Diana M. Girnita

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.

Disparate distribution of 16 candidate single nucleotide polymorphisms among racial and ethnic groups of pediatric heart transplant patients

Background. Allograft failure in African-Americans remains higher than in Caucasians. Single nucleotide polymorphisms (SNPs) have been associated with altered allograft outcomes. Methods. In this multi-center study we compared SNP frequencies in 364 pediatric heart recipients from three ethnic/racial groups: Caucasian (n=243), African-American (n=39), and Hispanic (n=82). The target genes were: tumor necrosis factor-&agr;, interleukin (IL)-10, IL-6, interferon (IFN)-&ggr;, vascular endothelial growth factor (VEGF), transforming growth factor-&bgr;1, Fas, FasL, granzyme B, ABCB1, CYP3A5. Results. Compared to Caucasians, African-Americans exhibited a higher prevalence of genotypes associated with low expression of IFN-&ggr; (24% vs. 45.7%, P<0.001) and IL-10 (33% vs. 57.1%, P=0.052). African-Americans also exhibited an increased prevalence of high IL-6 (82.9% vs. 38.1%; P<0.001). VEGF −2578 C/C and −460 C/C genotypes were found more frequently in African-Americans and Hispanics as compared to Caucasians (P<0.001). G/G genotype of Fas and T/T genotype of FasL were expressed more often by African-American recipients. The prevalence of Granzyme B (−295A/G) genotype was differentially distributed in the three groups. Compared with Caucasians, African-Americans were twice as likely to carry the ABCB1 2677 G/G genotype (78.6% vs. 33.7%, P<0.0025), and they were more frequent carriers of the CYP3A5 *1/*1 genotype (35.7% vs. 0.6% in Caucasians and 7.2% in Hispanics; P<0.001). Conclusion. African-Americans have a genetic background that may predispose to proinflammatory/lower regulatory environment, reduced drug exposure and immunosuppressive efficacy. In this ongoing multicenter study, these gene polymorphisms differences among ethnic/racial groups are being documented so that therapeutic strategies can be devised to optimize outcomes for pediatric transplant recipients.

Genetic polymorphisms impact the risk of acute rejection in pediatric heart transplantation: a multi-institutional study.

Objective. The objective of this study was to determine the association between the genetic polymorphisms of proinflammatory and regulatory cytokines and long-term rates of repeat and late acute rejection episodes in pediatric heart transplant (PHTx) recipients. Methods. Three hundred twenty-three PHTx recipients: 205 White non-Hispanic, 43 Black non-Hispanic, and 75 Hispanic were analyzed for time to first repeat and late acute rejection episodes by race, age at transplantation, and gene polymorphism (interleukin [IL]-6, −174 G/C, IL-10, −1082 G/A, −819 C/T, 592 C/A; vascular endothelial growth factor (VEGF) −2578 C/A, −460 C/T, +405 C/G; tumor necrosis factor alpha (TNF-&agr;)−308 G/A). Results. Recipient black race and older age at transplant were risk factors for both repeat and late rejections, though black race was more significantly related to late rejection (P=0.006). Individually, TNF-&agr; high, IL-6 high, VEGF high, and IL-10 low phenotypes did not impact the risk of repeat or late rejection. However, the combination VEGF high/IL-6 high and IL-10 low was associated with increased estimated risk of late rejection (P=0.0004) and only marginally with repeat rejection (P=0.051). In a multivariate analysis, adjusting for age and race, VEGF high/IL-6 high and IL-10 low still remained an independent risk factor for late acute rejection (RR=1.91, P<0.001). Conclusion. This is the largest multicenter study to document the impact of genetic polymorphism combinations on PHTx recipients’ outcome. The high proinflammatory (VEGF high/IL-6 high) and lower regulatory (IL-10 low) cytokine gene polymorphism profile exhibited increased risk for late rejection, irrespective of age and race/ethnicity.

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.

