©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (4): 6228-6239 (2013) Aberrant promoter methylation of the CD4 gene in peripheral blood cells of mastitic dairy cows X.S. Wang 1 , Y. Zhang 1 , Y.H. He 1 , P.P. Ma 1 , L.J. Fan 1 , Y.C. Wang 1 , Y.I. Zhang 1 , D.X. Sun 1 , S.L. Zhang 1 , C.D. Wang 1 , J.Z. Song 2 and Y. Yu 1 1 Key Laboratory of Agricultural Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Sciences and Technology, China Agricultural University, Beijing, China 2 Department of Animal & Avian Sciences, University of Maryland, College Park, MD, USA Corresponding author: Y. Yu E-mail: yuying@cau.edu.cn Genet. Mol. Res. 12 (4): 6228-6239 (2013) Received December 5, 2012 Accepted March 27, 2013 Published December 4, 2013 DOI http://dx.doi.org/10.4238/2013.December.4.10 ABSTRACT. Bovine mastitis is the most common and costly disease of dairy cattle. Cluster of differentiation 4 (CD4) is closely related to the immune response in mastitis. We quantiied promoter CpG methylation levels of the CD4 gene in Chinese Holsteins with clinical mastitis (CM) and in healthy controls; these levels were quantitatively detected with bisulite pyrosequencing assays and conirmed by cloning sequencing. We found that the bovine CD4 promoter had 16% more methyl groups in the cows with CM (75.0 ± 5.8%) compared to the controls (59.0 ± 8.5%). The decreased expression level of CD4 in CM cows may be downregulated by the increased DNA methylation levels in the CD4 promoter. Two-dimensional hierarchical clustering analyses showed large differences in promoter CD4 methylation between mastitic and healthy cows; the dendrogram clearly distinguished the cows with clinical mastitis from healthy controls based on methylation levels. The DNA methylation level of the CD4 gene was strongly inluenced by mastitis status in all comparisons. We suggest that