GENOMIC INSTABILITY IN FAMILIAL CANCER SYNDROMES

微卫星是基因组中由短核苷酸序列重复组成的DNA片段，重复了n次1至6个核苷酸。基因组中有成千上万个这样的基因，它们是由它们独特的侧翼区域单独定义的，因此可以通过聚合酶链反应（PCR）进行扩增和可视化。它们是多态的（一个等位基因上的重复次数），通常是稳定遗传的，尽管在一个等位基因上的序列重复次数会发生突变)，通常是稳定遗传的，尽管在相当高的频率（0.01-0.0001）下序列重复次数会发生突变。在某些类型的肿瘤中，由于错配修复基因的缺陷，它们已被证明是高度不稳定的。其他位点的缺陷也可能导致这种不稳定性。具体的微卫星轨迹不稳定性（MSI）的定量测量尚未开发。我们开发了一种程序，小池PCR （SP-PCR）能够进行定量，并与高通量荧光法相结合，以筛选微卫星位点频率的MSI。我们将把这些技术用于遗传多发性癌症易感李/弗鲁梅尼综合征（LFS）和Wilms肿瘤综合征（WT）的家庭，并回答一系列问题。不同类型的LFS肿瘤和WT肿瘤的不稳定性水平是什么？p53突变的肿瘤与未发现p53突变的家族肿瘤的不稳定性是否不同，这些差异是否反映了导致不稳定性的不同基因座？对于候选基因与19q相关的家族中的WT肿瘤，也会提出同样的问题。当LFS患者培养的成纤维细胞从衰老前期到永生化再到肿瘤形成时，何时表现出不稳定性？遗传致癌基因的患者与未受影响的同胞相比，“正常”周围组织和外周血淋巴细胞的不稳定性是否可检测到并在数量上有所不同？这些差异是否可以用于对致病突变未知的家庭进行症状前诊断？

Microsatellites are DNA stretches in the genome composed of repeats of short nucleotide sequences one to six nucleotides repeated n times. There are thousands of these in the genome and they are individually defined by their unique flanking regions and therefore amplifiable (by PCR) and so visualized. They are polymorphic (as number of repeats at an allele) and generally stably inherited, although shown to mutate in number of sequence repeats at an allele) and generally stably inherited, alth0ugh sown to mutate in number of sequence repeats at appreciable frequencies (0.01-0.0001). In certain types of tumors they have been shown to be highly unstable due to deficiencies in mismatch repair genes. Deficiencies at other loci may also enable such instability. Quantitative measures of specific microsatellite locus instability (MSI) have not been developed. We have developed a procedure, small pool PCR (SP-PCR) capable of carrying out that quantitation and coupled that with a high throughput fluorescent method to screen for MSI at frequencies at minisatellite loci. We will direct these techniques to families inheriting multiple cancer predisposition Li/Fraumeni Syndrome (LFS) and Wilms tumor syndrome (WT) and answer a series of questions. What is the level of instability in LFS tumors of different types and WT tumors? Is instability different in tumors with p53 mutation as opposed to tumors in families where p53 mutation have not been identified, and do those differences reflect different loci responsible for instability? The same question will be asked about WT tumors in families were the candidate gene is linked to 19q versus those where it is not. When is instability manifested in cultured fibroblasts from LFS patients as cells pass from pre-senescence to immortalization to tumor forming? Is instability detectable and quantitatively different in the "normal" surrounding tissue and peripheral blood lymphocytes of patients inheriting the gene responsible for cancer versus their unaffected sibs? Can those differences be used for presymptomatic diagnosis in families where the mutation responsible for the disorder is not known?