Study On Genetic Susceptibility Genes Of Familial Myeloproliferative Tumors By Whole Exome Sequencing

Objective:Myeloproliferative tumor(MPN)is a group of heterogeneous hematological malignancies,which is characterized by a class of clonal hematopoietic stem cell diseases with primary or multiple myeloid cells including erythroid,myeloid and megakaryocytic proliferation.The classic MPN(BCR/ABL negative MPN)is divided into three subtypes: polycythemia vera(PV),thrombocytopenia(ET)and myelofibrosis(PMF).Three factors witch driving MPN are JAK2,MPL and CALR mutations.Generally,thesemutations exist independently and repel each other.Previous studies have shown that MPN is mainly sporadic cases,with complex pathogenesis,and a few cases with family histrory.The familial MPN is a family with 2 or more MPN patients,which indicating that MPN has genetic susceptibility in its pathogenesis.Therefore,more and more researchers pay attention to the familialMPN,which provides the basis for further study of the pathogenesis of MPN.In our clinical work,we found 2 MPN inone family,one of them wasdiagnosed as PV with JAK2V617 F,the other was ET with JAK2V617 F.The purpose of this study is to find out the genetic susceptibility genes related to JAK2V617 Fby sequencing the exons of some members of this familial MPN,and to provide theoretical basis for elucidating the genetic mechanism of familial MPN.Methods:1.Pedigree analysis: Patients who were diagnosed as MPN in the department of Hematology,the Second Hospital of Shanxi Medical Universityfrom October 2016 to October 2018 were enrolled in our study.MPN patients with family history were screened out by asking family history.With informed consent of patients and their families,starting with the proband,all family members of the patients were traced back,and the healthy situation of each family member was investigated in detail and pedigree was drawn.2.Sample collection: The samples of hair follicle tissue(H)and peripheral blood(PB)were collected from two MPN patients,and the samples of peripheral blood from normal healthy members without clinical phenotype and JAK2V617 F mutation,MPL mutation orCALR mutation in the family were collected.3.Whole exome sequencing: The genomic DNA of each sample was extracted.Based on the Illumina sequencing platform,the Agilent SureSelectXT human all exon kit was used to capture and build the database.The paired end method was used to complete the sequencing analysis of 6 samples.The effective sequencing depth was Pb(200×),H(100×),and the average Q≥80%.By analyzing the original data of the whole exon group,we found the germline mutations that coexisted in two patients but did not exist in the healthy members.4.Verification of germline mutation: All the selected germline mutations were sequenced in the candidate family MPN patients and normal healthy members for verification.polyphen 2 and sift mutation prediction tools were also used to evaluate the related germline mutations.At the same time,the mutation of JAK2V617 F gene was detected in MPN samples collected from our hospital.Results:1.This family involves a total of 9 members of two generations,including 3 males and 6 females.One of the two MPN patients was a PV patient carrying a JAK2V617 F gene mutation,and one was an ET patient carrying a JAK2V617 F gene mutation.Seven other normal healthy members of the family,three of whom refused to cooperate with the study.2.Whole exome sequencing analysis finally obtained 5 germline susceptibility genes,which are 3 possible pathogenic missense variants ST6GALNAC4: c.C430T(p.R144X),PEX2: c.T227C(p.V76A)and MLLT10: c.1183G> A(p.Ala395Thr)and two splice sites CHMP4B: c.610 + 9T> C,TBC1D23: c.1687 + 5A> G.3.Design primers for the above-mentioned missense mutation sites,and verify them by Sanger sequencing in patients and normal healthy members of the family.As a result,only ST6GALNAC4: c.C430T(p.R144X)appeared in both patients.It does not appear in normal healthy members of the family.4.MPN specimens based on the initial JAK2V617 F gene mutation in the MPN specimen library of the Hematology Laboratory of our hospital from November 2016 to October 2019,a total of 307 cases.One generation sequencing was used to screen ST6GALNAC4 R144 X for 307 cases,and the results were all negative.Conclusions:1.ST6GALNAC4 was selected as a familial MPN susceptible germline variant by whole exome sequencing analysis.To our knowledge,this is the first study to link ST6GALNAC4 to malignant blood disorders.2.The ST6GALNAC4 gene is used as a susceptible germline variant of JAK2V617 F positive MPN,which needs to further expand the sample for verification and functional research.