The analysis of genomic imprinting in ovarian teratomas and choriocarcinoma

已经发现有几个基因受基因组印记的影响。它们包括IGF2和SNRPN等人类基因，这些基因仅由父亲等位基因表达，而H19则在大多数体细胞和胚胎外器官中由母亲等位基因表达。为了研究卵巢畸胎瘤中印迹的变异并确定印迹的状态，我们研究了三个人类印迹基因（IGF2、H19和SNRPN）在许多皮样囊肿中的表达水平和等位基因特异性表达。我们还检查了等位基因的甲基化状态，其中印记特异性差异甲基化已在人类中得到证实。卵巢良性畸胎瘤31例。共检查34个肿瘤，包括双侧和多发肿瘤。约三分之一至一半的肿瘤显示LOH，这是由减数分裂期间亲本基因组的减少引起的。在某些情况下，杂合性在一个位点（IGF2）保持，但在另一个位点（SNRPN）丢失更多。这可以用同源染色单体交叉的形成来解释。在良性畸胎瘤中，这三种基因的表达水平都很低，用Northern印迹法很难检测到。相反，未成熟畸胎瘤。H19高度表达，其水平几乎等于妊娠晚期胎盘的水平。未成熟畸胎瘤中IGF2表达水平较低，与皮样囊肿相似。虽然印迹基因的表达水平较低，但在所有畸胎瘤样本中均可通过RT-PCR扩增到cDNA。在14例成熟畸胎瘤中，IGF2的Apa I多态性位点信息丰富，8例肿瘤（57%）显示双等位基因表达。在H19中，有14例成熟畸胎瘤在Rsa I或Alu I多态性位点存在杂合，12例（86%）存在双等位基因表达。相比之下，SNRPN在所有信息性肿瘤中均为单等位基因表达（表1）。在两种基因均有单等位基因表达的肿瘤中，表达的等位基因与宿主的等位基因相同。我们还报道了双等位基因（IGF2/H19）和单等位基因（SNRPN）表达的肿瘤病例。检测H195'-启动子区的甲基化状态。该区域内的CpG位点已知是母系低甲基化的。成人体细胞组织半甲基化，精子DNA低甲基化，这与以前的报道一致。在卵巢畸胎瘤中，无论是成熟的（良性的）还是未成熟的，这个区域都是低甲基化的。同样，我们分析了IGF2外显子9区域的甲基化状态，该区域是母体等位基因差异低甲基化的地方。在这个位点，白细胞DNA是半甲基化的，而精子DNA是完全甲基化的。卵巢畸胎瘤只有低甲基化的等位基因。对于SNRPN，差异甲基化位点也在其母等位基因广泛甲基化的5'区。给出了Southern印迹的结果。我们在两例遗传结构不同的绒毛膜癌和7种绒毛膜癌细胞系中检测了人类印迹基因的等位基因特异性表达。病例1和所有四个信息细胞系在IGF2位点显示双等位基因表达。病例也显示H19的LOI，而Case2则没有。细胞系分析显示，3/4在H19位点显示loat。我们发现IGF2和H19的LOI通常与绒毛膜癌相关，这表明它与肿瘤发生有关。少

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