Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.