NBL2 is a tandem 1.4-kb DNA repeat, whose hypomethylation in hepatocellular 
carcinomas was shown previously to be an independent predictor of disease 
progression. Here, we examined methylation of all cytosine residues in a 0.2-kb 
subregion of NBL2 in ovarian carcinomas, Wilms' tumors, and diverse control 
tissues by hairpin-bisulfite PCR. This new genomic sequencing method detects 
5-methylcytosine on covalently linked complementary strands of a DNA fragment. 
All DNA clones from normal somatic tissues displayed symmetrical methylation at 
seven CpG positions and no methylation or only hemimethylation at two others. 
Unexpectedly, 56% of cancer DNA clones had decreased methylation at some 
normally methylated CpG sites as well as increased methylation at one or both of 
the normally unmethylated sites. All 146 DNA clones from 10 cancers could be 
distinguished from all 91 somatic control clones by assessing methylation 
changes at three of these CpG sites. The special involvement of DNA 
methyltransferase 3B in NBL2 methylation was indicated by analysis of cells from 
immunodeficiency, centromeric region instability, and facial anomalies syndrome 
patients who have mutations in the gene encoding DNA methyltransferase 3B. Blot 
hybridization of 33 cancer DNAs digested with CpG methylation-sensitive enzymes 
confirmed that NBL2 arrays are unusually susceptible to cancer-linked 
hypermethylation and hypomethylation, consistent with our novel genomic 
sequencing findings. The combined Southern blot and genomic sequencing data 
indicate that some of the cancer-linked alterations in CpG methylation are 
occurring with considerable sequence specificity. NBL2 is an attractive 
candidate for an epigenetic cancer marker and for elucidating the nature of 
epigenetic changes in cancer.