Germline Mutations In 40 Cancer Susceptibility Genes Among Chinese Patients With High Hereditary Risk Breast Cancer And Functional Study Of Detected PALB2 Missense Mutants

Breast cancer is the most common malignancy among women both in China and the West.It is estimated that about 27% of breast cancer incidences are attributed to hereditary factors.More than 25 breast cancer associated susceptibility genes have been identified,including genes with high penetrance(BRCA1,BRCA2,TP53,CDH1,PTEN,STK11,and NF1),moderate penetrance(PALB2,CHEK2,ATM,NBN,and Related Genes),and low penetrance.Over the last two decades,clinical genetic testing has become widespread.Recently,next-generation sequencing(NGS)has enabled massive parallel sequencing(MPS)at low cost,and multiple-gene panels are now commercially available for cancer risk assessment.Despite the technical efficiency is attractive,several challenges remain about the clinical value of multiple-gene panels for BC susceptibility,especially how to deal with the variants of uncertain significance(VUS).Multigene panel testing of BC predisposition genes is relatively rare in Chinese populations.Therefore,we assembled a large clinic-based cohort of Chinese high hereditary risk BC patients from different parts of mainland China to assess the frequency of detrimental germline mutations in 40 cancer predisposition genes.This is done in an effort to better illustrate the contribution of germline mutations in BC susceptibility genes among Chinese high hereditary risk BC patients,and to explore the questions about the clinical use of multiple-gene panels in BC.In the process of analysing the sequencing results,we found a lot of VUS in PALB2.Then we conducted a series of functional assay,in order to assess their pathogenicity and further explore the function of PALB2.This study is divided into two sections: 1.Germline mutations in 40 cancer susceptibility genes among Chinese patients with high hereditary risk breast cancer.2.Functional study of detected PALB2 missense mutants.Methods and material:Section 1:Between October 2015 and September 2016,patients with high hereditary risk BC from 26 hospitals of 14 provinces in China,were recruited.Patients who met one of the following criteria were included:(1)onset age ≤35 years(early-onset BC);(2)At least one first or second-degree relative with BC,ovarian cancer(OC),primary peritoneal cancer,or fallopian tube carcinoma;(3)Two primary BC;(4)Male BC;(5)BC with OC,fallopian tube carcinoma or primary peritoneal cancer;(6)Meeting two or more of the above criteria.Clinical information was abstracted from medical records and reviewed by breast pathologists.Fresh peripheral venous blood(5 ml)was collected from each participant.Complete exons of 40 Breast Cancer-related genes sequencing was performed at Annoroad Gene Technology(Beijing,China)on an Illumina Hi Seq 2500 platform(Illumina,San Diego,CA,USA).An automated sequencing data monitoring system was implemented for auto-launching the pipeline.An in-house QC tool was used for quality control statistics and pre-processing of raw sequence data.According to the American College of Medical Genetics(ACMG)guidelines,variants were classified as pathogenic,likely pathogenic,VUS,benign,and likely benign genetic variant.For VUS,the above mentioned in silico method was applied to predict its possible significance,but the result was not included in the final analysis of germline mutations.Section 2:In order to assess the pathogenicity of the PALB2 VUS detected in the previous section,we first utilized a lentivirus system to construct PALB2 knockdown HEK-293 T cell line and tested the knock-down efficiency with RT-PCR and Western Blot.Then site-directed mutagenesis was performed to generate missense mutation of PALB2 by using pc DNA3.1-Flag-PALB2 as template.p DR-GFP and p CBASce I plasmid were transfected into PALB2 knockdown HEK-293 T cell to establish Homologous recombination GFP reporter system.After missense mutation of PALB2 was introduced in the system,GFP-positive cells were scored by flow cytometry.For the mutation sites selected by above homologous recombination assay,a Co-Immunoprecipitation assay was performed to test their abilities to interact with BRCA1,BRCA2,RAD51,and PALB2.Main results and conclusions:Section 1:Germline mutations in 40 cancer susceptibility genes among Chinese patients with high hereditary risk breast cancer1.A total of 937 patients with BC were recruited,most of which were Han ethnic group(96.5%).According to the recruitment criteria,most