Detection Of Multiple Gene Mutations In Stool For Colorectal Cancer Screening

Colorectal cancer is the most common malignant tumor of the digestive tract in China,and the incidence and mortality has been increasing in recent years.Early screening is the most effective way to reduce the incidence and mortality of colorectal cancer.There is some limitations for most traditional screening methods,such as colonoscopy and fecal occult blood test(FOBT),and thus it's not suitable for large-scale promotion and application.Screening and diagnosis of colorectal cancer by fecal DNA is a promising method,but the relationship between the mutation of some related genes and the process of disease is not yet clear.In addition,the mutation frequencies of such genes is also varied between ethnic groups.In this study,we collected fecal DNA and tissue samples from different populations in South China,and then detected mutation frequencies of eight genes which are related to the development of colorectal cancer through high-throughput DNA sequencing.The correlation between mutation frequencies and disease type or processes of colorectal cancer was also analyzed,in order to develop an effective method for early screening of colorectal cancer.Methods: We collected fecal samples or tissue samples from normal people,patients with colorectal mucosa or colorectal cancer.After DNA was extracted using the QIAamp Fast DNA Stool Kit kit,primers were designed for 8 genes associated with colorectal cancer,and then PCR was performed,followed by DNA library construction and exon sequencing.The results were verified by Sanger sequencing.On the basis of this,we compared the SNP frequency of the eight genes in normal and different patients,and analyzed the correlation of mutation frequency and disease type or the process of colorectal cancer.In addition,we also analyzed the effect of the experimental conditions on the final results by changing the proportion of different components in the sample and adjusting the sample concentration.We also compared the SNP frequencies in different age groups and discussed the applicability of this screening method in different age groups.Results: 1.The mutations of APC and K-ras gene were most likely to be detected in stool samples of colorectal cancer patients.2,the mutated genes and mutation frequency varied in different patients,none mutation was shared by all the colorectal cancer patients,suggesting necessity for multi-gene detection.3,mutation of these genes can be also detected from the patients with other types of colorectal lesions.4,Mixing cancer tissue and normal tissue in different proportions resulted to different detected frequencies of mutation,suggesting that the number of shedding cells from tumor in fecal may affect the test results.5,the concentration of the sample less affected on the final sequencing results.6,the patient's age has little effect on the detection of mutations.Our results show that the early screening of colorectal cancer through fecal DNA is technically and methodologically feasible,however,much more theory research and data were necessary in order to find the right gene frequency for efficient and specific screening or diagnosis in Chinese population.