Genetic polymorphisms influence mycophenolate mofetil–related adverse events in pediatric heart transplant patients

BACKGROUND Mycophenolate mofetil (MMF) is an effective and commonly used immunosuppressant but has frequent adverse events. Genetic polymorphisms may contribute to variability in MMF efficacy and related complications. In this study we explore the distribution frequencies of common single nucleotide polymorphisms (SNPs) of IMPDH1, IMPDH2 and ABCC2 and investigate whether these SNPs influence MMF adverse events in 59 pediatric heart recipients. METHODS Genotypes were assessed by TaqMan analysis of: ABCC2 rs717620; IMPDH2 rs11706052; and IMPDH1 rs2288553, rs2288549, rs2278293, rs2278294 and rs2228075. Gastrointestinal (GI) intolerance was defined as diarrhea, vomiting, nausea or abdominal pain requiring dose-holding for >48 hours or MMF discontinuation. Bone marrow toxicity was evaluated using Common Terminology Criteria for Adverse Events Version 3 (CTCAE). RESULTS GI intolerance occurred in 21 patients, and 21 had bone marrow toxicity. The ABCC2 rs717620 A variant was significantly associated with GI intolerance leading to drug discontinuation (p < 0.001); the IMPDH1 rs2278294 A variant and rs2228075 A variant were also associated with greater GI intolerance (p = 0.029 and p = 0.002, respectively). The IMPDH2 rs11706052 G variant was associated with more frequent neutropenia requiring dose-holding (p = 0.046). CONCLUSIONS In this small sample of pediatric heart transplant patients receiving MMF, ABCC2, IMPDH1 and IMPDH2 SNPs were associated with MMF GI intolerance and bone marrow toxicity. Thus, genetic polymorphisms may directly influence MMF adverse events.

Effect of cytokine and pharmacogenomic genetic polymorphisms in transplantation

Consolidating the information that we have on pharmacogenetics and on cytokine genetics to produce patient-oriented individualized drug regimens is an important challenge in transplantation medicine. Using a multi-variant approach based on genetic profile and other relevant clinical factors a score system may be developed to predict the severity of rejection, infection, or other complications associated with transplantation. The ultimate goal of these studies is to improve patient outcome through individualized drug regimens.

A polymorphism linked to elevated levels of interferon-γ is associated with an increased risk of cytomegalovirus disease among Caucasian lung transplant recipients at a single center

BACKGROUND Single-nucleotide polymorphisms (SNPs) associated with active cytomegalovirus (CMV) infections after lung transplantation have not been identified. METHODS SNPs associated with varying levels of interferon (IFN)-γ (+874T/A), tumor necrosis factor-α (-308G/A), interleukin-10 (-1082G/A, -819C/T, -592C/A) and interleukin-6 (-174G/C) were characterized for 170 Caucasian lung transplant recipients who received alemtuzumab induction and valganciclovir prophylaxis against CMV. RESULTS Patients were followed for a median of 34 months post-transplant, and 66% (113 of 170), 24% (40 of 170) and 10% (17 of 170) had no CMV infection, CMV viremia and CMV disease, respectively. Median times to CMV viremia and disease were 7 and 10 months, respectively. For each gene, there was no significant deviation from Hardy-Weinberg equilibrium. Independent risk factors for the development of CMV disease were IFN-γ +874 T/T genotype (associated with high levels of IFN-γ production), CMV donor-positive/recipient-negative (D(+)/R(-)) serostatus and acute cellular rejection requiring augmented immunosuppression (p = 0.001, 0.003 and 0.049, respectively). The association between IFN-γ +874 T/T genotype and CMV disease was most striking among R(+) patients (p = 0.02). D(+)/R(-) serostatus was also a significant risk factor for CMV viremia (p = 0.0005). IFN-γ +874 T/T genotype was associated with significantly lower peak CMV viral loads (p = 0.03). There were no associations between tumor necrosis factor-α, interleukin-10 or interleukin-6 SNPs and CMV infections. CONCLUSION A genetic predisposition to elevated IFN-γ levels may play a dual role in controlling active CMV infection among lung transplant recipients receiving alemtuzumab induction and valganciclovir prophylaxis, limiting the extent of viral replication in serum but increasing the risk of CMV disease.

Gene polymorphisms impact the risk of rejection with hemodynamic compromise: a multicenter study.

