H-ras mutation detection in bladder cancer by COLD-PCR analysis and direct sequencing.

Objective: A sensitive mutation detection method called co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) was applied to improve the detection frequencies of expressive mutations in the H-ras gene, including exons 1 and 2, in a group of Chinese patients diagnosed with bladder cancer. Materials and Methods: The expressive mutations in the H-ras gene in 86 fresh tissues of human bladder cancer were identified by COLD-PCR or conventional PCR, followed by direct sequencing. Results: A high frequency of silent mutations of 29.1% (25 of 86) in exon 1 (c.81T>C, H27H) and activating mutations of 8.1% (7 of 86) were detected by COLD-PCR, yielding a 36% improvement in mutation detection compared with conventional PCR. No significant association was shown between activating mutations and clinicopathologic parameters, but the frequencies of silent mutations in recurrent tumors were higher than those in primary tumors (p = 0.034). Conclusions: COLD-PCR is a highly sensitive, reliable, and convenient clinical assay for mutation detection. The adoption of the method is straightforward and requires no additional reagents or instruments. Silent mutations might be important genomic alterations in bladder cancer, and play a role in bladder cancer recurrence. Copyright (C) 2012 S. Karger AG, Basel