of the participants included were early-onset BC patients(47.6%),followed by BC patients with BC/OC family history(34.7%)and bilateral BC(9.2%).223 subjects(23.8%)were found to carry pathogenic or likely pathogenic mutations.Of these,159 had BRCA1/2 mutations(17.0%),61 had non-BRCA1/2 gene mutations(6.5%)and 3 patients had both BRCA1/2 and non-BRCA 1/2 gene mutations(0.3%).2.A total of 53 deleterious BRCA1 mutations were detected in 82 individuals(8.8%),including about 94.3%(n=50)truncating mutations and less than 5.7 %(n = 3)known deleterious missense mutations.14 BRCA1 mutations were detected firstly in this research(26.4%).We also found 67 BRCA2 deleterious mutations in 81 participants(8.6%),all of which were truncating mutations.Among the pathogenic BRCA2 mutations,32 were novel(47.8%).For non-BRCA1/2 genes,55 mutations in 15 genes were detected in 64 patients(6.8%),which included about 76.3 %(n = 42)truncating mutations and 23.6 %(n = 13)deleterious missense mutations.22 variants were novel(40.0%).53(5.7%)patients carried mutations in non-BRCA1/2 breast cancer predisposition genes,including TP53(n=18),PALB2(n=11),CHEK2(n=6),ATM(n=6),BARD1(n=5),BRIP1(n=3),CDH1(n=2),and RAD50(n=2).In addition,7 participants were identified with mismatch repair(MMR)gene mutations and 5 individuals carried deleterious mutations in other 3 genes which were likely related to BC: NQO2(n=3),PPM1D(n=1)and ERBB2(n=1).There were other 8 participants with monoallelic MUTYH mutations who were not included in this analysis,for its reduced association with cancer risk.3.The mutation rate of BRCA1/2 tended to increase with age during BC diagnosis while in other genes the frequency was likely to decrease.For BRCA1/2,the prevalence of deleterious mutations was higher than average in patients with a personal or family history of BC/OC,but for non-BRCA1/2 genes,there was no such trend.BRCA1 mutations were enriched in patients with TNBC and BRCA2 mutations were associated with HR-positive BC.P53 mutations were more in patients with Her2 positive BC(5.2% vs 2.0%,p=0.001),especially in early-onset(≤35y)BC patients(12/167,7.2%).Mutations of PALB2,CHEK2,BRIP1,and MSH2 were likely to be associated with HR-positive BC,despite of no statistical difference.In summary,no factors predicted pathologic mutations in non-BRCA1/2 genes when treated as a whole.4.A total of 1352 VUS were identified in 908 women.64 unique missense mutations from 95 patients were predicted to be deleterious by in silico analysis.25 VUS were detected in PALB2 and then were analysed in next section.Section 2:Functional study of detected PALB2 missense mutants1.Homologous recombination assay showed that there was hardly any GFP positive cells in PALB2 null group while the rate of GFP positive cells was 1.36% in PALB2 WT group.For missense mutation,the rate of GFP positive cells was 0.28% in E1018 D group,0.89% in R37 H group and 0.92% in I966 T group,which were all lower than that in PALB2 WT group(p<0.05).This result suggested that these three missense mutations might be pathogenic.2.Co-Immunoprecipitation assay showed that,with E1018 D mutation,PALB2 could not combine with BRCA2.This was the reason why E1018 D could harm the homologous recombination function of the cell.Despite no statistical significance,the combination ability of PALB2 and RAD51 seemed to be decreased by R37 H mutation.Other than E1018 D and R37 H,neither I966 T nor P240 T could affect the interaction between PALB2 and other protein.Collectively,we performed the largest prospective study to date for evaluating the clinical use of NGS among Chinese high hereditary risk BC patients.Totally,23.8% of 937 patients carried putative germline deleterious mutations in 18 of 40 predisposition genes.BRCA1/2 gene mutation rates were 17.3%,while non-BRCA1/2 gene mutation rates were 6.8%.The frequency of BRCA2 mutation was comparable with BRCA1,moreover,TP53 and PALB2 also demonstrated a relatively high mutation rate.No factors were predicted for mutations in non-BRCA1/2 genes when treated as a whole,while TP53 mutations were associated with HER-2 positive BC and younger diagnosis at age,CHEK2 and PALB2 mutations enriched in patients with luminal BC.Our study demonstrates that multiple-gene sequencing has high clinical application value for cancer risk assessment in Chinese population.Further functional assay for PALB2 VUS identified a novel pathogenic missense mutation E1018 D,which could decrease cell homologous recombination function by disturbing combination ability of PALB2 and BRCA2.