Abstract
Background. Polymorphisms in genes, coding for proteins involved in immune response, or the pathogenesis of atherosclerosis may influence immunological and non-immunological mechanisms that lead to allograft loss. Vitamin D receptor (VDR) agonists reduce allograft rejection in animal models, and there are a number of functional polymorphisms in VDR. Methods. In all, 379 renal transplant recipients were genotyped for VDR (FokI & ApaI) polymorphisms, and the association of each genotype with renal allograft survival and acute rejection was determined. Results. There was significantly improved allograft survival for patients who were homozygous or heterozygous for the VDR FokI T allele (Hazard Ratio [HR] = 0.488, p < 0.001). Conclusion. The association of VDR FokI T allele with improved renal allograft survival is a unique observation. The finding is in keeping with data showing the prevention of chronic allograft rejection with the use of Vitamin D receptor agonists.
INTRODUCTION
Renal transplantation is the optimal choice of renal replacement for suitable patients, offering both improved survival[1] and quality of life.[2] Allograft loss can be secondary to a variety of immune and non-immune mechanisms. Polymorphisms in genes, coding for proteins involved in immune response, or the pathogenesis of atherosclerosis may influence immunological and non-immunological mechanisms that lead to allograft loss. We surmised that functional polymorphisms in genes coding for vitamin D receptors could impact graft survival or rates of acute rejection, based on available literature regarding their immune-regulatory function.
Calcitriol (1,25(OH)2D3), the activated form of vitamin D, is a seco-steroid hormone that has important effects on the growth and differentiation of many cell types, and pronounced immuno-regulatory properties[3],[4] and anti-tumor properties.[5] The biological effects of calcitriol are mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear hormone receptors.[6]
While there are many VDR polymorphisms, we studied two in particular: namely, FokI (rs10735810) and ApaI (rs7975232) variants. In VDR, the FokI polymorphism occurs as a result of a T/C transition at the first potential start codon site.[7] Where ACG replaces ATG, this start codon polymorphism leads to a protein that is three amino acids shorter. Thus, two variants of the protein can exist—a 427aa version corresponding to the T allele, and a 424aa version corresponding to the C allele. Some in vitro studies suggest that the shorter 424 aa protein has increased transcriptional activity.[8] Increased frequency of the CC genotype FokI polymorphisms was found in French patients with rheumatoid arthritis.[9] The ApaI variant is the most common variant in the Caucasian population, and is in close linkage disequilibrium with several other variants in Caucasians, including BsmI (rs1544410) and TaqI (rs731236) variants. In Crohn's disease, a link has been suggested between the FokI, TaqI, and ApaI polymorphisms and susceptibility to the disease.[10] VDR polymorphisms may influence the processes of allo-recognition, co-stimulation, and tolerance in the setting of renal transplantation.
In this study, our objective was to determine the frequency of VDR (FokI & ApaI) polymorphisms in a population of patients with functioning renal allografts and to determine if these polymorphisms were associated with allograft survival or acute rejection.
METHODS
Patient Characteristics
The study was carried out in Beaumont Hospital, the national renal transplant center in Ireland, with approval from the Ethics/Medical Research Committee. All renal transplant recipients older than 16 years were invited to partake in this study. Each patient was interviewed and examined. Data were collected on age, gender, duration since transplant, and duration exposed to each immunosuppressive drug. Data on the primary end-points of renal allograft survival, episodes of acute rejection, and secondary end-point of interval serum creatinine were obtained from medical records and from the Irish Transplant Registry. First transplants only from 1980 onward were analyzed for outcome.
Polymorphism Genotyping
Genomic DNA was isolated from a sodium tricitrate anticoagulated peripheral venous blood sample by a salting out method. The DNA samples were barcoded, de-identified, and stored at –20°C until genotyping was performed. Genotyping for the VDR (FokI & ApaI) polymorphisms was carried out using the AmplifluorTM method by K Biosciences (http://www.kbioscience.co.uk ). Genotyping was performed in 384-well microplates using a fluorescence resonance energy transfer (FRET)-based genotyping method. Amplification was initiated using allele-specific primers and a common downstream primer. The allele-specific primers were tailed with unique sequences that create corresponding complementary sequences in the two amplicons. In the second round of amplification, quenched Universal AmplifluorTM primers (in a hairpin formation) were used. These primers contain 3′ tails that specifically bind to the unique tailed sequences in the amplicons and continue amplification. In the final round of amplification, the action of the DNA polymerase opens up the hairpin structure, and the quencher and reporter moieties are spatially separated. The excited reporter moiety emits either red or green fluorescence, the color of which depends on which nucleotide is at the polymorphism site. The fluorescence is quantified by a microplate reader and then analyzed via an Excel macro to provide genotypes for each SNP.
