Loss of Heterozygosity Combined with Promoter Hypermethylation, the Main Mechanism of Human MutL Homolog (hMLH1) Gene Inactivation in Non-Small Cell Lung Cancer in a Chinese Population

Abstract

The mechanism of human MutL Homolog (hMLH1) gene transcriptional inactivation in non-small cell lung cancer (NSCLC) is still unclear. The aim of this study is to further investigate the main mechanism of hMLH1 gene inactivation in NSCLC samples of Chinese patients. This study was performed in surgically resected primary tumor and matched normal tissues from 116 NSCLC cases. The hMLH1 gene alterations examined included loss of heterozygosity (LOH) by D3S1612 locus PCR-electrophoresis-silver staining and promoter methylation by HpaII/ MspI-based PCR analysis. Loss of hMLH1 mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and loss of hMLH1 protein expression was studied by immunohistochemistry and Western blot. The frequencies of LOH and promoter hypermethylation of the hMLH1 gene were 68.1% (79/116) and 72.4% (84/116), respectively. Among the 79 hMLH1 LOH (+) cases, 68 (86.1%) showed hypermethylation, which was significantly higher than in the LOH (-) group. The frequencies of loss of hMLH1 mRNA expression and protein expression in NSCLC were 79.3% (92/116) and 76.7% (89/116), respectively. The frequency of 2-hit inactivation of hMLH1, 75.3% (67/89), by LOH combined with promoter hypermethylation was related to the loss of protein expression. Biallelic inactivation of the hMLH1 gene by LOH combined with promoter hypermethylation is likely to cause inactivation of hMLH1 protein and to play an important role in the development of NSCLC in the Chinese population.