Detection of truncating mutations of APC gene by digital protein truncation test labeled with fluorescent Lys-t-RNA

Abstract

Objective To investigate the relationship between truncating mutations of APC gene and sporadic colorectal cancer,and analyze the feasibility of non-radioactive protein truncation test (PTT) in the detection of mutations of APC gene.Methods Ninety-six samples of sporadic colorectal cancer tissues ( including 44 patients with colonic cancer and 52 patients with rectal cancer) were obtained from the General Hospital of Ningxia Medical University from September 2008 to September 2010.The mutation cluster region of the APC gene was screened using digital PTT labeled with fluorescent Lys-t-RNA,with a polymerase chain reaction fragment amplified from genomic DNA serving as a tenplate for in vitro translation.The occurrence of gene mutation was determined according to the emergence of truncated peptides.The mutation cluster region of the APC gene in 46 samples of colorectal cancer tissues was analyzed by direct sequencing.The detection rates of the 2 methods were compared by chi-square test.Results Thirteen (26％) truncated peptides were detected in the 50 samples of colorectal cancer tissues.The mutation type of 4 samples is nonsense mutation,which resulted in emergence of truncated gene products.Eleven (24％) truncated peptides were detected in the 46 samples of colorectal cancer tissues.There was no significant difference in the detection rates between PTT and direct sequencing ( x2 =0.033,P ＞ 0.05 ).Conclusions Truncating mutations of APC gene are common alterations in sporadic colorectal cancer in the Chinese.Digital PTT labeled with fluorescent Lys-t-RNA is rapid and high-sensitive in screening gene mutations. Key words: Colorectal neoplasms; APC gene; Protein truncation test; Mutation cluster region; Somatic mutation