Next Generation Sequencing Reveals Composition And Variation Of The B Cell Heavy Chain Receptor Repertoires In Patients With End-stage Renal Disease

Background and OBJECTIVE:Most studies for immune disorders of end stage renal disease(ESRD)patients have focused on immune of T lymphocyte in the past.In this study,We investigated the changes and composition of B lymphocyte heavy chain in patients with ESRD.Immune repertoire(IR)is the sum of all functional diversity B lymphocytes and T lymphocytes of an individual’s circulatory system at any given time.B cell antigen receptor(BCR)is a membrane immunoglobulin(SmIg)who can recognize B cell specific surface antigen and bind it.BCR is composed of two heavy chains including variable region(V region),the constant region(C region),a transmembrane region and cytoplasmic domain and two light chains including variable region and the constant region.V regions is composed of two domains of VH and VL and each of who consists of three complementarity determining regions(CDR1,CDR2 and CDR3)Composition.The composition and order of CDR amino acid are presenting high diversity.In each body,the diversity can reach up to 109～1012,which can make up of a huge capacity of BCR pool,Identify a variety of antigens and produce specific antibodies.Three CDR are involved in identification of the antigen.And they determine the antigen specificity of the BCR together.Immune repertoire has six major peptide chain,who are including light chain and heavy chain of BCR,a chain,3chain,γ chain and δ chain of TCR.The structural differences of each of immune protein are very small.But the variety of subtypes is very huge.The diversity of health plays a vital role in healthy people.The more subtypes of immune proteins,the more effective we can against pathogens.The less subtypes in the immune proteins,the more susceptible we get disease.In addition,age,environment,drug-induced diseases and many other factors also affect the diversity of the immune repertoire.CDR3 region of lymphocytes is the major site of antigen recognition,the length and diversity of which will affect the recognition and affinity of the antigen.Their specificity can be considered as genetic recombination and somatic hyper-mutation results.The varied features of CDR3 sequences provide a diverse selection for antigen in TCR repertoire.The analysis of the receptor pool of CDR3 can provide new ideas,new methods and tools which can better clinically monitor and analyze the mechanisms of birth and development of immune status and immune cells under physiological and pathological conditions.Therefore,this study only investigated BCR heavy chain CDR3 of ESRD patients.High-throughput sequencing(HTS)has revolutionized the field of genetics,permitting the investigation of complex sequencing targets at unprecedented depth with a reasonable cost.Here,we evaluate the diversity of the immune system of ESRD patients compared to normal controls by amplifying the complimentary-determining region(CDR)of B cell receptors(BCRs)using multiplex-PCR followed by high-throughput sequencing to explore the association between the immune repertoire and disease.We hope to find the disease-specific CDR3 sequence by comparing normal subjects and patients with immune repertoire.With HTS we can find disease-specific CDR3 suffering from the same disease(clinically defined)population.So these CDR3 sequences can be called representatives of the disease.And it can be found from the peripheral blood Biomarker.More importantly,the results will lead to the development of diagnostic,therapeutic or preventive strategies.Previous studies showed that the risk of ERDS detected in adults and children in B lymphocytes significantly reduced.Furthermore,it has been demonstrated in children with chronic renal failure in,CD5+ B cells and CD27 + inherent significantly reduced memory B cells.In a recent study,also confirmed in adults with ESRD have several other B-cell subsets significantly reduced this.Materials and Methods1.Research ObjectPatients with ESRD from Guilin People’s Liberation Army 181 Hospital Organ Transplant Center of the hospital,all patients met the American Kidney Foundation K/DOQI expert group criteria ESRD patients.Diagnostic criteria for ESRD patients:1 glomerular filtration rate(GFR)"15ml/min and most patients with symptoms of uremia 2.The need to start renal replacement therapy 10 cases of patients with ESRD(dialysis or kidney transplantation),3 females,7 males,aged 25-63 years,mean age 35.64± 11.27 years,duration 0 2-2.8 years.Healthy controls in 6 cases,3 females,3 males,aged 24-47 years,mean age 33.47±9.61 years for healthy people the same hospital in the same time period,does not meet the diagnostic criteria for ESRD,no significant history of disease,I no immediate family member or a history of ESRD.2.Research methodsWhole blood samples from 10 ESRD patients and 6 healthy individuals were collected at 181st Hospital of Guilin,China.And PBMCs were separated by Lymphocyte separation medium.We extract the DNA from the B lymphocytes,then use multiple PCR/5’RACE to capture CDR3,high-throughput sequencing platform by Illumina MiSeq.In order to build the immune repertoire,we got V-D-J gene frequency,clone frequency distribution,the polypeptide sequence number through statistical analysis.In this study,we mainly investigate B lymphocytes of ESRD patients and healthy people.We amplified Diversity complementarity determining region(CDR region)of B cell receptor(BCR)through 5’RACE or poly-PCR technology.Combined with high-throughput sequencing technology,a comprehensive assessment of the