Ian V. Hutchinson

IMPDH1 Gene Polymorphisms and Association With Acute Rejection in Renal Transplant Patients

Inosine 5′‐monophosphate dehydrogenase 1 (IMPDH1) catalyzes the rate‐limiting step of the de novo pathway for purine synthesis and is a major target of the immunosuppressive drug mycophenolic acid (MPA). Few variants of the IMPDH1 gene have been reported. The objective of this study was to identify and characterize IMPDH1 variants to determine whether genetic variation contributes to differences in MPA response and toxicity in transplant patients. Seventeen genetic variants were identified in the IMPDH1 gene with allele frequencies ranging from 0.2 to 42.7%. In this study, 191 kidney transplant patients who received mycophenolate mofetil were genotyped for IMPDH1. Two single‐nucleotide polymorphisms, rs2278293 and rs2278294, were significantly associated with the incidence of biopsy‐proven acute rejection in the first year post‐transplantation. Future studies of the multifactorial nature of acute rejection must consider IMPDH1 polymorphisms in MPA‐treated patients.

A novel variant L263F in human inosine 5'-monophosphate dehydrogenase 2 is associated with diminished enzyme activity.

Background and objective Inosine 5′-monophosphate dehydrogenase 2 is required for purine synthesis in activated lymphocytes. Variants in the IMPDH2 gene may account for the large inter-individual variability in baseline enzyme activity, immunosuppressive efficacy and side effects in transplant recipients receiving mycophenolic acid. Therefore, the objective of this study was to identify and functionally characterize IMPDH2 variants. Methods DNA samples from 152 solid organ transplant patients were screened at exons and exon/intron junctions of the IMPDH2 genes by PCR amplification followed by bidirectional direct DNA sequencing. Genetic variant was constructed by site-directed mutagenesis and transformed to an inosine 5′-monophosphate dehydrogenase-deficient strain of Escherichia coli h712. Proteins were purified to homogeneity and the enzymatic activity was measured by reduced nicotinamide adenine dinucleotide production. Results Nine genetic variants were identified in the IMPDH2 gene, with frequencies of the rarer alleles ranging from 0.5 to 10.2%. A novel nonsynonymous variant L263F was identified, and the kinetic assay demonstrated that the inosine 5′-monophosphate dehydrogenase activity of L263F variant was decreased to 10% of the wild-type. The Ki for mycophenolic acid inhibition of the L263F variant was comparable with the wild-type, and the variant Km for inosine 5′-monophosphate and nicotinamide adenine dinucleotide did not change significantly. Conclusions IMPDH2 has low genetic diversity, but the nonsynonymous variant L263F has a significant impact on inosine 5′-monophosphate dehydrogenase activity. This novel functional variant may be one of the factors contributing to the inter-individual difference of baseline inosine 5′-monophosphate dehydrogenase activity as well as drug efficacy and adverse events in transplant patients.

Impact of Epstein–Barr virus donor and recipient serostatus on the incidence of post-transplant lymphoproliferative disorder in kidney transplant recipients

BACKGROUND Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of transplantation. METHODS Using the OPTN/UNOS database, primary kidney recipients (2000-2009) were stratified according to transplant type (deceased donor, DD or living donor, LD), donor (D) and recipient (R) Epstein-Barr virus (EBV) serostatus (R+; D+/R- and D-/R-) and recipient age. Incidence and adjusted risk of PTLD and death were compared. RESULTS Of the 137 939 primary kidney recipients transplanted between 2000 and 2009, 913 subsequently developed PTLD. In 90 208 recipients with known EBV serostatus, we found a trend toward a decrease in PTLD incidence in years 2007-2009 when compared to 2000-2003. This was due to a significant decrease in PTLD incidence in EBV- recipients. Of those, 61 273 had a known donor serostatus and were further examined. In adults, PTLD incidence (in 1000 person-years) in DD and LD was 7.0 and 7.0 in D+/R-; 3.0 and 2.5 in D-/R- and 1.2 and 1.0 in R+, respectively. The hazard ratio (HR) for PTLD (R+ as reference) in D+/R- (6.2 in DD and 7.2 in LD) was double to thrice than for D-/R- transplants (2.4 in both DD and LD). In pediatric recipients, PTLD incidence in DD and LD was 15.9 and 17.3 in D+/R-; 12 and 18 in D-/R- and 1.2 and 2.2 in R+, respectively. The HR for PTLD was 17.4 and 6.9 in D+/R- and 15.9 and 7.6 in D-/R- in DD and LD, respectively. CONCLUSION A D+/R-, compared with a D-/R- transplant, may contribute to an increase in PTLD incidence of 35 and 42% in adult DD and LD transplants, respectively.

