Association of BAFF/BAFF-R Single Nuclear Polymorphisms with Renal Graft Function in Kidney Transplant Recipients

The BAFF/BAFF-R signal correlates with antibody-mediated rejection and renal function decline after renal transplantation. We investigated the relationship between BAFF/BAFF-R single nucleotide polymorphisms (SNPs) and renal graft function in kidney transplant recipients. Kidney transplant recipients (310) along with randomly selected healthy control individuals (51) were followed from September 2020 to March 2021 in Third A�liated Hospital of Soochow University. Anticoagulated peripheral blood and serum were collected from all individuals. Five BAFF/BAFF-R SNPs were genotyped by TaqMan quantitative PCR. According to the estimated glomerular �ltration rate (eGFR) cut-off value of 90 mL·min-1·1.73 (m2)-1, the recipients were divided into normal and abnormal eGFR groups. Analysis results revealed that BAFF rs9514828 CT genotype carriers had a signi�cantly higher incidence of abnormal graft kidney function than other genotype carriers (OR = 1.597, 95% CI = 1.012–2.519, P < 0.05) and had no signi�cant correlation with serum soluble BAFF level and positive rate of panel reactive antibodies; There was a strong linkage disequilibrium between the BAFF rs16972194, rs9514828, and rs16972197; Recipients woth haploid GCG had a signi�cantly higher incidence of normal graft function (OR = 0.381, 95% CI = 0.156–0.931, P < 0.05). This present results suggest that BAFF rs9514828 CT genotype may be a risk factor for kidney transplant recipients, resulting in reduced graft function, whereas the GCG haploid may play a protective role in normal graft function.


Introduction
Renal transplantation remains the most viable treatment option for patients with end-stage renal failure.However, the long-term outcomes of renal transplantation remain to be fully elucidated [1].Several factors are involved in renal allograft function, including the genetic factors of the recipient [2].Recent studies have made progress in determining the heritable variation associated with common complex diseases by exploring single nucleotide polymorphisms (SNPs) in the genome [3].
The human B-cell-activating factor (BAFF) gene belonging to the tumor necrosis factor family is located on chromosome 13q32-34, it has a length of 2.6 kb, and a speci c receptor, namely BAFF-R [4].The human BAFF-R gene is located on chromosome 22q13.1-13.31 and has a length of 4.5 kb.The BAFF/BAFF-R signal can regulate the survival and activation of B lymphocytes [5], and abnormal signals are closely related to the occurrence and development of several diseases.Additionally, the enhancement of the BAFF signal plays a role in renal transplantation rejection [6].
SNPs in the BAFF and BAFF-R genes have been found to be associated with certain diseases.For instance, BAFF rs16972197 C allele carriers have an increased risk of systemic myasthenia gravis [7], BAFF rs9514828 CT genotype carriers have a decreased risk of chronic lymphocytic leukemia [8], BAFF expression has been found to increase in patients with systemic lupus erythematosus (SLE) with the BAFF rs9514828 T allele [9], an increased frequency of the BAFF rs4000607 AA genotype has been found in Graves' patients in the UK [10], and BAFF-R rs61756766 GA+AA genotype carriers have an increased risk of multiple sclerosis [11].
Few studies have investigated genomic SNPs in kidney transplant recipients.We selected and analyzed ve SNPs, namely BAFF rs16972194, rs9514828, rs16972197, and BAFF-R rs61756766, and rs77874543.The genotype of each SNP was determined and analyzed with respect to the clinical indices of kidney transplant recipients.

Materials And Methods
This study was approved by the Ethics Committee of Third A liated Hospital of Soochow University and informed consent was obtained from the relevant research participants.During the study, followed-up kidney transplant recipients were randomly selected in Jiangsu from September 2020 to March 2021.Basic information such as age, sex, weight, and clinical indices, including routine blood examination, hepatic and renal function, and C-reaction protein, were collected.Peripheral blood was collected (2 ml) from kidney transplant recipients (EDTA anticoagulation), stored at 4 ℃, and analyzed for completion within one week.Additionally, 500 μl of serum was collected and stored at -80 ℃ for future use.
All renal transplant recipients were treated with the combined immunosuppression regimen, more speci cally, a combination of tacrolimus/cyclosporine A, mycophenolate/mycophenolate sodium, and glucocorticoids.
Healthy volunteers were routinely selected by medical personnel (excluded individuals were those with abnormal blood, urine, or stool test results, or abnormal cardiopulmonary, kidney, or liver function test results).Anticoagulated peripheral blood and serum were obtained from these healthy volunteers and stored for future use.
The whole genome DNA of each participant was extracted and puri ed following the extraction kit instructions (TaKaRa, Dalian China).DNA concentration and purity was detected using the NanoDrop 2000 (ThermoScienti c, MA, USA).The puri ed DNA (1.8 < 260/280 ratio < 2.2) was stored at -20 °C for later use.
Human BAFF PicoKine™ ELISA kits (BOSTER, Wuhan China) were used according to the manufacturer's instructions to detect the serum concentration of sBAFF.
The Lambda Antigen Tray TM Mixed kit (One Lamda, CA USA) was used, following the manufacturer's instructions to screen serum for donor-speci c anti-human leukocyte antigen (HLA) antibodies.
All data were analyzed using the SPSS 22.0.The continuous variables were expressed as mean ± standard deviation and analyzed by t-test, whereas the categorical variables were analyzed using the c 2 test.More speci cally, the c 2 test was used to compare the genotype and allele frequencies between three groups, namely, the recipient, recipient subsets, and the healthy control groups.
Statistical signi cance was set at P < 0.05.SNPstats (http://bioinfo.iconcologia.net/snpstats/start.htm) was used to estimate the Hardy-Weinberg equilibrium, and logistic regression was used to adjust for potential confounders that may have affected the differences between the three groups.Finally, linkage disequilibrium analysis and haploid analysis were performed using Haploview software (version 4.0).

