EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Key words : Adverse drug reaction, Allele frequency, Inosine triphosphatase, Pharmacogenetic gene sequence variants, Thiopurine metabolism

Kidney transplant is currently the gold standard therapy for patients with end-stage renal disease. Unfortunately, adaptive and innate immune respon-ses to graft alloantigens are the greatest threats to graft survival.¹ The introduction of several immuno-suppressive agents over the past decade has reduced the rate of acute rejection significantly and has improved short-term renal allograft survival.² In general, currently approved immunosuppressive agents are mostly used in the combined forms to treat renal transplant recipients. Such agents are commonly classified according to the respective mechanisms of action, such as calcineurin inhibitors (cyclosporin A, tacrolimus), inhibitors of purine synthesis (mycophenolate mofetil/mycophenolic acid and azathioprine), mechanistic target of rapamycin inhibitors (sirolimus and everolimus), and biologic drugs (belatacept).³ The thiopurine drugs 6-mercap-topurine and azathioprine are classic immuno-suppressants that are widely used for the treatment of various diseases, including childhood acute leukemia, inflammatory bowel disease, severe rheumatoid arthritis, dermatomyositis, and autoimmune hepatitis, as well as for the prevention of acute rejection after organ transplant.^4,5 Thiopurines are antimetabolites; that is, these drugs inhibit physiological processes by replacing the natural counterparts (guanosine nucleotides).^6,7 Inosine triphosphate pyrophosphates (ITPases) represent a ubiquitous group of protective enzymes encoded by the ITPA gene and function to prevent the incorporation of noncanonical (so-called rogue) purine nucleotides into DNA and RNA. Specifically, the ITPase catalyzes the hydrolysis of (deoxy) nucleoside triphosphates into the corresponding nucleoside monophosphate with the concomitant release of pyrophosphate.⁸ Recently, thiopurine drug metabolites such as azathioprine have been included in the lists of ITPase substrates. The ITPA gene is located on the short arm of chromosome 20 (20p13).⁹ Sumi and colleagues have previously determined the genomic structure of the ITPA gene, which consists of 8 exons and is about 13 kb long.¹⁰ Two single-nucleotide variants (SNVs, formerly SNPs) associated with ITPA deficiency have been identified in the ITPA gene: (1) the C-to-A substitution at nucleotide 94 (ITPA 94C>A) in exon 2; and (2) the A-to-C substitution at nucleotide 21 of the second intervening sequence (IVS2 +21A>C) in intron 2. Two SNVs affect the protein structure in several ways, including reduction of catalytic activity and reduction in stability and expression of the full-length transcript.¹¹ Cases that were homozygote for an open-reading frame 94C>A sequence variant had no observable enzyme activity. The activity of heterozygote cases was approximately 22.5% of the mean value of the control cases.¹² Recently, ITPA gene sequence variants were shown to be associated with ITPA deficiency, showing pharmacogenomic implications in patients treated with thiopurines.¹³ Furthermore, the ITPA c.94C>A sequence variant has been shown to be linked with the susceptibility to advanced drug reactions in azathioprine-treated patients.¹⁴ Approximately 1 in 10 patients treated with azathioprine will experience adverse side effects of the drug, the most problematic of which is bone marrow toxicity. There is evidence that sequence variants in the genes encoding thiopurine methy-ltransferase and ITPase predict adverse drug reactions to immunosuppression therapy.¹⁵ Several studies have investigated the association of the ITPA 94C>A sequence variant on the adverse effects of thiopurine.^16,17 Azathioprine is a thiopurine prodrug commonly used in patients with kidney transplant.^13,18 Although ITPA deficiency is not related to any defined pathology in humans, it was recently demonstrated that sequence variants in the ITPA gene associated with ITPA deficiency have pharmacogenomic implications for patients treated with thiopurine.¹³ The aim of our study was to evaluate the possible association of the ITPA 94C>A gene sequence variant in kidney transplant recipients treated with thiopurine drugs as a part of a standard immunosuppression regimen.

