methylation in white blood cells from adults with DS and normal controls and found recurrent abnormalities of gene methylation in DS, with several of the differentially methylated genes having roles in blood cells. Among the genes with hypo- or hyper-methylation in white blood cells or purified T-lymphocytes from adults with DS, compared to these same types of cells from normal adults, were TMEM131, TCF7, CD3Z, SH3BP2, EIF4E, SUMO3, CPT1B, NOD2/CARD15, NPDC1, and PLD6. Several of these genes showed not only different methylation but also different expression in DS versus control blood cells, which was recapitulated by exposing normal white blood cells to a demethylating drug. These findings show that altered DNA methylation of a specific group of genes is a fundamental cellular response to the gain of an extra chromosome 21 in humans. The abnormally methylated genes identified here may contribute to immune system abnormalities in people with DS.
PMCID: PMC2987931  PMID: 21124956
2.  TP73 allelic expression in human brain and allele frequencies in Alzheimer's disease 
BMC Medical Genetics  2004;5:14.
The p73 protein, a paralogue of the p53 tumor suppressor, is essential for normal development and survival of neurons. TP73 is therefore of interest as a candidate gene for Alzheimer's disease (AD) susceptibility. TP73 mRNA is transcribed from three promoters, termed P1 – P3, and there is evidence for an additional complexity in its regulation, namely, a variable allelic expression bias in some human tissues.
We utilized RT-PCR/RFLP and direct cDNA sequencing to measure allele-specific expression of TP73 mRNA, SNP genotyping to assess genetic associations with AD, and promoter-reporter assays to assess allele-specific TP73 promoter activity.
Using a coding-neutral BanI polymorphism in TP73 exon 5 as an allelic marker, we found a pronounced allelic expression bias in one adult brain hippocampus, while 3 other brains (two adult; one fetal) showed approximately equal expression from both alleles. In a tri-ethnic elderly population of African-Americans, Caribbean Hispanics and Caucasians, a G/A single nucleotide polymorphism (SNP) at -386 in the TP73 P3 promoter was weakly but significantly associated with AD (crude O.R. for AD given any -386G allele 1.7; C.I. 1.2–2.5; after adjusting for age and education O.R. 1.5; C.I. 1.1–2.3, N= 1191). The frequency of the -386G allele varied by ethnicity and was highest in African-Americans and lowest in Caucasians. No significant differences in basal P3 promoter activity were detected comparing -386G vs. -386A promoter-luciferase constructs in human SK-NSH-N neuroblastoma cells.
There is a reproducible allelic expression bias in mRNA expression from the TP73 gene in some, though not all, adult human brains, and inter-individual variation in regulatory sequences of the TP73 locus may affect susceptibility to AD. However, additional studies will be necessary to exclude genetic admixture as an alternative explanation for the observed associations.
PMCID: PMC420466  PMID: 15175114

Results 1-2 (2)