Statistical Analysis
Genotype and individual allele frequencies were calculated for all participants. The impact of being heterozygous or homozygous for the VDR FokI C/T and VDR ApaI G1281T polymorphisms was assessed with regard to allograft survival and acute rejection. In each case, the carriers of the common allele were compared to the common allele homozygote. The Pearson χ2 test was used to assess association between each genotype and rate of acute rejection. The log- rank test was applied to assess for a significant difference in allograft survival for each genotype of the three polymorphisms.
Cox Proportional Hazards methods were used to model the effects of each allele in both univariate and multi-factorial models. In the multi-factorial model, patients who possessed the T (heterozygous or homozygous) allele were compared to those who were homozygous for the C allele of the FokI polymorphism. Multivariate analysis was not done on the ApaI polymorphism, as there was no significant difference in outcomes in the univariate analysis. The multi-factorial model included factors commonly associated with decreased graft survival, such as episodes of acute rejection, recipient age, and gender. Hazard ratios (HR) defined the relative risk associated with a particular allele. A HR greater than 1 defined an increased risk, and a HR less than 1 a decreased risk of graft failure. In these models, patients who were homozygous or heterozygous for the minor allele were compared to those who did not possess the minor allele. A p value of less than 0.05 was deemed to be significant in all cases.
RESULTS
Baseline Characteristics
A total of 379 patients were included in the study. Of these patients, 352 were successfully genotyped for FokI polymorphisms and 361 for ApaI polymorphisms. All patients who were genotyped were included in the analysis. The mean age and gender distribution of patients were not significantly different between the allelic variants (see Table 1). The frequencies of the minor alleles (VDR [FokI T & ApaI A]; see Table 1) in our studied population were similar to those previously reported in Caucasian populations.[11],[12] Allelic distribution was in Hardy- Weinberg equilibrium for each polymorphism genotyped. The median graft survival was 18.6 years.
Table 1 Allelic Frequencies and Patient Characteristics
VDR FokI Polymorphism
There was no significant association between any of the VDR FokI genotypes and acute rejection (see Table 2). There was a significant increase in allograft survival in time period studied for the VDR FokI T allele (see Figure 1; p < 0.001). The increase in graft survival conferred by FokI T allele appears to have greatest impact from year 3 onwards (see Figure 1). Patients who were either homozygous or heterozygous for the VDR FokI T allele were combined and modeled for graft outcome in univariate and multifactorial models. Individually and in combination with other confounding variables, both models showed a strongly significant result (see Table 3).
Table 2 Acute rejection with VDR FokI & ApaI polymorphisms
Table 3 Univariate and multivariate analysis of factors affecting renal allograft survival
Published online:
07 July 2009Figure 1. Unadjusted renal allograft survival for Vitamin D receptor FokI polymorphisms. Log-rank test: p = 0.08 at 5 years, 0.009 at 10 years, < 0.001 at 15 years.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/irnf20/2007/irnf20.v029.i07/08860220701540417/20150616/images/medium/irnf_a_253902_uf0001_b.gif"})
Figure 1. Unadjusted renal allograft survival for Vitamin D receptor FokI polymorphisms. Log-rank test: p = 0.08 at 5 years, 0.009 at 10 years, < 0.001 at 15 years.
VDR ApaI Polymorphism
There was no significant association with any of the VDR ApaI genotypes with altered allograft survival or acute rejection rates (see Table 2 and Figure 2).
