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.
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.