eNOS gene polymorphism association with retinopathy in type 1 diabetes

Purpose: Nitric oxide (NO) is a major mediator in vascular biology, regulating blood pressure and regional blood flow. NO and the enzymes required for its production may contribute to the aetiology of vascular pathologies. In diabetes, over-production of NO might play a role in the development of diabetic nephropathy, while reduced NO production may be related to the development of diabetic retinopathy and neuropathy, where VEGF (vascular endothelial growth factor) levels are increased in a counter regulatory manner. Among the three nitric oxide synthase (NOS) enzymes most attention has focussed on endothelial NOS (eNOS) because of its relevance to angiopathies. Methods: In this study the influence of a single nucleotide polymorphism at position -786 in the eNOS gene, where there is a C/T base substitution, on development of type 1 diabetes mellitus (T1DM) and its microvascular complications was studied in 249 British Caucasian type 1 diabetics using a case-control association design. Genotyping was carried out using PCR-RFLP technique. Results: There was a significant association between the polymorphism -786*C/T and both T1DM and diabetic retinopathy. The distribution of eNOS gene polymorphism genotype frequencies showed a significant difference observed between diabetic patients and healthy controls [CC+CT vs. TT p = 0.05, OR = 1.5 95%CI(0.9–2.5)]. The genotype frequencies for eNOS gene polymorphism was also significantly different between diabetic retinopaths and healthy controls [CC+CT vs. TT p = 0.0000 OR = 3.4 95%CI(1.9–6.1) No significant differences for eNOS allele and genotype frequencies were found in other groups compared to the controls. Conclusion: Therefore, eNOS gene variation may be a factor in the genetic propensity to T1DM and diabetic retinopathy that may have a prognostic value or may suggest interventional approaches to regulate eNOS in patients with diabetes.

Gene polymorphism of vascular endothelial growth factor −1154 G>A is associated with hypertensive nephropathy in a Hispanic population

The aim of this study was to determine the association between hypertensive nephropathy and gene polymorphisms of vascular endothelial growth factor (VEGF) in a self-reported Hispanic patient group. A total of 155 Hispanic living kidney donors as controls and a total of 86 Hispanic kidney transplant patients, whose renal failure was attributed to hypertensive nephropathy after ruling out diabetes mellitus or other causes, were genotyped for four different single nucleotide polymorphisms of VEGF: −2578 C>A (rs699947), −1154 G>A (rs1570360), −460 C>T (rs833061), and +936 C>T (rs3025039). The homozygous mutant type (AA) of VEGF −1154 G>A (rs1570360) was found with significantly higher frequency in the hypertensive nephropathy patients than in controls. On the other hand, homozygous wild type (GG) was found less frequently in the hypertensive nephropathy patient group than in the control group. Linkage disequilibrium (LD) analyses revealed a high degree of LD among VEGF −2578 C>A (rs699947), VEGF −1154 G>A (rs1570360), and VEGF −460 C>T (rs833061). The haplotype analysis revealed that two haplotypes, CGTC and CATC (in the order of VEGF −2578 C>A (rs699947), −1154 G>A (1570360), −460 C>T (rs833061), and +936 C>T (3025039)), were significantly associated with hypertensive nephropathy in Hispanic patients. Hence, the −1154 G>A polymorphism (rs1570360) and two haplotypes (CGTC and CATC) of VEGF appear to be associated with hypertensive nephropathy in Hispanic patients who developed end-stage renal disease requiring kidney transplant.

Influence of Cyclooxygenase-2 (COX-2) gene promoter-1195 and allograft inflammatory factor-1 (AIF-1) polymorphisms on allograft outcome in Hispanic kidney transplant recipients

