Detection of DNA mismatch repair proteins in fresh human blood lymphocytes--towards a novel method for hereditary non-polyposis colorectal cancer (Lynch syndrome) screening.
Detection of DNA mismatch repair proteins in fresh human blood lymphocytes--towards a novel method for hereditary non-polyposis colorectal cancer (Lynch syndrome) screening.
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DOI：
10.1186/1756-9966-30-100
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发表时间：
2011-10-21
期刊：
影响因子：
--
通讯作者：
Fields JZ
中科院分区：
文献类型：
--
作者：
Hassen S;Boman BM;Ali N;Parker M;Somerman C;Ali-Khan Catts ZJ;Ali AA;Fields JZ
一种广泛的基于人群的检测方法，在林奇综合征(LS)患者患上癌症之前对他们进行检测，将通过预防癌症来节省生命和医疗保健资金。LS是由DNA错配修复(MMR)基因的胚系突变引起的，特别是涉及MSH2或MLH1的蛋白质截断突变。我们发现来自LS患者的永生化淋巴细胞降低了全长MLH1或MSH2蛋白的水平。因此，通过检测淋巴细胞中MMR蛋白水平的降低，在广泛的人群基础上检测LS患者可能是可行的。因此，我们确定是否也可以在新鲜淋巴细胞中检测到MSH2和MLH1蛋白。用两种商业上可买到的单抗建立了定量蛋白质印迹分析，这两种单抗是检测全长MLH1或MSH2的特异性抗体。为了直接确定这些MMR蛋白水平的比例，我们在多重类型的蛋白质印迹中使用了这两种抗体。在新鲜淋巴细胞中，MLH1和MSH2水平通常检测不到，但如果新鲜淋巴细胞首先用PHA刺激，则很容易检测到。正常人新鲜淋巴细胞的MMR比值约为1.0。在LS高危患者(N>；50)的新鲜淋巴细胞中，MMR比率呈双峰分布(范围：0.3-1.0)。发现可以在新鲜淋巴细胞中测量MMR蛋白水平，并考虑到具有杂合子MMR突变的细胞全长MMR蛋白水平降低，这表明我们的免疫检测方法可以进步到筛查LS高危人群的定量测试。
A broad population-based assay to detect individuals with Lynch Syndrome (LS) before they develop cancer would save lives and healthcare dollars via cancer prevention. LS is caused by a germline mutation in a DNA mismatch repair (MMR) gene, especially protein truncation-causing mutations involving MSH2 or MLH1. We showed that immortalized lymphocytes from LS patients have reduced levels of full-length MLH1 or MSH2 proteins. Thus, it may be feasible to identify LS patients in a broad population-based assay by detecting reduced levels of MMR proteins in lymphocytes. Accordingly, we determined whether MSH2 and MLH1 proteins can also be detected in fresh lymphocytes. A quantitative western blot assay was developed using two commercially available monoclonal antibodies that we showed are specific for detecting full-length MLH1 or MSH2. To directly determine the ratio of the levels of these MMR proteins, we used both antibodies in a multiplex-type western blot. MLH1 and MSH2 levels were often not detectable in fresh lymphocytes, but were readily detectable if fresh lymphocytes were first stimulated with PHA. In fresh lymphocytes from normal controls, the MMR ratio was ~1.0. In fresh lymphocytes from patients (N > 50) at elevated risk for LS, there was a bimodal distribution of MMR ratios (range: 0.3-1.0). Finding that MMR protein levels can be measured in fresh lymphocytes, and given that cells with heterozygote MMR mutations have reduced levels of full-length MMR proteins, suggests that our immunoassay could be advanced to a quantitative test for screening populations at high risk for LS.
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DOI：
10.1016/j.lab.2003.10.001
发表时间：
2004-01-01
期刊：
JOURNAL OF LABORATORY AND CLINICAL MEDICINE
影响因子：
--
作者：
Fields, JZ;Gao, ZP;Boman, BM
通讯作者：
Boman, BM
影响因子：
3.9
作者：
Coolbaugh-Murphy, Mary I.;Xu, Jing-Ping;Ramagli, Louis S.;Ramagli, Brian C.;Brown, Barry W.;Lynch, Patrick M.;Hamilton, Stanley R.;Frazier, Marsha L.;Siciliano, Michael J.
通讯作者：
Siciliano, Michael J.
影响因子：
4.3
作者：
Agarwal, Rakhee;Mumtaz, Hamid;Ali, Nawab
通讯作者：
Ali, Nawab
影响因子：
14.9
作者：
Gu, LY;Hong, Y;Li, GM
通讯作者：
Li, GM
影响因子：
45.3
作者：
Hampel, Heather;Frankel, Wendy L.;de la Chapelle, Albert
通讯作者：
de la Chapelle, Albert