diversity of the immune system,we can know the relationship of immune repertoire and ESRD deeply.Results and conclusion:In this study,we analysis BCR heavy chain(IGH)of 10 cases of renal transplantation preoperative with end-stage renal disease(ESRD)patients and 6 healthy controls in the same period in NGS sequencing,we did comparison of the CDR3,V,D,J,and V-J distribution,as well as the calculation of Shannon entropy(Shannon entropy,SE),a high cloning(Highly enpended clone,HEC)compared with the control group.ESRD patients’ clonal of B lymphocytes have a higher degree of amplification than healthy people.B lymphocytes have a lower immune repertoire diversity in RSRD patients.We sequenced immune repertoire of a patient from the DNA sequence,AA sequence and combination VJ level,to develop biomarkers for early diagnosis of diseases.Mature B cells from peripheral blood move out into the spleen,lymph nodes,mainly in the spleen nodules,splenic cord and lymphoid nodules,lymph lymphoid nodules Basso and gastrointestinal mucosa.With stimulation of antigen,B cells proliferate and differentiate into plasma cells,then synthetic antibodies which play humoral immune function.The CDR3 region of BCR is the unique molecular structure for representing the different population of B cells.In our study,we used a next-generation sequencing protocol to investigate characteristics and polymorphisms of the B cell receptor complementarity-determining region 3(BCR CDR3)gene in peripheral blood mononuclear cells(PBMCs)from an ESRD group(n = 10)and an NC group(n = 6).The vast majority of B cell clones in BCR sequence are of lower frequencies.And these clones show no clonal expansion.In this study,some high clones(Highly enpended clone,HEC)are found in each individual BCR H chain CDR3.These clones may be formed in the complex environment of antigen or pathogen stimulation.In our study we found that ESRD patients and NC group have the phenomenon of clonal expansion.And the extent of clonal expansion of ESRD patients is higher than NC group.Multifactorial,genetic,sex hormones,and environmental factors may cause ESRD disease.And toxins was gathered which will affect the immune status of ESRD patients.During B lymphocytes development,V,D,J gene segments of BCR will be rearranged.Different number of nucleotides may be randomly inserted into V-J segments or V-D-J segments.And Different number of nucleotides may be randomly deleted in V-J segments or V-D-J segments.Through this way,a highly diversity variable CDR3 region can be formed,which are quite different in length and amino acid sequences.Namely CDR3 sequence determines a unique BCR clone type,so we can judge the clonal diversity of B lymphocytes by detecting the the length of CDR3.However,in our study,we compare normality of CDR3 length distribution between ESRD patients and NC groups.It can be seen in patients with ESRD,head of distribution tend to be skewed distribution,and the NC group tend to be normal distribution.But there is no statistically significant difference.We also compare Shannon entropy and HEC of each sample between ESRD patients and NC group.From the view of number and proportion,there are differences between the two groups.However,may be due to the small sample size,statistical significance was not significant.We also systematically analyze V,D,J and V-J segments.We extract peripheral blood mononuclear cells in 6 healthy volunteers and 10 patients with ESRD.Then we do T test of BCR H chain V,D,J and V-J segments frequency of use.We found 5 up-regulated genes:IGHV1-24,V-J combination(IGHV3-9,IGHJ1),V-J combinations(IGHV1-46,IGHJ3),V-J combination(IGHV3-48,IGHJ1),V-J combination(IGHV2-I,IGHJ3);9 down-regulated genes:IGHV3-20,IGHD4/OR14-4a,IGHD4/OR14-4b,IGHJ5,V-J combination(IGHV3-20,IGHJ5),V-J combinations(IGHV3-49,IGHJ5),V-J combinations(IGHV3-64D,IGHJ3),V-J combinations(IGHV3-20,IGHJ4),V-J combinations(IGHV1-69,IGHJ1).These abnormal expression of BCR H chain CDR3 of V,D,J and V-J combinations sub-family gene may be involved in the development of ESRD disease.Multifactorial,genetic,sex hormones,and environmental factors may cause ESRD disease.And toxins was gathered which will affect the immune status of ESRD patients.In this process,these up-regulated genes IGHV1-24,VJ combination(IGHV3-9,IGHJ1),V-J combinations(IGHV1-46,IGHJ3),V-J combinations(IGHV3-48,IGHJ1),V-J combination(IGHV2-I,IGHJ3)may be involved in some BCR-specific clonal proliferation of B lymphocytes and undermining BCR diversity.The BCR diversity play an essential role in healthy people.The more subtypes of immune proteins,the more effective we can against pathogens.The less subtypes in the immune proteins,the more susceptible we get disease.The down-regulated genes IGHV3-20,IGHD4/OR14-4a,IGHD4/OR14-4b,IGHJ5,V-J combination(IGHV3-20,IGHJ5),V-J combination(IGHV3-49,IGHJ5),V-J combinations(IGHV3-64D,IGHJ3),V-J combination(IGHV3-20,IGHJ4),V-J combinations(IGHV1-69,IGHJ1)may be involved in inhibiting BCR certain specific clonal proliferation of B lymphocytes.And some such subtypes significantly reduced.It also coincided with a number of previous studies confirmed that the B lymphocytes in patients with ESRD may significantly reduced.In conclusion,we demonstrated a successful approach for determining the entire diversity of the immune repertoire at sequence-level resolution.Future investigation should be aimed at better understanding the role of the BCR repertoire in immune responses,autoimmunity and alloreactivity,thus unlocking the potential to optimize T cell clone-type selection from the available repertoire for therapeutic benefit.