Cyclooxygenase-2 (COX-2) alleles have been associated with allograft outcomes in kidney transplant recipients; however, these alleles may be in linkage with other genes. Human allograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein and is produced by macrophages. Its synthesis is regulated by several cytokines, including interferon gamma. We investigated whether polymorphisms of gene encoding COX-2 and AIF-1 were associated with allograft outcomes among Hispanic renal transplant recipients (RTRs). A total of 527 de novo RTRs of Hispanic ethnicity were included in this study transplanted at St. Vincent Medical Center (SVMC) during 2000-2009. Patients were genotyped for the following: COX-2 (-1195C>T rs689466, intron 6 rs2066826) and AlF1 (rs2269475). Analysis of the results showed that COX-2-1195 CC genotype (OR=1.92, CI%=1.00-3.67, p=0.04) were more frequent, but COX-2-1195 CT genotype was less frequent in kidney allograft acute rejection in comparison with control group (OR=0.59, CI%=0.38-0.91, p=0.017). The genetic variant TT/CT of the AIF-1 gene was associated with a lower risk of rejection (OR=0.63, CI%=0.41-0.98, p=0.038). No association of COX-2 (rs2066826) was observed with allograft rejection. We are unable to find statistically significant association between COX-2 and AIF-1 gene polymorphisms and allograft survival. The -1195C>T in the COX-2 promoter and AIF-1 gene polymorphisms could be a potential predictor of allograft rejection in our Hispanic kidney transplant recipients.

TGF-β1 and IGF-I gene variations in type 1 diabetes microangiopathic complications

BackgroundGrowth factors are generally believed to have a perpetuating role in the development of diabetic complications, However there is ample of evidence of a protective or therapeutic potential for some of them. IGF-I, according to some reports, may contribute to complication development, although a protective role for IGF-I has been claimed for all late diabetic complications, making it an exception among growth factors. Transforming growth factor (TGF)-β1 as a pleiotropic cytokine is a key player in immunoregulation. Dysregulation of TGF-β1 in diabetes has been addressed as a leading event of kidney pathologies, while there is no similar pivotal role for TGF-β1 in diabetic retinopathy or neuropathy. An association study was conducted to evaluate the distinctive roles of TGF-β1 and IGF-I in T1DM microvascular complications by gene variation-based regulatory mechanisms that are operational in modulation of both in situ and systemic levels of the gene product.MethodsTwo polymorphisms of the IGF-I gene at positions −383*C/T and −1089*C/T and two functional TGF-β1 gene polymorphisms, including codons 10 (+869*C/T) and 25 (+915*G/C) were examined in 248 British Caucasian T1DM patients and 113 healthy controls.ResultsThe distribution of IGF-1 gene polymorphisms did not reflect any significant association with different endpoints among the cases or different subgroups (complication triad) and controls. For TGF-β1 gene codon 25 polymorphism the low producer variant (allele C) were more frequent in cases than controls, which is compatible with the anti-inflammatory role of TGF-β1 and for codon 10 polymorphism the frequency of allele C was highest in retinopaths and, on the contrary and expectedly, nephropathy was more frequently accompanied by allele T (high producer). The frequency of allele G (high producer) of codon 25 polymorphism was slightly higher in the complication free group than in other subgroups.ConclusionAlthough there were some differences in distribution of allele and genotype frequencies of TGF-β1 gene polymorphism in diabetes microvascular complications the differences were not statistically significant. Regarding IGF-1 our result firstly questions the functionality of the employed polymorphic marker and secondly may entail that the main regulator of IGF-I functionality resides elsewhere rather than the IGF-I gene itself, such as post-transcriptional regulation.

Impact of NF-κB gene polymorphism on allograft outcome in Hispanic renal transplant recipients

BACKGROUND The dimeric NF-κB transcription factors play critical roles in diverse cellular processes including adaptive and innate immunity, cell differentiation, proliferation and apoptosis. It regulates the expression of numerous genes that play a key role in the inflammatory response during kidney allograft rejection. This study aims to determine the association of NF-κB gene polymorphisms with allograft outcomes in the Hispanic renal transplant recipients. METHODS A total of 607 Hispanic renal transplant recipients at St. Vincent Medical Center between 2001 and 2010 were included in this study. The NF-κB genotypes were studied along with clinical data. In the case of NF-κB genotypes, the following single nucleotide polymorphisms (SNPs) were included: NF-κB1 (rs3774959, rs3774932, rs3774937, rs230526, rs230519), NF-κB2 (rs1056890, rs7897947, rs12769316) and NF-κB inducing kinase (NIK) (rs9908330, rs7222094). The association of each genotype with renal allograft survival and acute rejection was evaluated. RESULTS NF-κB1 (rs3774937) CC genotype showed protective association with allograft rejection (OR=0.66, 95% CI=0.44-0.99, p=0.04). There was a significant increase in allograft survival time associated with the NF-κB1 (rs3774959) A allele (OR=0.76, 95% CI=0.60-0.98, p=0.03) while GG genotype was associated with a higher risk of graft failure (OR=1.51, 95% CI=1.02-2.21, p=0.03). There were no associations between polymorphic markers in NF-κB2 and NIK genes with allograft survival or acute rejection. Among non-genetic factors, we found that the use of tacrolimus, a deceased donor, delayed graft function and acute rejection were associated with allograft failure. CONCLUSION The result of present study suggests that NF-κB1 gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.

