Altered DNA Methylation in Leukocytes with Trisomy 21

Kristi Kerkel,Nicole Schupf,Vundavalli Murty,Kota Hatta,Ali Torkamani,Deborah Pang,Richard P Mayeux,Alexander Kratz,Wayne Silverman,Benjamin Tycko,Nicholas J Schork,Edmund C Jenkins,Mark Minden,Warren B Zigman,B Anne Croy,Martha Salas

doi:10.1371/journal.pgen.1001212

Abstract

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.

Highlights

It is 5 decades since Down syndrome (DS) was first shown to result from trisomy 21 [1,2], and some progress has been made toward understanding the genes that contribute to the complex array of DS phenotypes – mostly by studying the effects of the trisomy on transcriptional profiles in humans and mice and by creating transgenic and trans-chromosomal mouse models [3,4]
DNA methylation is a mechanism that might contribute to these abnormalities
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 different methylation and 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

Summary

Introduction

It is 5 decades since Down syndrome (DS) was first shown to result from trisomy 21 [1,2], and some progress has been made toward understanding the genes that contribute to the complex array of DS phenotypes – mostly by studying the effects of the trisomy on transcriptional profiles in humans and mice and by creating transgenic and trans-chromosomal mouse models [3,4]. There are multiple blood cell-related phenotypes including leukemoid reactions and childhood leukemias, macrocytosis with or without anemia, a markedly increased incidence of autoimmune disorders, and increased susceptibility to recurrent bacterial and viral infections [5,6,7,8,9,10] All of these abnormalities must reflect the downstream responses of human cells and tissues to the chromosome 21 aneuploidy. Gain of methylation at cytosines in CpG dinucleotides in promoter-associated CpG islands (CGI’s) can enforce dosage compensation in X-inactivation, and methylation in other types of CG-rich sequences including intragenic sequences and insulator elements can affect expression and functional gene dosage at imprinted loci With these simple ideas in mind we set out to ask whether gains or losses of genomic DNA methylation might occur as a downstream consequence of trisomy 21 in blood cells from adults with DS. We show that a small group of genes, distributed across various chromosomes and not over-represented on chromosome 21, are consistently altered

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Discussion

Conclusion