A proof-of-principle demonstration of a novel microarray-based method for quantifying DNA methylation levels.
Demethylation of CD11a (ITGAL; GeneID:3683; HGNC: 6148) and CD70 (TNFSF7; GeneID:970; HGNC:11937) regulatory regions in CD4(+) T cells contributes to the development of autoreactivity and autoantibody overstimulation in systemic lupus erythematosus (SLE). In this study, we present a novel approach for measuring the methylation status of CD11a and CD70 promoter sequences. The procedure combines the standard method of bisulfite conversion of methylated CpG pairs with high-throughput oligonucleotide microarray-based technology that allows for rapid quantification of deoxycytosine and deoxymethylcytosine content in bisulfite-treated DNA samples. The microarrays were first used to generate a standard curve from fully methylated and fully unmethylated DNA samples using a one-dimensional linear regression equation that calculated fluorescence emission as a function of methylation levels. The methylation status of the CD70 and CD11a promoters in SLE and control CD4(+) T cell samples were measured, and the microarray prediction was found to be highly accurate when compared to bisulfite sequencing. Furthermore, the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples. These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid, and cost effective diagnostic and prognostic test for SLE.
Zhang
Xiujuan
X
Department of Dermatology, Epigenetic Research Center, Second Xiangya Hospital, Central South University, No. 139 Renmin Middle Rd., Changsha, Hunan, 410011, People's Republic of China.
Zhou
Dongrui
D
Zhao
Ming
M
Luo
Yongqi
Y
Zhang
Peng
P
Lu
Zuhong
Z
Lu
Qianjin
Q
D000328:Adult; D001483:Base Sequence; D040881:CD11a Antigen / Q000378:metabolism; D053282:CD27 Ligand / Q000378:metabolism; D019175:DNA Methylation; D017931:DNA Primers; D005260:Female; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000378:metabolism*; D008297:Male; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic
D040881:CD11a Antigen; D053282:CD27 Ligand; D017931:DNA Primers