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.