Application Of Next-Generation Sequencing Technology In Detection Of Germline Mutation In Breast Cancer Susceptibility Genes

Objective:Hereditary breast cancer account for about 5% to 10% of the total number of breast cancer patients.Pathogenic mutations in breast cancer susceptibility genes BRCA1/2 are closely related to the occurrence of hereditary breast cancer.Meanwhile,more highly penetrant cancer susceptibility genes were invovled in breast carcinogenesis.In this study,we screened whole exon regions of 6 highly penetrant breast cancer susceptibility genes using next-generation sequencing technology to discover germline mutations in the six genes and evaluate their relationship with clinicopathologic features of breast cancer patients.Methods:A customized amplicon-based target sequencing panel was designed by next-generation sequencing technique using the Illumina Miseq sequencing platform to enrich all exon regions of 6 highly penetrant breast cancer susceptibility genes.DNA samples from blood of 146 breast cancer patients,71 healthy women with high risk factors of breast cancer and 55 healthy women without family history from November 2013 to July 2015 in Tianjin Medical University Cancer Hospital were collected and sequenced.Based on the test results,the correlation between germline mutations and respective clinicopathologic features of breast cancer patients was evaluated.Results:1.In this study,We performed multiple gene sequencing using breast cancer amplicon-based target sequencing panel to achieve an average sequencing depth of 587× for all target regions.Sequencing data for 92% samples was finished with Q30 percentage upper than 80%.In all samples,More than 85% of sequencing reads were mapped to targetted region for all samples,with average percentage 89.7%.More than 95% of the target exon regions were covered by sequenced data for all samples,with average percentage 98.1%.The sequencing depth for each amplicon across different samples had excellent replicability and there was high consistency of sequencing data distribution between amplicons.2.A total of 67 variants were identified including 50 missense mutations,8 nonsense mutations and 9 indels.And 21 variants(31.3%)were not described in Ex AC,Clin Var or db SNP database,described as newly detected variant types.There were 85.1%(57/67)mutations only found in patients and high risk individuals.There were 37 high risk pathogenic germline mutations in 67 mutations.3.Mutation state of breast cancer patients were correlated with the risk of axillary lymph nodes involvement and TNM stage,and the difference had statistical significance(P<0.05).There were no significant correlations among age,family history of malignancy,histological grade and the expression of ER,PR,H er-2,Ki67 and molecular typing(P> 0.05).Then among patients with mutations,the pathogenic group was shown more frequency than the nonpathogenic group in family history and in the rate of triple negative phenotype and the difference had statistical significance(P<0.05).There were no significant correlations among age,axillary lymph nodes involvement,TNM stage,histological grade and the expression of ER,PR,Her-2 and Ki67(P> 0.05).Conclusion : 1.This study demonstrated the feasibility and utility of this amplicon-based target sequencing panel in screening germline mutations in breast cancer related genes by next-generation sequencing.This method provides an approach for us to assess the genetic risk of the breast cancer.2.Some novel variants were found,extending the breast cancer susceptibility gene mutational spectrum,but the further studies were needed for elaborating the impact of these novel variants on the genetic risk of breast cancer.3.Mutation-positive breast cancer patients had a higher risk of axillary lymph nodes involvement and were higher at TNM stage compared with mutation-negative patients,which suggested a poor prognosis.4.Patients with pathogenic mutations have a higher proportion of family history of malignant tumors,indicating a higher genetic risk of suffering from breast cancer.At the same time,it is more likely to be a triple negative phenotype.Thus,the findings in this study would also provide insights for the treatment of breast cancer patients,especially for triple negative breast cancer patients.