| Objective:1.The characteristics of IGH repertoires in bone marrow and peripheral blood of patients with Ig A and Ig G type Multiple myeloma(MM)and volunteers with iron deficiency anemia(IDA)were analyzed: 1)To provide basic data for the classification of myeloma BCR repertoires;2)Bioinformatics methods were used to screen potential myeloma sequences in MM patients;3)Analysis of CDR3 clones of different types of myeloma provides new ideas for the development of disease-targeting drugs.2.The expression of genes related to the developmental differentiation of bone marrow and peripheral blood B lymphocytes in patients with Ig A-type and Ig G-type myeloma were analyzed by transcriptome comparison to provide a research basis for exploring the immune activation mechanism related to myeloma.Methods:1.From the Department of Hematology of the affiliated hospital,6 patients with MM(3each of Ig A and Ig G types)were screened according to the diagnostic criteria proposed by the International Myeloma Working Group(IMWG)and the 2020 Chinese guidelines for the diagnosis and treatment of multiple myeloma,and in compliance with the principle of informed consent of volunteers.2.5ml of bone marrow and peripheral blood were collected from 6 patients with multiple myeloma and 3 iron deficiency anemia volunteers.Bone Marrow Mononuclear Cells(BMMCs)in each bone marrow sample and mononuclear cells(Peripheral Blood Mononuclear Cells,PBMCs)in peripheral blood samples were isolated,and total RNA was extracted from each sample.3.IGH repertoire construction and sequencing:Total RNA of each sample was reverse transcribed to c DNA,The repertoire construction was performed by 5′RACE technology,and IGH repertoire gene sequences were determined using Illumina Nova Seq platform.4.Transcriptome sequencing: m RNA was isolated and enriched from the total RNA of each sample,the m RNA was reverse transcribed into c DNA,the database is constructed by PCR,and the transcriptome gene expression level of each sample was determined using Illumina Nova Seq 6000 sequencing platform.5.Analysis of sequencing results:(1)IGH repertoires characterization: comparison of the diversity of bone marrow and peripheral blood IGH repertoires,CDR3 length distribution and amino acid(AA)fetching,germline gene family fetching,somatic hypermutation(SHM),nucleotide insertions and deletions,Analysis of the number of AA changes due to somatic hypermutation,and oncogenic cloning.(2)Transcriptome analysis: To compare and analyze the differentially expressed genes in the bone marrow and peripheral blood transcriptomes of Ig A-type and Ig G-type MM patients and IDA volunteers,to screen for B cell development,differentiation,activation and proliferation-related genes,and to analyze the characteristics of B related genes expression regulation in both types of myeloma.Results:1.Composition of IGH repertoire: The proportion of Ig A sequence in Ig A-type MM group was significantly higher than that in the Ig G-type MM group and IDA group(P <0.0001).The proportion of Ig G sequences was significantly higher in the Ig G-type MM group than in the Ig A-type MM and IDA group(P < 0.0001).The dominant subtype sequences in the IGH subtype ratio data of patients with both types of MM were consistent with clinical typing.2.Diversity of IGH repertoire: The inverse Simpson’s diversity index(1/DS)of the bone marrow IGH repertoires of Ig A-type,Ig G-type MM patients and IDA group was 1.2,1.2and 2845.6.The 1/DS of the IGH repertoires in peripheral blood was 1.6,1.2 and 206.7 for the three groups.The diversity of the bone marrow and peripheral blood IGH repertoires of both Ig A and Ig G types of MM was significantly lower than that of the IDA group.3.CDR3 length distribution and amino acid sampling:There was no statistical difference in the mean length of CDR3 in the bone marrow and peripheral blood IGH repertoires of both Ig A-type and Ig G type of MM and IDA group.CDR3 length distribution of Ig A type MM was dominated by 11 and 17 amino acid lengths.Ig G type MM was dominated by 15 and 16 amino acid lengths.The CDR3 length distribution of the IDA group was similar to the Gaussian distribution.There was no significant difference in CDR3 amino acid fetching between the groups.4.Usaging of IGHV,IGHD,IGHJ gene families,insertion and trimmed of IGH repertoire,the number of AA changes due to SHM:(1)There is a bias for IGHV,IGHD,and IGHJ gene family usage in bone marrow and peripheral blood IGH repertoires of MM patients.(2)There was no statistical difference in the average number of inserted and trimmed nucleotides in bone marrow and peripheral blood IGH repertoires among the three groups.The proportion of insertion and trimmed of different numbers of nucleotides differed significantly between the three groups.(3)The mean number of amino acid changes in the FR1,CDR1,FR2,CDR2 and FR3 regions of the IGH sequences of Ig A,Ig G-type MM and IDA groups were not statistically different.The sequence proportions of the number of amino acid changes differed significantly between the groups.5.Screening and characteristic analysis of oncogenic clones: 10 potentially oncogenic clones were obtained from the first 100 sequences of MM groups by bioinformatics method.The CDR3 sequences analysis of the first 10 clones showed that "CARGLIIGHF DSW" 、 "CARGAASTASGSRGFIDYW" 、 "CARDIPYYGSGLAFDNW" amino acid sequences were expressed in several MM patients.6.Regulation of B-cell development,differentiation,activation and proliferationrelated gene expression:In bone marrow,the genes of POU2F2,IRF4,ICAM1,CD40,CD38,BCL2,BLNK,NFKBIA,NFKBIE,MEF2C,FLT3,TICAM1 and JUN were upregulated and the genes of IGLL1 and CXCL12 were down-regulated in the Ig A MM group compared with the IDA group.Compared with the IDA group,POU2F2,IRF4,ICAM1,NFKBIE,JUN,MEF2C and TICAM1 were up-regulated in Ig G MM group,while FLT3was down-regulated.CXCL12 gene was down-regulated and BCL2 gene was up-regulated in Ig A MM group compared with Ig G MM group.Peripheral blood regulatory genes overlapped with bone marrow.These genes regulating B cells were up-regulated and overexpressed as genes that induced or promoted B cell proliferation in both types of MM.Genes promoting B cell apoptosis are down-regulated and underexpressed.Ultimately,NF-κB,NFAT and AP-1 signaling pathways were activated to promote the development,differentiation and proliferation of B cells and inhibit their apoptosis.Conclusions:1.The IGH repertoires in bone marrow of multiple myeloma not only identifies the staging of myeloma,but also deciphers the characteristics of B cell clonal proliferation.2.The diversity of bone marrow and peripheral blood IGH CDR3 repertoires in Ig A-type and Ig G-type MM groups was significantly lower than that in the IDA group.The distribution and structure of CDR3 clones showed significant differences among the three groups,which were caused by VDJ gene rearrangement,nucleotide insertion and deletion and somatic hypermutation.3.10 potentially oncogenic clones were obtained from the MM groups,which can provide reference data for the exploration of new drug targets for the individualized treatment of MM.4.Myeloma polygenes are involved in the regulation of B-cell proliferation and apoptosis.CXCL12 and BCL2 genes are differentially expressed in the bone marrow of Ig A-type MM and Ig G-type MM,which may be related to the differential formation of the IGH repertoires between the two types of Myeloma. |
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