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，显示为增变因子效应的结果-具有突变的基因或基因的遗传降低了细胞纠正DNA损伤的能力。在HNPCC中，错配修复（MMR）基因的显性遗传缺陷导致受影响组织中正常等位基因功能丧失后微卫星不稳定。通过在一组微卫星基因座上进行PCR并在至少40%的此类基因座上观察突变片段，可以在肿瘤组织DNA中观察到这一点。通过肿瘤的免疫组织化学染色（IHC）鉴定哪一个MMR基因可能是负责的，然后鉴定与疾病分离的MMR基因中的显著突变，来获得此类家族中疾病的分子基础。当MMR基因突变在洛缺失后敲除了IHC可检测酶的水平，并且当所产生的MSI处于足够高的水平以通过简单PCR检测时，这很好地起作用。然而，MMR突变可能产生IHC可检测的蛋白质，这是低效的，但并非完全无效。这种“亚型”突变将允许突变的微卫星片段积累，但在标准PCR检测不到的水平。与该假设一致，HNPCC已被证明在其癌症不符合MSI-IHC-MMR突变标准的家庭中分离。他们遗传疾病的分子基础可以通过在单分子水平上对来自样品的许多等份DNA进行PCR来接近。通过适当的统计，可以量化标记微卫星基因座处的突变片段，并生成该样品的MSI表型（所有基因座上突变等位基因的加权频率）。这种小池PCR（SP-PCR）在此将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（或其缺乏）的MSI表型。我们还打算确定该程序的能力，以确定个人在家庭中的风险，没有MMR突变已被确定，并确定“散发”患者携带生殖系突变易患癌症。最后，我们打算确定遗传的生物体显性/细胞隐性生殖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.