Results
All kidney transplant recipients were Han Chinese from the Jiangsu Province.We enrolled 310 kidney transplant recipients (198 men, 112 women) aged 44.32 ± 19.21 years and 51 healthy control individuals (26 men, 25 women) aged 48.38 ± 12.23 years.
The Hardy-Weinberg genetic distribution test results revealed that all the kidney transplant recipient genotypes were in accordance with genetic equilibrium (P > 0.05), suggesting that the recipients enrolled in this present study were likely representative of the population (Table 2).
Thereafter, we analyzed the association between BAFF/BAFF-R SNPs genotypes and eGFR using logistic regression analysis after adjusting for sex.The results indicated that recipients with the rs9514828 CT genotype had a signi cantly higher incidence of abnormal graft function than those with the rs9514828 (CC+TT) genotype in the hybrid model (OR = 1.597, 95% CI = 1.012-2.519,P < 0.05).However, no signi cance differences were found for the rs16972194 and rs16972197 genotypes in the implicit, dominant, and heterozygous models.The BAFF-R rs61756766 genotype in all recipients comprised the GG mutation, and no statistical differences were observed for the BAFF-R rs7787543 genotype and eGFR under all the genetic models (P > 0.05) (Table 3).
The SNP alleles of the recipient subsets and the control group were also studied, however, signi cant allele differentiation was not observed (Table 4).
We compared the clinical data of recipients with the BAFF rs9514828CT (CC+TT) genotype and all the other genotypes in the hybrid model.The clinical data included the following parameters: sex, age, duration after kidney transplantation, sBAFF, and positive rate of PRA.No signi cant differences were found between the genotypes and sBAFF, positive rate of PRA, and other indices for the recipient subsets and control groups (P > 0.05).Additionally, the indices coincided with rs16972194 and rs16972197 carriers.The sBAFF concentration in kidney transplant recipients were notably higher than those in the healthy control individuals, but the difference was not statistically signi cant (P > 0.05) (Table 5).
The Haploview software (version 4.0) was used for linkage disequilibrium and haplotype analyses.The results revealed that the D' value was > 0.8 for BAFF rs16972193, rs9514828, and rs16972197, suggesting a strong linkage disequilibrium among the three SNPs (Figure 1).The same analyses showed that three haploids could be formed, namely GCG, GTG, and ACC.Finally, the logistic analysis results revealed a signi cant difference in the number of kidney transplant recipients with the GCG haploid between the eGFR normal and abnormal groups (P < 0.05), suggesting that kidney transplant recipients with the GCG haploid had a higher eGFR or a signi cantly lower incidence of abnormal graft function than those with other haploids (Table 6).

