The analysis of genomic imprinting in ovarian teratomas and choriocarcinoma

已经发现几个基因受到基因组印记。它们包括人类基因，如IGF 2和SNRPN，它们仅从父系等位基因表达，以及H19，它在大多数体细胞和胚外器官中从母系等位基因表达。为了研究卵巢畸胎瘤中印记的变化并确定印记状态，我们研究了三种人类印记基因（IGF 2，H19和SNRPN）在一些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。共检查了34个肿瘤，包括双侧和多发性肿瘤。大约三分之一到一半的肿瘤表现出洛缺失，这是由减数分裂期间双亲基因组的减少引起的。在某些情况下，杂合性保持在一个基因座（IGF 2），但 ...更多信息 在另一个（SNRPN）。这可以用同源染色单体交换的形成来解释。在良性畸胎瘤中，所有三个基因都以非常低的水平表达，这是很难检测到的北方印迹。相反，未成熟畸胎瘤。H19高度表达，其水平几乎等于晚期妊娠胎盘的水平。IGF 2在未成熟畸胎瘤中的表达较低，与皮样囊肿中的表达水平相似。虽然印迹基因的表达量很低，但RT-PCR可从所有畸胎瘤标本中扩增出cDNA。在14个成熟畸胎瘤中，在IGF 2的阿帕I多态性位点有信息，8个肿瘤（57%）显示双等位基因表达。在H19中，14个成熟畸胎瘤在Rsa I或Alu I多态性位点杂合，12个肿瘤（86%）中观察到双等位基因表达。相比之下，SNRPN表达在所有提供信息的肿瘤中是单等位基因的（表1）。在任一基因中显示单等位基因表达的肿瘤中，表达的等位基因型与宿主的等位基因型相同。展示了显示双等位基因（IGF 2/H19）和单等位基因（SNRPN）表达的肿瘤实例。检测H195 '启动子区甲基化状态。已知该区域内的CpG位点是母系低甲基化的。成年体细胞组织半甲基化，精子DNA低甲基化，这与以前的报告是一致的。在卵巢畸胎瘤中，无论是成熟的（良性）还是未成熟的，这个区域都是低甲基化的。类似地，分析IGF 2外显子9区域的甲基化状态，其中母体等位基因是差异低甲基化的。在该位点，白细胞DNA被半甲基化，而精子DNA被完全甲基化。卵巢畸胎瘤只有低甲基化等位基因。对于SNRPN，差异甲基化位点也在其5 '-区域被证明，其中母体等位基因被广泛甲基化。Southern杂交结果。本研究检测了两例不同遗传组成的绒毛膜癌和七个绒毛膜癌细胞系中人类印记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显示IGF 2位点的双等位基因表达。病例还显示H19的LOI，但病例2没有。细胞系分析显示3/4的细胞在H19位点显示LOI。我们发现IGF 2和H19的LOI通常与绒毛膜癌相关，提示其与肿瘤发生相关。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