HLA-B*46:01:01:01 and HLA-DRB1*09:01:02:01 are associated with anti-rHuEPO-induced pure red cell aplasia

Treatment of anemia in patients with chronic kidney disease (CKD) with recombinant human erythropoietin (rHuEPO) can be disrupted by a severe complication, anti-rHuEPO-induced pure red cell aplasia (PRCA). Specific HLA genotypes may have played a role in the high incidence of PRCA in Thai patients (1.7/1,000 patient years vs. 0.03/10,000 patient years in Caucasians). We conducted a case–control study in 157 CKD patients with anti-rHuEPO-induced PRCA and 56 controls. The HLA typing was determined by sequencing using a highly accurate multiplex single-molecule, real-time, long-read sequencing platform. Four analytical models were deployed: Model 1 (additive: accounts for the number of alleles), Model 2 (dominant: accounts for only the presence or absence of alleles), Model 3 (adjusted additive with rHuEPO types) and Model 4 (adjusted dominant with rHuEPO types). HLA-B*46:01:01:01 and DRB1*09:01:02:01 were found to be independent risk markers for anti-rHuEPO-induced PRCA in all models [OR (95%CI), p-values for B*46:01:01:01: 4.58 (1.55–13.51), 0.006; 4.63 (1.56–13.75), 0.006; 5.72 (1.67–19.67), 0.006; and 5.81 (1.68–20.09), 0.005; for DRB1*09:01:02:01: 3.99 (1.28–12.49), 0.017, 4.50 (1.32–15.40), 0.016, 3.42 (1.09–10.74), 0.035, and 3.75 (1.08–13.07), 0.038, in Models 1–4, respectively. HLA-B*46:01:01:01 and DRB1*09:01:02:01 are susceptible alleles for anti-rHuEPO-induced PRCA. These findings support the role of HLA genotyping in helping to monitor patients receiving rHuEPO treatment.

PRCA is caused by EPO-induced antibodies that neutralize all exogenous drugs and cross-react with endogenous EPO, leading to undetectable serum levels of EPO, ineffective erythropoiesis, and blood transfusiondependent to relieve anemic symptoms 8,11,14,15 .Factors related to an increase in the incidence of PRCA included stabilizer, immunogenic polysorbate 80 that was used in human serum albumin-forming micelles, and leachates released by an uncoated rubber prefilled syringe stopper that can interact with polysorbate 80 that aggravating immune reactions 8,[15][16][17] .Other factors were problems related to EPO product quality, the cold chain, and the subcutaneous route of administration 5,6,8,17 .Furthermore, immunogenicity is partly dependent on the type of genetic variations in human leukocyte antigen (HLA) 17 .Previous studies have shown that the mechanism of PRCA may be related to antibody response in genetically susceptible patients (HLA-DRB1*09) [2][3][4][5]7,9 .
In addition to the factors mentioned above, numerous rHuEPO biocopy products are widely used in Thailand and other developing countries.Our previous report showed that rHuEPO biocopy products were associated with neutralizing antibody production and PRCA 19 .The rHuEPO biocopy products differ in a substantial amount of their protein fragments 21 , which can also play a crucial role in anti-rHuEPO-induced PRCA.However, this factor has not yet been thoroughly clinically evaluated.
This study investigated associations of HLA genotypes, sequenced by highly accurate long-read HLA sequencing, with anti-rHuEPO-induced PRCA in Thai patients with CKD.Relationships were also adjusted for the source of rHuEPO products (innovator and biocopy).

Baseline characteristics and frequency of HLA alleles in cases and controls
A total of 213 participants (157 cases and 56 controls) were enrolled.As expected, the cases had a significantly shorter exposure time to rHuEPO compared to the controls.All participants had Telfon-coated prefilled rHuEPO administered subcutaneously.Two brands of innovator rHuEPO were prescribed to 14 cases (8.92%), while none of the controls received innovator rHuEPO.At least nine brands of rHuEPO biocopy were prescribed in both cases and controls (Table 1).