Materials and Methods

This retrospective case-control study was performed in the Transplant Center of Nemazi Hospital, affiliated with Shiraz University of Medical Sciences. In this study, we collected data for 140 recipients of kidney transplant who were seen at Transplantation Center of Namazi Hospital (Shiraz, southern Iran) from 2011 to 2015, as well as data for 100 healthy individuals (as a normal population, for the control group). In our study, the recipients were divided into 2 groups: kidney transplant recipients with rejection and kidney transplant recipients without rejection. The Ethics Committee of Shiraz University of Medical Sciences approved the protocol, which conformed to the ethical guidelines of the 1975 Helsinki Declaration. Written informed consent was obtained from all participants. According to the rules of the transplant ward of Namazi Hospital, all transplant recipients received organs from deceased donors. Tests for human leukocyte antigen (HLA) typing, lymphocyte cross-matching, and ABO blood compatibility were performed for all kidney transplant recipients. Expert pathologists, according to Banff criteria, certified the rejection state of the second group after analysis of kidney biopsies from the patients. Rejection episodes were identified by an expert nephrology team, based on approved clinical diagnostic criteria and confirmed by needle biopsy and elevated levels of serum creatinine and blood urea nitrogen.¹⁹ The routine immunosuppression regimen for all recipients consisted of cyclosporine (5 mg/kg initially, then a maintenance dosage of 2-2.5 mg/kg; cyclosporine level was 50-150 ng/mL), prednisolone (120 mg/d initially, tapered to 10 mg/d), and mycophenolate mofetil (1000 mg twice daily) plus azathioprine.

DNA extraction and genotyping

To obtain genomic DNA from patients and control participants for polymerase chain reaction (PCR), we used a DNA extraction kit (DNG-Plus, CinnaGen) according to the manufacturer’s instructions to extract DNA from buffy coat samples. The DNA quantity was measured with a spectrophotometer (NanoDrop) using 5 μL of DNA and 45 μL of double-distilled water. Given that the optimum DNA quantity for PCR was considered 100 ng, individual volumes of extracted DNA for each sample were calculated. This SNV was studied using a PCR-restriction fragment length polymorphism method as previously described.13 The forward PCR primer was 5'-CAGGTCGTTCAGATTCTAGGAGAAAAG-3', and the reverse primer was 3'- GAACAGGGTGTGA ACGAGAAGAAGAAC-5'. After PCR, the products were digested with the restriction enzyme XmnI, and the amplified products were monitored by agarose gel electrophoresis and DNA safe staining.

Statistical analyses

All statistical analyses were performed using SPSS software (version 16). The frequencies of the alleles/genotypes were compared in the patients with or without transplant kidney and in the control participants by the chi-square test or the Fisher exact test. In addition, the relationship between genetic sequence variant and the incidence of acute kidney rejection was estimated by odds ratio. We used the Bonferroni adjustment for multiple comparisons.

Demographic data and clinical characteristics of patients

Our analysis included 140 patients. The rejection group included 70 patients, composed of 40 male patients and 30 female patients, with a mean age of 40.23 ± 13.36 and 40.11 ± 15.43 years, respectively. The nonrejection group included 70 patients, composed of 45 male patients and 25 female patients, with a mean age of 38.63 ± 17.38 and 34.97 ± 16.86 years, respectively. According to the rules of the transplant ward of Namazi Hospital, all transplant recipients received organs from deceased donors. Tests for HLA typing and ABO blood compatibility were performed for all kidney transplant patients.

