Geisler John P, Goodheart Michael J, Sood Anil K, Holmes Richard J, Hatterman-Zogg Melanie A, Buller Richard E
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA.
Cancer. 2003 Nov 15;98(10):2199-206. doi: 10.1002/cncr.11770.
hMLH1, the human MutL homologue, has been linked to microsatellite instability (MSI) in gastrointestinal tumors. However, to the authors' knowledge, the role of hMLH1, the other mismatch repair genes (MMR), and MSI in ovarian carcinoma has not been well defined. The purpose of the current study was to determine the relation between MSI of ovarian carcinoma and MMR gene expression, hMLH1 and hMSH2 hypermethylation, and hMLH1 and hMSH2 null mutations.
hMLH1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and amplification of cDNA using a housekeeping gene (glycerol 3-phosphate dehydrogenase) as a control for mRNA quality and quantity. Methylation-specific PCR (MS-PCR) was used to correlate methylation of the hMLH1 and hMSH2 CpG islands with mRNA expression status. Similar techniques were used to evaluate the concomitant expression of five other MMR: hMSH2, hMSH3, hMSH6, PMS1, and PMS2. Microsatellite instability was studied using the National Cancer Institute consensus markers (D2S123, D5S346, D17S250, BAT25, and BAT26) and NM23 as described previously.
One hundred twenty-five primary tumors were analyzed. High-frequency MSI (MSI-H) was found in 21 tumors (16.8%). hMLH1 mRNA was absent in 10 of these 21 tumors (47.6%). In each case, coordinated hypermethylation of both regions A and C of the promoter was identified. Microsatellite stable and low-frequency MSI tumors all were found to express not only hMLH1 but the other MMR genes as well (P < 0.001). Absence of expression of hMSH2 and the four other MMRs occurred in tumors with absent hMLH1 mRNA expression because of CpG island hypermethylation. No absence of expression of hMSH2, hMSH3, hMSH6, PMS1, or PMS2 was found to occur in tumors expressing hMLH1. None of the 11 MSI-H tumors without promoter hypermethylation demonstrated a null mutation in hMLH1 or hMSH2.
A molecular mechanism to explain > 50% of the MSI-H phenotype in ovarian carcinoma cases was demonstrated. MSI-H may occur because of MMR defects, especially hMLH1 promoter hypermethylation. Additional mechanisms are required to explain the balance between the cases of MSI-H as well as the phenomenon of MSI-L tumors.
人MutL同源物hMLH1与胃肠道肿瘤中的微卫星不稳定性（MSI）有关。然而，据作者所知，hMLH1、其他错配修复基因（MMR）以及MSI在卵巢癌中的作用尚未明确界定。本研究的目的是确定卵巢癌的MSI与MMR基因表达、hMLH1和hMSH2高甲基化以及hMLH1和hMSH2无义突变之间的关系。
采用逆转录聚合酶链反应（RT-PCR）检测hMLH1 mRNA，并以管家基因（甘油3-磷酸脱氢酶）扩增cDNA作为mRNA质量和数量的对照。甲基化特异性PCR（MS-PCR）用于将hMLH1和hMSH2 CpG岛的甲基化与mRNA表达状态相关联。采用类似技术评估其他5种MMR（hMSH2、hMSH3、hMSH6、PMS1和PMS2）的伴随表达。如前所述，使用美国国立癌症研究所的一致性标志物（D2S123、D5S346、D17S250、BAT25和BAT26）和NM23研究微卫星不稳定性。
分析了125例原发性肿瘤。21例肿瘤（16.8%）中发现高频MSI（MSI-H）。这21例肿瘤中有10例（47.6%）未检测到hMLH1 mRNA。在每种情况下，均发现启动子A区和C区均存在协同高甲基化。微卫星稳定和低频MSI肿瘤均不仅表达hMLH1，还表达其他MMR基因（P < 0.001）。由于CpG岛高甲基化导致hMLH1 mRNA表达缺失的肿瘤中，hMSH2和其他4种MMR也未表达。在表达hMLH1的肿瘤中未发现hMSH2、hMSH3、hMSH6、PMS1或PMS2表达缺失。11例无启动子高甲基化的MSI-H肿瘤中，均未发现hMLH1或hMSH2无义突变。
证实了一种分子机制可解释卵巢癌病例中>50%的MSI-H表型。MSI-H可能是由于MMR缺陷，尤其是hMLH1启动子高甲基化所致。需要其他机制来解释MSI-H病例与MSI-L肿瘤现象之间的平衡。
