Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene,ALX3,that is hypermethylated in neuroblastoma

Abstract

Restriction landmark genome scanning (RLGS) allows comparative analysis of several thousand DNA fragments in the genome and provides a means to identify CpG islands that are altered in tumor cells as a result of amplification, deletion, or methylation changes. We have developed a novel informatics tool, designated virtual genome scan (VGS), that makes it possible to predict automatically the sequence of fragments in RLGS patterns by matching to the human genome sequence. A combination of RLGS and VGS was utilized to identify changes of chromosome 1-derived fragments in neuroblastoma. A NotI-EcoRV fragment was found to be absent frequently in neuroblastoma cell line RLGS patterns. Sequence prediction by VGS as well as cloning of the fragment showed that it contained a CpG island that is part of the human orthologue of the hamster homeobox gene Alx3. Expression analysis in a panel of human and mouse tissues showed predominant expression of ALX3 in brain tissue. Methylation-sensitive sequence analysis of the promoter region in neuroblastoma cell lines indicated that methylation of specific sequences correlated with repression of the ALX3 gene. Expression was re-induced after treatment with the methylation inhibitor 5-aza-2'-deoxycytidine. Promoter methylation analysis of ALX3 in primary neuroblastoma tumors, using methylation-sensitive polymerase chain reaction, found preferential ALX3 methylation in advanced-stage tumors. The VGS approach we have implemented in combination with RLGS is useful for the identification of genomic CpG island-related methylation changes or deletions in cancer.