Genetic polymorphisms of UGT1A8, UGT1A9 and HNF-1α and gastrointestinal symptoms in renal transplant recipients taking mycophenolic acid.

Mycophenolic acid (MPA), a widely used immunosuppressant, has a complex metabolism that involves a number of enzymes. Some of its metabolites are thought to be the cause of gastrointestinal (GI) side effects. In this study, we investigated whether polymorphisms of UDP-glucuronosyltransferases (UGT1) A8, 1A9, and hepatocyte nuclear factor (HNF1α) genes or pharmacokinetic parameters of mycophenolic acid (MPA) were associated with the severity of GI symptoms in patients receiving MPA therapy. A total of 109 kidney transplant patients taking mycophenolic acid (MPA) derivatives were genotyped for UGT1A8, 1A9 and HNF1α genes. Among these, a total of 15 patients were participants in the pharmacokinetic study. Severity of GI symptoms was assessed using a validated Gastrointestinal Symptom Rating Scale (GSRS). The overall and subscale GSRS scores were measured at 1 week (baseline), 2 weeks, 3 months and 6 months post-transplantation. In the case of the pharmacokinetic study, EC-MPS was administered and a total of nine blood samples were obtained at -1, 0, 0.5, 1, 2, 4, 6, 8, and 12h. Genotypes of UGT1A8 were significantly associated with the overall GSRS scores at week 1 (p=0.02) and week 2 (p=0.036). Subscales were only statistically significant for constipation at week 1 (p=0.002) and indigestion at week 2 (p=0.02), while UGT1A9 was only significant for the constipation at week 1 (p=0.04). HNF1α genotypes were significantly different at week 1 in the overall GSRS (p=0.004), and for abdominal pain (p=0.04), acid reflux (p=0.036) and constipation subscales (p=0.04). In addition, abdominal pain was statistically significantly different at 3 months and 6 months after transplantation (p=0.03 and 0.02, respectively). In the case of the pharmacokinetic study, we have found some correlations between MPAC0 and constipation (p=0.02) where MPAAUC was correlated with acid reflux (p=0.02) and constipation (p=0.012), MPAGCL/F was correlated to acid reflux, indigestion, constipation and the sum of the GSRS scores (p=0.037, p=0.032, p=0.033 and p=0.04, respectively). Multinomial regression analysis for MPAGCL/F showed a statistical significance for the subscale indigestion and the sum of the GSRS (p=0.033 and p=0.037, respectively). Our data suggests that among patients receiving MPA the UGT1A9 alleles might play a role in determining the severity of early GI side effects, while the HNF1α allele appears to be associated with a later effect as well as early side effects. Our data also showed that some kinetic parameters might predict MPA side effects.

Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients

Granzyme B (GZMB) and perforin 1 gene (PRF1) are key effector molecules of cytotoxic T lymphocytes, in causing acute and chronic solid organ transplant rejection. In this study, we analyzed the impact of GZMB and PRF1 polymorphism on kidney allograft outcomes. In all, 527 de novo kidney Hispanic allograft recipients were genotyped for PRF1 (rs10999426, rs35947132) and GZMB (rs8192917, rs7144366). PRF1 (rs10999426, rs35947132) G alleles and GG genotypes were negatively associated with allograft rejection, demonstrating protection against allograft rejection (OR = 0.61, p = 0.005 for rs1099946; OR = 0.4, p = 0.01 for rs 35947132). On the other hand, the GA heterozygosity of PRF1 was found marginally associated with the rejection group (OR = 1.53, p = 0.05 for rs10999426; OR = 2.24, p = 0.07 for rs35947132). There was a significant increase in allograft survival in time period studied for the PRF1 (rs10999426) GG genotype, while the GA heterozygosity was associated with graft failure. We found no association for polymorphic markers in GZMB gene with allograft rejection. Survival was significantly improved for patients who were homozygous TT for the GZMB (rs8192917) (TT vs. CC/TT, p = 0.041). The result suggests that PRF1 and GZMB gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.