The analysis of genomic imprinting in ovarian teratomas and choriocarcinoma

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Several genes have been discovered to be subject to genomic imprinting. They include human genes such as IGF2 and SNRPN,which are expressed exclusively from the paternal allele and H19, which is expressed from the maternal allele in most somatic and extra-embryonic organs. In order to examine the variance of imprinting in ovarian teratomas and to identify the imprinting status, we investigated expression level and the allele-specific expression of three human imprinted genes (IGF2, H19, and SNRPN) in a number of dermoid cysts. We also examined the methylation status of the alleles, where imprint-specific differential methylation has been demonstrated in humans. Thirty one cases of benign ovarian teratomas. (A total of 34 tumors including bilateral and multiple tumors) were examined. About one-third to half of the tumors showed LOH,which is caused by the reduction of either parental genome during meiotic division. In some cases, heterozygosity was maintained at one locus (IGF2) but was … More lost at another (SNRPN). This can be explained by the formation of crossing over of homologous chromatids. In benign teratomas, all three genes were expressed at very low levels, which is hardly detectable by Northern blotting. In contrast an immature teratoma. H19 was highly expressed, at levels almost equal to that of the third trimester placenta. IGF2 expression in an immature teratoma was low and similar to the level in dermoid cysts. Although the expression of the imprinted genes was low, cDNA could be amplified by RT-PCR from all samples of teratomas. In fourteen mature teratomas that were informative at the Apa I polymorphic site of IGF2, eight tumors (57%) showed biallelic expression. In H19, fourteen mature teratomas which were heterozygous either at the Rsa I or the Alu I polymorphic sites and biallelic expression was observed in 12 tumors (86%). In contrast, SNRPN expression was monoallelic in all the informative tumors (Table 1). In the tumors that showed monoallelic expression in either of the genes, the expressed allelotype was the same as that of the host. Examples of tumors showing biallelic (IGF2/H19) and monoallelic (SNRPN) expression were presented. The methylation status of H195'-promoter region was examined. The CpG sites within this region is known to be maternally hypomethylated. The adult somatic tissue was hemimethylated and sperm DNA was hypomethylated, which is compatible with previous reports. In ovarian teratomas, either mature (benign) or immature, this region is hypomethylated. Similarly, the methylation status of IGF2 exon 9 region, where the maternal allele is differentially hypomethylated, was analyzed. At this site, leukocyte DNA was hemimethylated, whereas sperm DNA was totally methylated. Ovarian teratomas had only hypomethylated alleles. For SNRPN,differentially methylated site is also demonstrated at its 5'-region, where the maternal allele is extensively methylated. The results of Southern blotting is presented. At this site,We have examined the allele specific expression of human imprinted genes in two cases of choriocarcinoma which had different genetic constitutions and seven choriocarcinoma cell lines. Case 1 and all four informative cell lines showed biallelic expression at the IGF2 site. Case also showed LOI for H19 but Case2 did not. Analyzes in cell lines revealed that 3/4 showed LOIat the H19 site. Our finding that the LOI of IGF2 and H19 is commonly associated with choriocarcinomas suggests its relevance to tumorgenesis. Less