Effect of rs1127354 sequence variant on kidney rejection

The results showed a statistically significant difference between the control group and the nonrejection group regarding the rs1127354 genotype and allele frequency. The control group and 2 experimental groups did not show deviation from Hardy-Weinberg equilibrium. The results showed no significant associations for the rs1127354 sequence variant between the control group and the rejection group. Also, there was no significant difference between the rs1127354 sequence variant with kidney graft in the rejection group and the nonrejection group. The genotype frequencies in the rs1127354 sequence variant in the rejection group were CC = 40, CA = 2, and AA = 28, whereas the nonrejection group showed CC = 36, CA = 3, and AA = 31. The frequencies of alleles C and A were 137 and 63 in the control group and 75 and 65 in the nonrejection group, respectively. The frequencies of alleles C and A in the rejection group were 82 and 58, respectively (Table 1). After classification of recipients according to age, a significant association was observed between age in patients with kidney transplant (with or without transplant rejection). There was a significant association between age ranges (31-45 years and 46-60 years) with the rejection group (Table 2). There was a significant difference between blood groups in kidney transplant recipients with rejection and nonrejection. The results revealed a statistically significant difference between the A-positive blood group and rejection (Table 3). Also, the results revealed no significant differences between sex and underlying disease in kidney transplant recipients (with or without transplant rejection).

The enzyme ITPase is encoded by the human ITPA gene. Five SNVs in the human ITPA gene have been recognized, and 2 of these are associated with IVS2+21A>C). These changes interact and affect branch points, which results in missplicing of exons 2 and 3 and leads to shortening of polypeptide stretches in the enzyme.^11,20 The other 3 coding region ITPA sequence variants are silent alterations (138G>A, 561G>A, and 708G>A).^x10 Homozygosity of the ITPA 94A allele, which results in deficiency of ITPase enzyme activity in erythrocytes and lymphocytes, is observed in nearly 1 in 1000 people among the White population.²¹ The results of our present study showed a significant correlation between the CC genotype of the ITPA 94C>A sequence variant and kidney transplant in the control and nonrejection groups. Furthermore, our results indicated a significant association between age range (31-45 and 46-60 years) with the rejection group. To the best of our knowledge, our present study on the ITPA 94C>A sequence variant in kidney transplant patients with and without rejection is the first of its kind in Iran. Therefore, we cannot compare our results with other studies. The frequency of the ITPA 94C>A variant has been investigated in various populations. A sequence variant occurred with a frequency of 0.06 in a White population in the United Kingdom,²⁰ 0.05 in an African population, and a higher frequency of 0.11 to 0.15 in 2 Asian populations.²² The study of Marsh and colleagues²³indicated that the frequency of the 94C>A sequence variant is higher in Asian populations (11%-19%) than in White populations (5%-7%). Previous studies revealed significant correlations between the ITPA 94C>A sequence variant and the onset of adverse reactions such as infiuenza-like symptoms, rash, and pancreatitis in patients treated with thiopurine.^13,17 In the study of Marinaki and colleagues, the ITPA 94C>A genotype was significantly related to infiuenza-like symptoms, rash, and pancreatitis.¹³ The association between the ITPA 94C>A genotype and infiammatory bowel disease has been shown.²⁴ The study by Xiong and colleagues demonstrated a reduction of ITPA activity in patients with the 94C>A variant (P < .01). Patients with the ITPA 94C>A homozygous allele are at high risk to develop azathioprine-related gastrointestinal toxicity and influenza-like symptoms.²⁵ Heidari and colleagues showed a significant difference between CA genotype frequency of the ITPA 94C>A sequence in the rejection and nonrejection groups of liver transplant recipients. Also, a meaningful difference was observed between this sequence variant and allelic frequency in the liver rejection group versus the control group.²⁶

The ITPA 94C>A sequence variant is probably a common sequence variant in the Iran population, and it may have an important role to prevent graft rejection in middle-aged patients versus the control group. Further study is needed, with a larger population and with different ethnicities. The ITPA 94C>A sequence variant is an important factor in kidney transplant recipients treated with thiopurine drugs. This study was performed on a few patients and control participants. To the best of our knowledge, this is the first report on kidney graft rejection and ITPA SNVs. Further studies are required to confirm and extend the present results.

Volume: 21 Issue: 8 August 2023

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