The Microsatellite Instability Phenotype

描述（由申请人提供）：遗传性非息肉病性结肠癌（HNPCC）是一种癌症综合征，被证明是突变效应的结果-基因遗传或突变基因减少了细胞纠正DNA损伤的能力。在HNPCC中，错配修复（MMR）基因的主要遗传缺陷导致受影响组织中正常等位基因功能丧失后的微卫星不稳定。在肿瘤组织DNA中，通过对一组微卫星位点进行PCR，并在至少40%的此类位点上观察到突变片段，可以看到这一点。通过肿瘤的免疫组化染色（IHC）确定哪个MMR基因可能负责，然后确定该MMR基因与疾病分离的显著突变，从而在这些家族中得出疾病的分子基础。当MMR基因的突变在LOH后敲除ihc可检测酶的水平，并且当所得的MSI达到足够高的水平，可以通过简单的PCR检测到时，这种方法就可以很好地工作。然而，MMR突变可能产生IHC可检测的蛋白，这是低效的，但并非完全无效。这种“半胚性”突变将允许突变的微卫星片段积累，但其水平无法通过标准PCR检测到。与这一假设相一致，HNPCC已被证明在癌症不符合MSI-IHC-MMR突变标准的家庭中有很大比例的分离。他们的遗传疾病的分子基础可以通过在单分子水平上进行PCR从一个样品的许多等分的DNA接近。通过适当的统计，可以量化标记微卫星位点上的突变片段，并生成该样本的MSI表型（突变等位基因在所有位点上的加权频率）。这种小池PCR （SP-PCR）将在这里用于确定肿瘤和正常（pbl）组织中相对于HNPCC家系中MMR突变类型（或缺乏）的MSI表型。我们还打算确定该程序在未发现MMR突变的家庭中识别高危个体的能力，以及识别携带易患癌症的种系突变的“散发性”患者的能力。最后，我们打算确定遗传生物体显性/细胞隐性种系突变对后代的影响。

DESCRIPTION (provided by applicant): Hereditary non-polyposis colon cancer (HNPCC) is a cancer syndrome shown to be the result of a mutator effect - inheritance of gene or genes with mutations detracting from the cells' ability to correct damage to DNA. In HNPCC, dominantly inherited defects in mismatch repair (MMR) genes lead to microsatellite instability after loss of the normal allele function in affected tissue. This is seen in tumor tissue DNA by conducting PCR across a set of microsatellite loci and observing mutant fragments at a minimum of 40% of such loci. The molecular basis of the disease in such families is arrived at by identifying which MMR gene might be responsible by immunohistochemical staining (IHC) of the tumor, and then identifying a significant mutation in that MMR gene segregating with the disease. This works fine when the mutation in the MMR gene knocks out the level of IHC-detectable enzyme after LOH and when the resultant MSI is at high enough level to be detected by simple PCR. However, MMR mutations might produce IHC detectable proteins, which are inefficient but not totally ineffectual. Such "hypomorphic" mutations would allow mutant microsatellite fragments to accumulate but at levels not detectable by standard PCR. Consistent with that hypothesis, HNPCC has been shown to segregate in a significant proportion of families whose cancers do not meet the MSI-IHC-MMR mutation criteria. The molecular basis of their genetic disease can be approached by conducting PCR at the single molecule level in many aliquots of DNA from a sample. With appropriate statistics one can quantify mutant fragments at marker microsatellite loci and generate an MSI phenotype (weighted frequency of mutant alleles over all loci) for that sample. Such small pool PCR (SP-PCR) and will be used here to determine the MSI phenotypes in both the tumor and normal (PBLs) tissues relative to the type of MMR mutation (or lack thereof) in HNPCC pedigrees. We also intend to determine the ability of the procedure to identify individuals at risk in families where no MMR mutation has been identified, and to identify "sporadic" patients carrying germline mutation predisposing to cancer. Finally, we intend to determine the impact of inherited organism-dominant/cell recessive germline mutations on future generations.