Associations of HLA alleles and anti-rHuEPO-induced PRCA
Table 5 shows the significant HLA alleles associated with anti-rHuEPO-induced PRCA.In the no covariate analysis models (Models 1 and 2 When data were adjusted by the types of rHuEPO as a covariate (Models 3 and 4, Table 5), some HLA alleles lost significance, leaving anti-rHuEPO-induced PRCA highly associated with the presence of HLA-B*46:01  1).

Discussion
We successfully conducted a case-control study to identify genetic variation in HLA as a significant risk factor for anti-rHuEPO-induced PRCA in patients with CKD using the highest accuracy long-read sequencing technique.Regardless of the types of rHuEPO, CKD patients with HLA-B*46:01:01:01 or HLA-DRB1*09:01:02:01 had a three to sixfold increased risk of developing anti-rHuEPO-induced PRCA.
HLA associations of HLA with antibody-positive PRCA have been reported in other studies [2][3][4]7 . Futhermore, previous data in Thai patients with CKD (22 cases) have strongly suggested the association of HLA-DRB1*09 and anti-rHuEPO-induced PRCA 4 .Here, our study was carried out in a larger cohort size (157 cases, not including 22 cases in the previous study 4 ), with the highest resolution of the HLA genotyping platform, confirming the risk of anti-rHuEPO-induced PRCA in patients with HLA-DRB1*09 and, more specifically, HLA-DRB1*09:01:02:01. Our study also adds the new information that HLA-B*46:01:01:01 is also associated with a very high risk of anti-rHuEPO-induced PRCA.
A report in Caucasians (24 cases) also showed a significant association between HLA-DRB1*09 and antibodypositive PRCA 3 .However, the allele frequency of HLA-DRB1*09 alleles in Caucasians was lower than the data observed in our Thai cohort (cases/controls = 12.70/5.92%(Table 3) and 9.6/ 0.7% for this study and Fijal et al. 3 , respectively).Differences in HLA-DRB1*09 allelles in the background of Thai and Caucasian populations could    www.nature.com/scientificreports/partly explain the low incidence of anti-rHuEPO-induced PRCA in western countries.A study in Chinese patients suggested the association of HLA-DRB1*12:02 with PRCA 7 .Here, HLA-DRB1*12:02:01:01 was found highly in our cases and controls (16.94% vs 18.34%, Table 3) but no association with anti-rHuEPO-induced PRCA was shown in our analyses.Furthermore, other HLA alleles (HLA-A*25, HLA-B*53, HLA-C*12, HLA-DRB1*04, HLA-DQB1*03, and HLA-DQB1*06) were also identified as risk factors for PRCA 3 .It should be noted that in our data there were no HLA-A*25 and HLA-B*53 (Table 2).The mechanism of PRCA in patients with CKD that are correlated with HLA needs to be clearly defined.However, the immunogenic antibody activated by rHuEPO was related to T cell activation 23,24 .HLA Class II, including HLA-DR, HLA-DQ, and HLA-DP, are expressed primarily in antigen-presenting cells that uptake, process, and present the antigen as a peptide epitope in naive T cells with a Class II molecule of the major histocompatibility complex (MHC) on its surface.Binding between the MHC class II epitope complex and the T cell receptor activates the T cell to release cytokines to trigger the differentiation of B cells into plasma cells.Plasma cells then secrete antibodies against the corresponding epitope 23 .Our study revealed that HLA-B*46:01:01:01 and HLA-DRB1*09:01:02:01 alleles demonstrated strong interactions with crucial residues of rHuEPO, such as R103, T107, L108, R143, and R150 (Table 8, Fig. 2).These results are consistent with those reported in a previous www.nature.com/scientificreports/study 25 .Consequently, the favorable interactions observed between rHuEPO and the mentioned HLAs could potentially stimulate the immune defense system.

Table 4 .
Frequency of HLA-DQB1 and HLA-DPB1 alleles in cases and controls.

Table 5 .
Significant HLA alleles associated with anti-rHuEPO-induced PRCA.rHuEPO recombinant human erythropoietin.Additive: homozygous for the tested allele was coded 2, heterozygous was coded 1, and other alleles were coded 0. Dominant: homozygous and heterozygous for the tested allele were coded 1, and other alleles were coded 0. Types of rHuEPO: innovator and biocopy.