Mismatch repair gene expression defects contribute to microsatellite instability in ovarian carcinoma.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA. john-geisler@indianagynonc.com
John P
JP
Geisler
Michael J
MJ
Goodheart
Anil K
AK
Sood
Richard J
RJ
Holmes
Melanie A
MA
Hatterman-Zogg
Richard E
RE
Buller
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch / Q000235:genetics*; D002277:Carcinoma / Q000235:genetics*; D002352:Carrier Proteins; D019175:DNA Methylation; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000235:genetics* / Q000494:pharmacology*; D009687:Nuclear Proteins; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein