Analysis For Homogeneity And Heterogeneity Of BCR-H CDR3 Repertoires In The Peripheral Blood Of Twins

Objective: We collected peripheral blood samples from healthy monozygotic(MZ)twins,dizygotic(DZ)twins and non-twins(NT)aged 20-50 years to analyze the homogeneity and heterogeneity of BCR-H pseudogene CDR3,out of frame CDR3,and productive CDR3 repertoires,as well as the dynamic changes of the BCR-H CDR3 repertoires before and after 5 years.This study initially discussed the formation mechanism of the BCR-H CDR3 repertoires,provides a basis for further research on the mechanism and application of BCR rearrangement.Methods:1.We collected respectively 10 ml peripheral blood from each healthy twin volunteer(D,e,F,G,h,i),isolated PBMCs and extracted total DNA.Meanwhile,we designed and synthesized BCR-H CDR3 repertoires primers(7 upstream primers containing total IGHV family,9 IGHJ downstream primers containing functional genes and pseudogenes).The total DNA as a template was used for PCR amplification,and recovering the PCR products.After quality inspection,the purified PCR products was used for Illumina Solexa high-throughput sequencing of BGI(Beijing Genomics institution)2.We collected respectively 10 ml peripheral blood from each healthy twin volunteer(A,B,c),isolated PBMCs and extracted total DNA in 2012 and 2017.And then send the total DNA to Adaptive Biotechnologies immune SEQ company in 2012,and to BGI in 2017.The two companies used DNA as template to establish BCR-H CDR3 repertoires by PCR technology,and sequenced with Illumina Solexa high-throughput technology(the experiment in 2012 was completed by the research team).3.We sent DNA samples from 9 pairs of healthy twin volunteers(A,B,c,D,e,F,G,h,i)to Beijing Bo'ao Jingdian Biotechnology Co.Ltd.to detect HLA-A/B/C/DRB1 by PCR-SBT.4.We analyzed the sequencing results of BCR-H CDR3 repertoires:(1)Comparativeanalysis for the homogeneity and heterogeneity of pseudogene CDR3,out of frame CDR3 and productive CDR3 sequences among MZ twins(D1/D2,F1/F2,G1/G2),DZ twins(e1/e2,h1/h2,i1/i2),and NT(D1/e1,D1/F1,D1/G1,D1/h1,D1/i1)groups;(2)Comparative analysis for dynamic changes of BCR-H CDR3 repertoires among MZ twins(A1/A2,B1/ B2),DZ twins(c1/c2)and NT(A1/B1,A1/c1,A1/c2,B1/c1,B1/c2)before and after 5 years.Results:1.The HLA typing results of twins showd that twins A,B,D,F,and G were monozygotic twins,and twins c,e,h,and i were dizygotic twins.2.There was no significant difference in BCR-H CDR3 repertoires diversity among MZ twins,DZ twins and NT,but HEC statistics among the three groups showed that MZ twins and DZ twins were different from NT.There was no significant difference in the diversity of BCR-H CDR3 repertoires between before and after 5 years.3.In pseudogene,out of frame and productive BCR-H CDR3 repertoires,the families with different IGHV usage have the smallest difference between twins;the changes before and after 5 years also showed that MZ twins were the most similar,especially in pseudogene BCR-H CDR3 repertoires.There was no difference in the frequency of IGHJ usage among MZ twins,DZ twins and NT in pseudogene and out of frame BCR-H CDR3 repertoires,but there was difference in the frequency of IGHJ1 usage in productive BCR-H CDR3 repertoires,while showed that twins were the most similar.In pseudogene and out of frame BCR-H CDR3 repertoires,the changes of IGHV and IGHJ usage before and after 5 years were the most similar in MZ twins,but there was no difference among MZ twins,DZ twins and NT in productive BCR-H CDR3 repertoires.4.There was no significant difference in amino acid usage,amino acid length distribution and amino acid overlap of BCR-H CDR3 repertoires among MZ twins,DZ twins and NT,and there was no significant difference in the changes before and after 5 years.5.In the productive BCR-H CDR3 repertoires,the proportion of insertion and deletion of different nucleotides at the V(D)J junction of MZ twins and DZ twins was more similar than that of NT.There was no difference among MZ twins,DZ twins and NT in the ratio ofinsertion and deletion of different nucleotides at V(D)J junction before and after 5 years.In the pseudogene and out of frame BCR-H CDR3 repertoires,there was no difference among MZ twins,DZ twins and NT in the ratio of insertion and deletion of different nucleotides at V(D)J junction,but the changes before and after five years were different.Conclusion:1.(1)Compared with NT(D1/e1,D1/F1,D1/G1,D1/h1,D1/i1),the IGHV usage of BCR-H CDR3 repertoires was more homogeneous in MZ twins(D1/D2,F1/F2,G1/G2)and DZ twins(e1/e2,h1/h2,i1/i2).(2)In the pseudogene BCR-H CDR3 repertoires,there was no differences of IGHJ usage among MZ twins,DZ twins,and NT groups.(3)In the productive BCR-H CDR3 repertoires,there were no differences in amino acid usage,amino acid length distribution,amino acid overlap,and nucleotide insertion and deletion among MZ twins,DZ twins,and NT groups.The IGHV usage has a certain genetic bias during the initial rearrangement of V(D)J,while IGHJ usage does not.This bias may affect the composition and characteristics of the naive B cell repertoires in the periphery of the individual,and further affect the individual's immune response to peripheral antigens.2.(1)In the pseudogene BCR-H CDR3 repertoires,compared with NT(A1/B1,A1/c1,A1/c2,B1/c1,B1/c2)and DZ twins(c1/c2),the IGHV usage in MZ twins(A1/A2,B1/B2)showed more consistent changes in before and after 5 years,while the IGHJ usage in MZ twins,DZ twins and NT had no difference.This further suggests that individual genetic factors may have some influence on the IGHV usage during the initial rearrangement.(2)In the productive BCR-H CDR3 repertoires,there was no significant difference among MZ twins,DZ twins and NT in amino acid usage,amino acid length distribution,amino acid overlap and nucleotide insertion and deletion before and after five years.This suggests that long-term environmental antigen stimulation may "dilute" the genetic bias of the peripheral blood total B cell repertoires,which provides a basis for further analysis of the differences between the naive and memory B cell repertoires of twins.