FKBP-CaN-NFAT pathway polymorphisms selected by in silico biological function prediction are associated with tacrolimus efficacy in renal transplant patients
Graphical abstracts
Introduction
Tacrolimus (TAC, FK506), a calcineurin inhibitor (CNI), is widely used as a first-line immunosuppressive agent for renal transplantation (Barbarino et al., 2013). Appropriate use of TAC can significantly improve the short-term and long-term survival of kidney grafts. However, clinical application of TAC is limited by the narrow therapeutic index and large inter-patient pharmacokinetic (PK) and pharmacodynamic (PD) variability.
In clinical practice, therapeutic drug monitoring (TDM) is mandatory to ensure that TAC receivers achieve the recommended therapeutic target range (Van Gelder et al., 2014). However, the current TDM strategy does not always elicit consistent drug responses in transplant patients. Indeed, an inter-individual variability in the pharmacodynamic effects of TAC is evident. As previously reported (Barbarino et al., 2013; Fioretto et al., 2011), in the first few years after renal transplantation, the clinical pharmacodynamic variability of TAC increases the risks of acute rejection, acute nephrotoxicity, infections and abnormal renal allograft function tendency (Elens et al., 2013; Seyhun et al., 2015; Wu et al., 2017).
The immunosuppressive effects of TAC are largely mediated by inhibition of the calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway (Barbarino et al., 2013). Upon entry into T-cells, TAC binds to the FK506 binding protein (FKBP), particularly FKBP12, with high affinity (Kang et al., 2008). The TAC-FKBP complex subsequently interacts with CaN, inhibiting NFAT nuclear translocation. Through this mechanism, downstream activation of a series of immunity-related cytokines by members of the NFAT family is suppressed (Barbarino et al., 2013; Liu et al., 1991; Shaw et al., 1995). Interactions between proteins in the activation pathway of TAC are likely to determine drug response, and analysis of the corresponding genes may help to distinguish the various clinical drug responses.
FKBP12 and its isoform FKBP12.6 are encoded by FKBP1A and FKBP1B, respectively (Kang et al., 2008; MacMillan, 2013). CaN is a dimeric protein composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA has three isoforms (CNAα, CNAβ, CNAγ), encoded by the PPP3CA, PPP3CB and PPP3CC genes, respectively. CNB has two isoforms (CNB1 and CNB2), encoded by the PPP3R1 and PPP3R2 genes, respectively (Hogan et al., 2003; Liu et al., 1991). However, only PPP3CB and PPP3R1 are extensively expressed in T- and B-cells (Klee et al., 1998; Kung et al., 2001). Likewise, while the NFAT family of transcription factors (TFs) consists of five closely related proteins (NFAT1-NFAT5), only NFAT1 and NFAT2, which are encoded by NFATC2 and NFATC1, are expressed primarily in immune cells (Hogan et al., 2003; Rao et al., 1997).
To date, most previous studies on the pharmacogenetics of TAC have focused on PK-relevant genes, while the potential influence of genetic variants in the PD pathway (FKBP-CaN-NFAT pathway) on TAC efficacy have not been comprehensively inspected. In 2014, Noceti et al. reported strong and statistically significant associations between polymorphisms in the CaN pathway and TAC PD parameters in 30 healthy adult volunteers in Uruguay (Noceti et al., 2014). These variants included the IL2RA haplotype, which was associated with variability in the basal activity of IL-2+CD4+ and CD25+CD3+, PPP3CA rs45441997, which was related to variability in the maximal inhibition of IL-2+CD4+, and PPIA rs8177826 C>G, which was correlated with NFAT1 IC50 (50% inhibitory concentration) variability. In contrast, Pouché et al. explored 23 polymorphisms in the PD pathway of TAC in patients of European descent and detected no positive variants (Pouché et al., 2016). However, the range of polymorphisms investigated in previous studies was comparatively limited, and the applicability of the results to Han Chinese people is unknown.
In our previous study (Wu et al., 2019), we investigated the clinical significance of 77 tag-SNPs in the FKBP-CaN-NFAT pathway with minor allele frequency (MAF)>0.1. In the present study, we expanded the range of candidate SNPs by setting a lower MAF limit (MAF≥0.01). As a result, a total of 1284 SNPs in the FKBP-CaN-NFAT pathway have been extracted for bioinformatic function prediction. Compared with traditional tag-SNP extraction, in silico prediction of biological function provides more accountable evidence for causal SNP detection. A grading system was employed to comprehensively assess and prioritize the clinical significance and putative biological function of the positive SNPs detected in our study. Our aim was to identify new regulatory SNPs, that could serve as PD-associated pharmacogenetic biomarkers for the prediction of individual responses to TAC, and thus aid clinical management of renal transplant patients.
Section snippets
Population information
In this retrospective study, we enrolled 140 Chinese renal transplant patients of Han ethnicity, who had received their first kidney transplant between January 2003 and August 2015, and who had been followed up for at least five years at Huashan Hospital, Fudan University. Patients under 18 years old or patients who underwent other organ transplantation were excluded from the study.
Clinical characteristics collected during the follow-up included: age and body weight at the time of the
Clinical characteristics
A total of 140 Chinese renal transplant patients of Han ethnicity who received TAC as an immunosuppressive drug were enrolled and followed up in our study. The follow-up time for patients was ranging from 2 to 13 years after transplantation, 64.3% (90) of whom were over 5 years. The clinical characteristics of these patients are shown in Table 1. The average age of all patients was 38.54 ± 9.86, and 98 patients (70%) were men. Induction therapy was applied in 122 patients (87.14%). Of these, 86
Discussion
To the best of our knowledge, this is the first study to apply systematic bioinformatic prediction for 1284 SNPs of six key genes in the pharmacodynamic pathway of TAC. Our study identified 109 SNPs that may affect gene function, and their association with the clinical endpoints in transplant patients taking TAC was explored. We also comprehensively assessed and prioritised the clinical significance and biological function of the 44 critical SNPs. Our study demonstrated that NFATC2 rs150348438,
Conclusion
In this study, we screened out 44 clinically significant SNPs from 109 bioinformatically positive SNPs in the FKBP-CaN-NFAT pathway. These 44 critical SNPs were prioritised based on bioinformatic predictions and clinical relevance. We demonstrated that NFATC2 rs150348438, rs6013219, rs1052653, and NFATC1 rs754093, ranking high in scoring, significantly affected the post-transplant eGFR and the incidence of pneumonia, acute rejection, and nephrotoxicity in renal transplant patients taking TAC.
Credit author statement
Zheng Xinyi: Conceptualization, Methodology, Data curation, Software, Formal analysis, Visualization, Writing- Original draft preparation. Huai Cong: Writing - Review & Editing. Xu Qinxia: Data curation. Xu Luyang: Data curation. Zhang Ming: Resources. Zhong Mingkang: Project administration, Funding acquisition. Qiu xiaoyan: Conceptualization, Methodology, Writing - Review & Editing, Supervision.
Xinyi Zheng and Xiaoyan Qiu conceived the study. Xinyi Zheng, Qinxia Xu and Luyang Xu participated
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgements
Dr. Xiaoyan Qiu was supported by the Shanghai “Rising Stars of Medical Talent” Youth Development Clinical Pharmacist Program (No. 2018-1). Prof. Mingkang Zhong was supported by the 2019 Key Clinical Program of Clinical Pharmacy (No. shslczdzk06502).
Compliance with ethical standards
The study was performed in accordance with the Declaration of Helsinki and its amendments. Protocols were approved by the Ethics Committee of Huashan Hospital, Fudan University and written informed consent was obtained from all subjects.
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