Molecular genetic study on the relation of parental alleles to the loss of tumor suppressor genes in gliomas.

为了探讨人类胶质母细胞瘤中常见的染色体17p杂合性缺失(LOH)与父亲或母亲哪个等位基因的关系，我们分析了青年和儿童胶质瘤患者肿瘤和外周血白细胞基因组DNA的限制性片段长度多态性(RFLP)。迄今为止，我们收集了15例从肿瘤、患者白细胞和父母白细胞中提取基因组DNA的病例。然而，在这15例病例中，我们没有发现患者白细胞来源DNA中的染色体17p杂合性缺失(LOH)与亲代DNA相比的情况。与WT1基因相比，染色体17p上的p53基因LOH在胶质瘤的发生中存在，但在生殖系中不存在。但由于限制性内切酶片段长度信息量的限制，RFLP分析可能遗漏TP53基因附近基因组DNA的细微变化，这一假设有待证实。此外，减数分裂过程中染色体基因的重组可能会导致LOH。为了解决这个问题，我们采用一种新的酵母双杂交系统检测突变型P53基因在mRNA和基因组DNA水平上的异常。该方法可以通过体内表达P53基因和构建ADE2基因报告质粒来定量检测P53基因突变。到目前为止，我们在年轻患者(30岁以下)中发现了4例P53基因突变，但这些突变在白细胞来源的基因组DNA中没有复制。结论：P53基因突变主要发生在胶质瘤细胞中，而不发生在亲代胚系中，除特殊的Li-Fraumeni综合征外。

To investigate which of paternal or maternal allele is related to the loss of heterozygosity (LOH) of chromosome 17p that is frequently observed in human glioblastomas, we analyzed restriction fragment length polymorphism (RFLP) of genomic DNA derived from tumor and peripheral blood leukocytes in young adults and children with glial tumor.So far, we have collected 15 cases in which genomic DNA was extracted from the tumor, patient leukocytes, and parents' leukocytes.In these 15 cases, however, we have not found a case which shows LOH of chromosome 17p in patient's leukocyte-derived DNA as compared to the parental DNA.As an explanation for this, it is considered that LOH of p53 gene on chromosome 17p occurs in glioma-oncogenesis but not in germ-line, contrasting to the WT1 gene.However, this supposition remained to be confirmed, since RFLP analysis can miss a minute change of genomic DNA adjacent to the TP53 gene, because of the limitations in informativeness of fragment length by a limited number of restriction enzyme digestion.Also it is possible that recombination of chromosomal genes in meiosis can mas the LOH.To solve this problem, we employed a new assay of mutant p53 using yeast two-hybrid system to detect abnormality of p53 gene at both mRNA and genomic DNA levels.This assay can quantify them mutation of p53 gene as loss of the transcriptional activity by in vivo expression of p53 and reporter plasmid construct of ADE2 gene.So far, we have found 4 tumors with p53 gene mutation in young patients (below 30 year-old), but these mutations have not been reproduced in the leukocyte-derived genomic DNA.In conclusion, p53 gene mutation is considered to occur primarily in the glioma cells but not in parental germ-line, except for special cases of Li-Fraumeni syndrome.