Published online:
07 July 2009Figure 2. Unadjusted renal allograft survival for Vitamin D receptor ApaI polymorphisms. Log-rank test: p = NS.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/irnf20/2007/irnf20.v029.i07/08860220701540417/20150616/images/medium/irnf_a_253902_uf0002_b.gif"})
Figure 2. Unadjusted renal allograft survival for Vitamin D receptor ApaI polymorphisms. Log-rank test: p = NS.
DISCUSSION
The data presented here indicate that having a VDR FokI T allele confers a significant advantage in terms of graft survival. VDR is expressed in most cell types of the immune system, in particular macrophages and dendritic cells, as well as in both CD4+ and CD8+ T lymphocytes.[13] VDR agonists have been shown to cause inhibition of T cell proliferation,[14] induction of hyporesponsiveness to allo and self-antigens,[15] inhibition of IL-2 production,[15] inhibition of IFN-γ production,[16] and inhibition of Th1 cell development.[17] The FokI T/C start codon polymorphism may affect the influence of VDR on the processes of allo-recognition, co-stimulation, and tolerance.
VDR agonists have been shown to reduce short and long term allograft rejection in animal models when used as sole immunosuppressive agents.[18],[19] They have also had additive immunomodulatory effects when used in combination with cyclosporine.[20] In a retrospective study, patients receiving 1,25(OH)2D3, in addition to standard immunosuppressive treatment, showed decelerated renal graft loss.[21] VDR agonists as immunosuppressive agents would be an attractive option, as they would theoretically offer some degree of bone protection as well as reduce the need for calcineurin inhibitors. Non-hypercalcemic VDR agonists[18],[19] have shown similar immunomodulatory properties without the side-effect of hypercalcemia. If VDR agonists were used as routine immunosuppressant agents, they may well have different efficacy in patients with different VDR FokI polymorphisms. It would be worthwhile examining the effect of these polymorphisms on the immunomodulatory function of VDR agonists to determine if renal transplant recipients of a certain genetic background may markedly benefit from VDR agonist therapy. We are currently continuing this study in a prospective fashion on patients from time of transplant in order to confirm these findings.
This research was supported with grants from Irish Nephrology Society, Irish Transplant Foundation, Punchestown Kidney Research Fund (Irl), Amgen Foundation, Roche Ireland Pharmaceuticals, and Fujisawa Ireland Pharmaceuticals.
| Variant | Frequency | Mean age (SD) | Gender, male/female(%) | Medianfollow-up time (years) |
|---|---|---|---|---|
| VDR FokI | ||||
| C:C | 145 (41.2%) | 40.7 (17.6) | 61.4 / 38.6 | 7.9 |
| C:T | 163 (46.3%) | 40.3 (15.1) | 65.6 / 34.4 | 9.9 |
| T:T | 44 (12.5%) | 39.0 (13.5) | 65.9 / 34.1 | 10.1 |
| VDR ApaI | ||||
| G:G | 73 (20.2%) | 39.0 (15.8) | 61.6 / 38.4 | 9.9 |
| G:T | 181 (50.1%) | 40.9 (16.7) | 64.6 / 35.4 | 9.1 |
| T:T | 107 (29.7%) | 37.1 (14.6) | 65.4 / 34.6 | 8.8 |
| Variant | Frequency of acuterejection | p (Pearsonχ2 test) |
|---|---|---|
| VDR FokI | ||
| C:C | 26/145 (17.9%) | p = 0.916 |
| C:T | 29/163 (17.8%) | |
| T:T | 9/44 (20.4%) | |
| VDR ApaI | ||
| G:G | 12/73 (16.4%) | p = 0.082 |
| G:T | 27/181 (14.9%) | |
| T:T | 27/107 (25.2%) |
| Exposure factor | p value | Hazard ratio | Lower 95% CI | Upper 95% CI |
|---|---|---|---|---|
| Univariate analysis | ||||
| VDR FokI T | <0.001 | 0.488 | 0.335 | 0.710 |
| Multivariate analysis | ||||
| VDR FokI T | <0.001 | 0.505 | 0.348 | 0.735 |
| Acute rejection | 0.021 | 1.689 | 1.083 | 2.635 |
| Recipient age | 0.167 | 0.853 | 0.681 | 1.069 |
| Recipient gender | 0.734 | 1.069 | 0.727 | 1.571 |
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