Discussion
With the development of genome-wide association studies, millions of mutations have been discovered, most of which are single nucleotide substitutions [13].These mutations are also referred to as SNPs.SNPs can occur in any region of the genome which is likely due to various selection pressures that generate single-base mutations in speci c regions of the genome, with varying frequencies [14].There are two types of SNPs, namely synonymous and non-synonymous SNPs.Synonymous SNPs do not alter encoded amino acids, whereas non-synonymous SNPs do alter amino acids and may ultimately in uence certain protein properties or functions.Additionally, studies have found that non-synonymous SNPs may also play a regulatory role by interacting with noncoding RNAs [15].
To date, more than 15,000 SNPs of the BAFF gene have been identi ed, yet fewer than 20 of these SNPs have been intensively studied.In this present study, ve BAFF/BAFF-R SNPs were selected, including three BAFF SNPs located in the promoter region and two non-synonymous BAFF-R SNPs.Considering the role of BAFF/BAFF-R signaling in antibody-mediated rejection, the primary purpose of this study was to analyze the correlation between these ve SNPs and chronic rejection in kidney transplant recipients.
Since the number of pathologically con rmed patients presenting with chronic rejection was limited, we used eGFR as an indicator of graft renal function during the data analyses.
Ultimately, we found that having the BAFF rs9514828 CT genotype may be a risk factor for Han Chinese from the Jiangsu Province, resulting in reduced renal allograft function after kidney transplantation, however, no signi cant correlation with sBAFF or positive rate of PRA was found.Gottenberg et al. [16] studied 162 French patients with Sjogren's syndrome and found that patients with the rs9514828 TT genotype had higher levels of sBAFF, which was associated with anti-SSA and anti-SSB antibody levels.Furthermore, Kawasaki et al. [17] studied Japanese patients with SLE and rheumatoid arthritis and found that rs9514828 TT genotype carriers tended to have higher levels of anti-Sm antibodies, and patients with the -871 T allele had signi cantly higher levels of BAFF mRNA in monocytes.However, several studies related to rs9514828 have not obtained signi cant results [18,19].Kompoti et al. [20] found that severely ill patients with the rs61756766 C allele had a lower risk of acquiring sepsis in the ICU, but no association was found between rs61756766 and susceptibility to sepsis in trauma and surgical patients in Greece.In a study of Spanish patients with immunoglobulin A vasculitis, BAFF, APRIL, and BAFF-R SNPs, including BAFF-R rs77874543, were not found to be associated with the pathological mechanisms of the disease [21].We also examined two non-synonymous BAFF-R SNPs, but did not obtain statistically signi cant results.We postulate that the different signi cant genotype results found in these studies may be due to variation in pathological mechanisms or ethnic differences.
The analyses of clinical data for signi cantly differentiated genotypes in this present study included assessing the association between sBAFF and positive rate of PRA, however, no signi cant results were obtained.
Currently, only two reports related to BAFF SNPs and kidney transplantation have been published on PubMed.One study assessed Hispanic white kidney transplant recipients and found that BAFF rs12583006 was associated with a higher level of the class II donor speci c antibody [22].The other study investigated BAFF system SNPs (including BAFF, three BAFF receptor SNPs, and APRIL SNPs) in Hispanic white kidney transplant recipients, and found that the APRIL rs3803800 AA genotype may be high risk factor for acute rejection.Compared to GG/GA carriers, recipients with this genotype had a signi cant acute rejection free time [23].
Interestingly, despite both studies investigating kidney transplant recipients, contrasting results were found.This begs the question: are these different ndings related to geographical, or ethnic differences?Ultimately, more data needs to be gathered before drawing such conclusions.
Graft renal function is affected by several factors, and immune rejection caused by alloantigens should be one of the important ones [24], and some evidences suggested that the rate of graft loss caused by antibody-mediated rejection is more than 50% [25].The successful development of novel immunosuppressants has many bene ts for transplant recipients.Belimumab, a speci c anti-BAFF monoclonal antibody, has been approved by the US Food and Drug Administration for the treatment of SLE, however, unexpected effects have been observed in some patients [26].Moreover, belimumab did not meet certain clinical expectations in a phase II trial in kidney transplant recipients [27].Some studies suggest that differences in belimumab e cacy may be due to individual genetic factors [28].Ultimately, further research is required to determine whether BAFF SNPs affect the action of BAFFspeci c antibody reagents.Kidney transplant recipients with the GCG haplotype may be prone to abnormal graft function.Indeed, all clinical indices of recipients with the rs16972194 and rs16972197 SNPs were coincident (for all seven kidney transplant recipients).This nding is consistent with the linkage disequilibrium analysis results and our initial hypotheses.
At present, research methods concerning SNPs have seen an increase in quantity and ease of use, resulting in an improved understanding of SNPs.Remarkable strides in SNP research have taken place since the construction of an SNP database [29], completion of protocols for a genome-wide, preponderant SNP network [30], and development of a model to predict tacrolimus levels in pediatric solid organ transplant recipients, by integrating clinical data with genetic factors [31].The effect of the interaction between different SNPs, and between SNPs and non-coding RNAs (such as miRNA and lncRNA) on the translation, expression, and function of relevant genes forms an important component of SNP research [32,33].
SNPs represent stable genetic alterations in the genome [34].The present study partially elucidated the correlation between genomic SNPs found in kidney transplant recipients in the Jiangsu Province.We aim to increase the sample size and screen for BAFF SNPs related to chronic rejection, and combine this informaion with the pathological diagnosis.Ultimately, this work will help to establish individual treatment protocols after kidney transplantations to obtain satisfactory long-term outcomes based on genetic variation.

Declarations
Authors' contributions (I) HX and XH designed and supervised the project, (II) JC and XH performed the experiments, (III) HX, XH, JC and XR analyzed and interpreted data, (IV) JC and XR collected blood and plasma samples and clinical data, (V) Manuscript writing: all authors, (VI) All authors read and approved the nal manuscript.

Tables
Linkage disequilibrium analysis of three BAFF SNPs Linkage disequilibrium analysis of BAFF rs16972194, rs9514828, and rs16972197 implemented using the Haploview software (version 4.0).The D' value between three SNPs was equal to 1. Generally, a D' value > 0.8 suggests strong linkage disequilibrium between the SNPs.We conclude that strong linkage disequilibrium exists between these three BAFF SNPs.

Table 6 .
Haploid analysis of three BAFF SNPs