Background. Rejection with hemodynamic compromise (RHC) is associated with high mortality in heart recipients. This study investigates the association between genetic polymorphisms and RHC in pediatric heart recipients. Methods. Data from 532 pediatric heart recipients from six centers in the Pediatric Heart Transplant Study were analyzed for time to RHC by recipient race, age at transplantation, and genotype at 13 genetic polymorphisms (TNF-&agr; A-308G, IL-6 G-174C, INF-&ggr; T+874A, IL-10 G-1082A, C-819T, and C-592A; FAS A-670G, FASL C-843T, and ACE I/D; and VEGF A-2578C, C-1451T, C+405G, and -2549 I/D). Results. RHC occurred in 126 (23.7%) patients during the study period. Adjusting for age and race, IL-10 G-1082A, FAS A-670G, and ACE I/D genotypes were associated with RHC. IL-10 G-1082A GG genotype was associated with decreased risk of RHC with an adjusted hazard ratio (HR) of 0.49 (95% confidence interval [CI], 0.27–0.90; P=0.020). FAS A-670G AA genotype was associated with increased risk of RHC with an adjusted HR of 1.84 (95% CI, 1.25–2.69; P=0.002). ACE II genotype was associated with decreased risk of RHC with an adjusted HR of 0.58 (95% CI, 0.36–0.95; P=0.031). Conclusions. Recipients with a high anti-inflammatory and immune-regulatory genetic profile (high interleukin-10) were protected from RHC. Conversely, recipients with a pro-apoptotic genetic profile (high Fas) or high angiotensin-1-converting enzyme producing genotype were at increased risk of RHC. This represents progress toward understanding the genetic risk factors of posttransplantation outcomes in pediatric heart recipients.

Association of genetic polymorphisms and risk of late post-transplantation infection in pediatric heart recipients

BACKGROUND Late infections are common causes of morbidity and mortality after pediatric heart transplantation. In this multicenter study from 6 centers, we investigated the association between genetic polymorphisms (GPs) in immune response genes and late post-transplantation infections in 524 patients. METHODS Late infection was defined as a clinical infectious process occurring >60 days after transplantation and requiring hospitalization, intravenous antimicrobial therapy, or a life-threatening infection requiring oral therapy. All patients provided a blood sample for GP analyses of 18 GPs in cytokine, growth factor, and effector molecule genes by single specific primer-polymerase chain reaction and/or sequencing. Significant associations in univariable analyses were tested in multivariable Cox regression models. RESULTS Late infection was common, with 48.7% of patients experiencing ≥ 1 late infection, 25.2% had ≥ 1 late bacterial infection, and 30.5% had ≥ 1 late viral infection. Older age at transplantation was a protective factor for late infection, both bacterial and viral (hazard ratio [HR] 0.89-0.92 per 1-year age increase, p < 0.001). Adjusting for age, race, and transplant etiology, late bacterial infection was associated with HMOX1 A+326G AG and GG genotypes (HR, 2.41, 95% confidence interval [CI] 1.35-4.30; p = 0.003) and GZMB A-295G AA genotype (HR, 1.47; 95% CI; 1.03-2.1; p = 0.036). Late viral infection was associated with FAS A-670G GG genotype (HR, 1.42; 95% CI, 1.00-2.00; p = 0.050) in the adjusted model and with CTLA4 A+49G AA and AG genotypes (HR, 1.49; 95% CI, 1.02-2.19; p = 0.041) in univariable analysis. CONCLUSION We found an association between late bacterial infection and GP of HMOX1, which may control macrophage activation. A weaker association was also found between late viral infection and GP of CTLA4, a regulator of T-cell activation. This represents progress toward understanding the clinical and genetic risk factors of outcomes after transplantation.

Clinical Impact of Cytokine and Growth Factor Genetic Polymorphisms in Thoracic Organ Transplantation

Demographic and clinical risk factors may only partially predict short- and long-term outcomes after thoracic transplantation. The interindividual variability seen in rejection profiles could be related to the recipients or donors genetic background. Rejection, either acute or chronic, elicits an alloimmune response that involves a complex network of cytokines, growth factors, adhesion molecules, and other molecules, which may modulate the immune response toward rejection or, conversely, mediate graft acceptance. Herein, the authors discuss the current evidence regarding the importance of genetic polymorphisms as independent predictors of allograft outcome. They believe that pretransplant genotype profiling of patients, in combination with other relevant clinical information, might be useful to predict the risk for posttransplant adverse events and also to facilitate the implementation of individualized immunosuppression.