Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder

Abstract

Background: Mutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer. Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder (UCB).

Aim: To determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.

Methods: This study was performed to determine the mutational spectrum at the hotspot regions of H-Ras, K-Ras and N-Ras genes by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing followed by their clinical impact (if any) by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.

Results: None of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene; codon 61 of N-Ras gene and codons 12, 13 of K-Ras gene by PCR-RFLP. Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras, N-Ras and K-Ras genes in the tumor and control bladder mucosal specimens.

Conclusion: Our findings on the lack of mutations in H-Ras, K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression, inherent genetic susceptibility, tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

Keywords: Coding exons; Oncogenic activation; Point mutations; Polymerase chain reaction - restriction fragment length polymorphism; Ras genes; Urothelial carcinoma of bladder.

Figure 1

H-Ras gene point mutation analysis in patients diagnosed with urothelial bladder cancer. A: Polymerase chain reaction (PCR) - restriction fragment length polymorphism of codon 12: Undigested amplified PCR product (420-bp); MspI-cut PCR product (390-bp and 30-bp); Lanes 1, 3, 5, 7, 9, 11 and 13: Undigested products; and Lanes 2, 4, 6, 8, 10, 12 and 14: Digested products. Lanes 1 and 2 represent the band pattern in normal bladder mucosal tissues whereas lanes 3, 4; 5, 6; 7, 8; 9, 10; 11, 12; and 13, 14 represent the band patterns in tumor specimens; B: Direct DNA sequencing of H-Ras coding exon 1 in bladder tumors and normal bladder tissues. Codons 12 and 13 are highlighted; C: Direct DNA sequencing of H-Ras coding exon 2 in bladder tumors and normal bladder tissues. Codon 61 is highlighted. TS: Tumor specimen; WT: Wild-type.

Figure 2

N-Ras gene point mutation analysis in patients diagnosed with urothelial bladder cancer. A: Polymerase chain reaction (PCR) - restriction fragment length polymorphism of codon 61. Undigested amplified PCR product (65-bp); MscI-cut PCR product (44-bp and 21-bp); Lanes 1, 3, 5, 7, 9, 11, and 13: Undigested products; and Lanes 2, 4, 6, 8, 10, 12 and 14: Digested products. Lanes 1 and 2 represent the band patterns in normal bladder mucosal tissues whereas lanes 3, 4; 5, 6; 7, 8; 9, 10; 11 12; and 13, 14 represent the band patterns in tumor specimens; B: Direct DNA sequencing of N-Ras coding exon 1 in bladder tumors and normal bladder tissues. Codons 12 and 13 are highlighted; C: Direct DNA sequencing of N-Ras coding exon 2 in bladder tumors and normal bladder tissues. Codon 61 is highlighted. TS: Tumor specimen; WT: Wild-type.

Figure 3

K-Ras gene point mutation analysis in patients diagnosed with urothelial bladder cancer. A: Polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) of codon 12. Undigested amplified PCR product (144-bp); BstI-cut PCR product (115-bp and 29-bp); Lanes 1, 3, 5, 7 and 9: Undigested products and Lanes 2, 4, 6, 8 and 10: Digested PCR products. Lanes 1 and 2 represent the band patterns in normal bladder tissues whereas lanes 3, 4; 5, 6; 7, 8; and 9, 10 represent the band patterns in tumor specimens; B: PCR-RFLP of K-Ras codon 13. Undigested amplified PCR product (144-bp); Hph I-cut PCR product (101-bp and 43-bp); Lanes 1, 3, 5, 7, 9, and 11: Undigested products and Lanes 2, 4, 6, 8 and 10: Digested PCR products. Lanes 1 and 2 represent the band patterns in normal bladder tissues whereas lanes 3, 4; 5, 6; 7, 8; 9, 10; and 11 represent the band patterns in tumor specimens; C: Direct DNA sequencing of K-Ras coding exon 1 in bladder tumors and normal bladder tissues. Codons 12 and 13 are highlighted; D: Direct DNA sequencing of K-Ras coding exon 2 in bladder tumors and normal bladder tissues. Codon 61 is highlighted. TS: Tumor specimen; WT: Wild-type.