Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma.
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.
Wimmer
Katharina
K
Institut für Medizinische Biologie, Universität Wien, Vienna, Austria. katharina.wimmer@univie.ac.at
Zhu Xx
Xiao-xiang
XX
Rouillard
Jean Marie
JM
Ambros
Peter F
PF
Lamb
Barbara J
BJ
Kuick
Rork
R
Eckart
Markus
M
Weinhäusl
Andreas
A
Fonatsch
Christa
C
Hanash
Sam M
SM
D000328:Adult; D000818:Animals; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D006224:Cricetinae; D053938:DNA Fragmentation; D019175:DNA Methylation / Q000187:drug effects; D005260:Female; D005801:Genes, Homeobox / Q000187:drug effects; D015894:Genome, Human; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
C485163:ALX3 protein, human; C113607:Alx3 protein, mouse; D018398:Homeodomain Proteins