Detection of CpG island hypermethylation of caspase-8 in neuroblastoma using an oligonucleotide array.
BACKGROUND
The caspase-8 gene (CASP8) is frequently inactivated in unfavorable neuroblastomas through DNA methylation. The present study utilized oligoarrays to evaluate the methylation status of a CpG island located between exons 2 and 3 of caspase 8 in neuroblastomas.


METHODS
DNA derived from 70 neuroblastomas was amplified by PCR after bisulfate modification and subjected to analysis on a self-made oligoarray that utilized a polycarbodiimide-coated slide to detect methylation of six intragenic CpG islands of caspase 8. In 30 cases, the methylation status was also analyzed by sequencing. In six cases, the PCR product was cloned into a vector and analyzed.


RESULTS
Among the 70 tumor-derived DNAs, methylation was not detected in 18 cases, one methylated CpG was found in 12 cases, two in 18 cases, three in 3 cases, four in 8 cases, five in 1 case and six in 10 cases. All methylated CpG loci detected by sequencing were detected by oligoarray, but some methylated CpGs in three loci were detected by oligoarray alone. In these discrepant loci, methylation was detected in some clones after subcloning, indicating that the oligoarray might be more sensitive than sequencing. The CASP8 expression level was depressed in the tumors having two distinct CpG doublets. These results were significantly correlated with MYCN amplification and with clinical outcomes.


CONCLUSIONS
A significant difference in the methylation status within the CpG island of CASP8 was shown between favorable and unfavorable subtypes, and CASP8 methylation detected by oligoarray may be useful in the clinical evaluation of neuroblastomas.
Kamimatsuse
Arata
A
Department of Surgery, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.
Matsuura
Kaoru
K
Moriya
Shogo
S
Fukuba
Ikuko
I
Yamaoka
Hiroaki
H
Fukuda
Emi
E
Kamei
Naomi
N
Hiyama
Keiko
K
Sueda
Taijiro
T
Hiyama
Eiso
E
D000293:Adolescent; D000328:Adult; D053181:Caspase 8 / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns / Q000235:genetics; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011379:Prognosis; D015996:Survival Rate; D055815:Young Adult
D004247:DNA; D053181:Caspase 8