METHYLATION SIMILARITIES OF 2 CPG SITES WITHIN EXON-5 OF HUMAN H19 BETWEEN NORMAL-TISSUES AND TESTICULAR GERM-CELL TUMORS OF ADOLESCENTS AND ADULTS, WITHOUT CORRELATION WITH ALLELIC AND TOTAL LEVEL OF EXPRESSION

Testicular germ cell tumours (TGCTs) of adolescents and adults morphol ogically mimic different stages of embryogenesis. Established cell lin es of these cancers are used as informative models to study early deve lopment. We found that, in contrast to normal development, TGCTs show a consistent biallelic expression of imprinted genes, including H19, i rrespective of histology. Methylation of particular cytosine residues of H19 correlates with inhibition of expression, which has not been st udied in TGCTs thus far. We investigated the methylation status of two CpG sites within the 3' region of H19 (exon 5:positions 3321 and 3324 ) both in normal tissues as well as in TGCTs. To obtain quantitative d ata of these specific sites, the ligation-mediated polymerase chain re action technique, instead of Southern blot analysis, was applied. The results were compared with the allelic status and the total level of e xpression of this gene. Additionally, the undifferentiated cells and d ifferentiated derivatives of the TGCT-derived cell line NT2-D1 were an alysed. While peripheral blood showed no H19 expression and complete m ethylation, a heterogeneous but consistent pattern of methylation and level of expression was found in the other normal tissues, without a c orrelation between the two. The separate histological entities of TGCT s resembled the pattern of their nonmalignant tissues. While the CpG s ites remained completely methylated in NT2-D1, H19 expression was indu ced upon differentiation. These data indicate that methylation of the CpG sites within exon 5 of H19 is tissue dependent, without regulating allelic status and/or total level of expression. Of special note is t he finding that, also regarding methylation of these particular sites of H19, TGCTs mimic their non-malignant counterparts, in spite of thei r consistent biallelic expression.