The Identification And Mechanism Of POLQ Mutation In Hereditary Colorectal Cancer

Objective: Colorectal cancer is the result of a combination of internal and external factors,in which genetic factors play an important role.Genetic predisposition can be observed in 5%-15% of patients with colorectal cancer.Accurate identification of genetically susceptible individuals is very important,but the clinical manifestations of these patients are complex and varied,so the exploration of genetic defects in these syndromes is an important step forward in the study of the disease.This classification of clinical syndromes based on genetic information can improve the interference of changeable clinical characteristics on diagnosis and treatment.Meanwhile,the study of pathogenicity of genetic defects can also provide theoretical basis for the clinical management of related genetic syndromes.Although some hereditary colorectal cancer patients and families have been effectively managed,there are still many patients with typical clinical characteristics that cannot be explained,and these patients may ultimately miss the optimal treatment opportunity due to the lack of molecular diagnosis.Therefore,this study intends to explore new pathogenic genes of hereditary colorectal cancer through next-generation sequencing,and further explore the pathogenic mechanism of potential pathogenic genes,to provide a new theoretical basis for the identification,management,and treatment of hereditary colorectal cancer families.Method: In the genetic screening stage,we conducted whole exome sequencing on the core members of 12 families that met the diagnosis of hereditary colorectal cancer and excluded genes related to Lynch syndrome by clinical molecular diagnosis.Through bioinformatics analysis and co-isolation verification,the candidate pathogenic genes and mutation information were preliminarily identified,and independent families and sporadic colorectal cancer samples were verified.The mechanism of the pathogenic genes of hereditary colorectal cancer identified in the stage of genetic screening was further studied.Firstly,the cell mutation model and mutation overexpression plasmid were constructed by CRISPR/Cas9 technique in vitro.Based on the effect of mutation mode on gene expression and function,combined with DNA damage response related experiments and tumor biology experiments,the key mechanism of pathogenic gene mutation involved in tumorigenesis was explored,and related recovery experiments were carried out with chemical inhibitors.Finally,through the verification of clinical mutation carrier research data,the pathogenic mechanism of the candidate gene was determined.Results: In the genetic screening phase of this study,we carried out whole exome sequencing in 12 families with hereditary colorectal cancer diagnosis and clinical molecular diagnosis excluding genes related to Lynch syndrome.It was found that one family carried a germline mutation of POLQ and was co-isolated with the disease status.Later,through the verification of the probands of 74 independent families,it was found that the second family carried the same mutation of the same gene and was co-isolated from the disease state.Through the verification of 310 cases of sporadic colorectal cancer,we identified a new possible pathogenic gene of hereditary colorectal cancer syndrome.Its allele shows a genetic pattern,the phenotype has a high penetrance,and the tumor is mainly located in the colon and rectum.A total of 10 people in the two families carried POLQ p.Arg1953*heterozygous mutations,of which 7 had clinical manifestations such as multiple colorectal adenomas and colorectal cancers,and most of the patients were less than 60 years old.In addition,carriers were also prone to extra-colonic tumors such as endometrial carcinoma and ureteral epithelial cancer.The classification of clinical manifestations belongs to polyp syndrome,and the phenotype partially overlaps with MUTYH polyposis,Polymerase proofreading polyposis and Lynch syndrome.POLQ mutant tumors showed microsatellite stability and high tumor mutation burden.In summary,based on genetic screening and validation data,we recommend that all individuals or families with unexplained multiple colorectal adenomas,early-onset colorectal cancer or endometrial cancer should be tested for POLQ Arg1953*.Families with POLQ polyposis syndrome should be included in the management of clinical screening of hereditary colorectal cancer,and it is necessary to conduct intensive colonoscopy and endometrial examination for carriers.We have studied the pathogenic mechanism of POLQ polyposis syndrome.POLQ is a multifunctional DNA damage response enzyme,which independently regulates the Polymerase theta-mediated end joining,plays a key role in the repair of DNA double strand breaks,and also plays a role in DNA repair pathways such as base excision repair and interstream cross-linking repair.We successfully constructed POLQ p.Arg1953* and control site p.Gln1949* heterozygous and homozygous mutant cell lines by CRISPR/Cas9.It was found that the prematurely terminated mutation triggered nonsense-mediated m RNA degradation resulting in a decrease in the initial expression of POLQ,and no truncated protein residue was detected in homozygous mutant cells.Further tumor biological experiments showed that heterozygous mutant cells showed stronger ability of proliferation,clone formation and tumorigenesis under the action of DNA damage factors such as IR and UV.This shows that the function of damage repair plays an important role in the formation of tumor phenotype.Next,we study how POLQ mutations participate in tumorigenesis by changing the function of DNA damage reponse.First,by detecting the expression of DNA damage markers γ-H2 AX and POLQ protein at different time and different induction doses,we found that different from the expression pattern of wild-type cells gradually increasing with time,heterozygous mutant cells showed a new expression pattern of early haploid overexpression in the process of DNA damage response.This new expression pattern shows stronger repair ability of DNA damage.Through the detection of repair pathways of DNA double strand breaks,we found that the abnormally enhanced DNA damage repair ability of mutant cells was achieved through overactivated TMEJ pathway.The error-prone repair of TMEJ pathway is also the main reason for the high tumor mutation burden in mutant patients.Finally,according to the results of POLQ haploid overexpression,we treated with POLQ inhibitor Novobiocin(NVB),and found that the tumor-promoting phenotype of mutant cells induced by DNA damage could be reversed by NVB.Conclusions: Our study found that germline mutation of POLQ predispose to colorectal adenomas and carcinomas,which may be a new pathogenic gene of hereditary colorectal cancer.POLQ p.Arg1953* heterozygous mutations change the expression patterns involved in DNA damage response,over-activate TMEJ repair pathway,abnormally enhance DNA repair function,resulting in genomic mutation accumulation involved in tumorigenesis.NVB may be a potential adjuvant in the treatment of patients with POLQ p.Arg1953*.