Genetic Lineage Study Of Lynch Syndrome Based On Second Generation Sequencing

BckgroundLynch Syndrome(LS),which is also called heredity nonpolyposis colorectal cancer(HNPCC),is the most common family colorectal disease with autosomal dominant inheritance characteristics,caused by germline mutations in mismatch repair(MMR)genes including the MSH2,MLH1,MSH6 and PMS2,leading to microsatellite instability(MSI).Clinically,Lynch Syndrome is characterized by high incidences of early-onset colorectal cancer especially right hemicolon as well as endometrial,ovarian,gastrointestinal malignancies.In addition,there is another subtype called Familial Colorectal Cancer Type X(FCCTX),which meet the clinical diagnose criteria but absent from the MMR gene mutation showing microsatellite stability,the pathogenesis and the genetic basis is still unknown and concluded in highly heterogeneity,needed to be investigated deeply.The diagnose criteria of Lynch Syndrome used in clinical are Amsterdam II and Bethesda criteria,which still lead to a high rate of missed diagnosis in LS and FCCTX,there is still numerous MMR gene mutations waiting to be discovered.In order to dig into the pathogenesis and genetic basis of LS and FCCTX,thus guide the clinical diagnosis and treatment,this program aim to do genetic lineage research in LS pedigrees collected from clinical using the next generation sequencing,and establish the clinical Lynch Syndrome database to standardize the clinical treat and prognosis surveillance.Objective1.Collect the recent 28 months patients who underwent colorectal and endometrial cancer surgery and fulfilled the Immunohistochemical dMMR,select 23 of them who occupied LS suspicious factors,call back to hospital and collect the blood sample.2.Fulfill the genetic screening for the proband of suspicious Lynch Syndrome pedigree with a Panel using next generation sequencing.Finish the co-segregation test among the MMR mutation(+)pedigree using sanger sequencing3.Fulfill the Whole Exome Sequencing in pedigree who failed to detect MMR gene mutation in Panel screening but meet the clinical criteria of LS owning characteristic family history4.Establish the Lynch Syndrome database for PUMCH and guide clinical diagnosis,treatment and surveillance in the future.Methods1.Review all the medical record and make telephone follow-up of patients whose pathology immune-histochemical staining shows defective MMR expression after receiving colorectal and gynecology surgery in PUMCH during October,2014 to February,2017,together with collecting suspicious Lynch Syndrome pedigree during clinical work from 2015 till now.We identified 23 highly suspicious LS patients and called them back to do reexamination and blood sample taken to extract DNA and fulfill the genetic Panel screening using next generation sequencing.2.For those patients who discovered novel or known MMR gene mutation,we tried our best to get blood sample from all the patients and normal members among the pedigree to fulfill the co-segregation test and affected member screening by extract DNA,design primers and sanger sequencing technic.If all the affected members in the pedigree own the same gene mutation with the proband,at the same time,all or majority of the normal members in the pedigree are absent of this suspicious gene mutation,we can almost confirm the pathogenicity of this mutation.3.For those patients who failed to find out MMR gene mutation in genetic Panel screening but own characteristic family history,we collected blood samples from all the affected family members and part of the normal members to fulfill the exome sequencing.4.To establish the database,we reached a cooperation with Yidu Cloud company and set up the PUMCH Lynch Syndrome database,which already collected 23 suspicious or diagnosed Lynch Syndrome pedigrees and will be applied to clinical work for longer function fulfillment,including standardized diagnosis and treatment,longer follow-up,early prevention and well surveillance.Results1.We discovered 11 gene mutaions in 23 probands,includeing 4 MSH2 gene mutations,3 MLH1 gene mutations(in 4 pedigrees),1 PMS2 gene mutation,1 BRCA2 gene mutation and 1 PALB2 mutation,among which,the c.1676-1679delTAAA(p.Asn560Lysfs*29)of MSH2 in pedigree 1 and c.3054G>C(p,E1018D)of PMS2 in pedigree are novel gene mutations2.In pedigree 1 and 5,all the affected family members occupied the deleterious gene mutation with all the normal family members occupied the wild type,which demonstrated the co-segregation characteristic and further demonstrated the pathogenicity.3.In pedigree 12,we fulfilled whole exome sequencing among 11 family members,including 6 affected members and 5 healthy members.By comparing to normal population database(1KGP,ESP6500,ExAC,BGlinhouse 1500),we discovered all of the 6 affected members occupied BCLAF1,SDK1,PSPH and KCNJ18 mutation,satisfying the freq<0.005,impact=HIGH/MODERATE,SIFT,POLYPHEN indicated harmful.After reviewing associated publications,we mainly focused on BCLAF1 and PSPH in the future research.4.We established a Lynch Syndrome Clinical Database which has already collected 23 Lynch Syndrome patients,for the future standardized treatment and follow-up surveillance.ConclusionsLynch Syndrome is mainly caused by Mismatch Repair Gene mutation,we found out a high cost-effective flow to prevent,diagnose and treat the colorectal cancer,by fulfilling the colorectal genetic Panel to the proband and demonstrate the co-segregation character using sanger sequencing in all the other affected and healthy pedigree members.Additionally,the result in MMR pathology immunohistochemical can’t 100%predict the MMR gene mutation,we still need the genetic test to figure out the Lynch Syndrome.It’ s definitely a beneficial revolution to set up the standardized treatment to Lynch Syndrome pedigree.