Methylation patterns in human androgen receptor gene and clonality analysis

Tumor clonality, an important issue in tumor biology, has been analyzed: us ing X-chromosome inactivation studies based on the differential methylation patterns of active and inactive alleles, Recently, a PCR-based androgen re ceptor gene (AR) analysis method was developed that takes advantage of high ly polymorphic CAG repeats and nearby HpaII and HhaI sites in exon 1 of AR at the Xq13 region. However, the data from this assay, which is now widely used, are sometimes uninterpretable and irreproducible For some currently u nclear reason. To determine that reason, we analyzed a panel of lung cancer cell lines, using HpaII or HhaI restriction enzymes, methylation-specific PCR, and bisulfite genomic sequencing of the polymorphic CAG repeat site of AR exon 1, including neat-by CpG sites. We found direct evidence of a vari able methylation pattern at the restriction sites that prevented proper enz yme cleavage in two lung cancer cell lines (NCI-H292 and NCI-H1944) obtaine d from female patients who had a polymorphic CAG repeat in AR exon 1. Our d ata suggest that methylation patterns at the CpG sites of AR exon 1 are com plicated and vary among different individuals. Therefore, the reliability o f the PCR-based clonality analysis may require further evaluation.