Recipient Genotype Research Articles

PNPLA3 as a liver steatosis risk factor following living-donor liver transplantation for hepatitis C.

Liver steatosis frequently occurs following liver transplantation (LT) and can affect patient outcome. Here, we aimed to clarify the steatosis and steatohepatitis risk factors that apply after living-donor LT for chronic hepatitis C. We retrospectively examined 43 transplant recipients and donors, and tested for single nucleotide polymorphisms in the PNPLA3 gene. Liver biopsies taken 1year after transplantation and yearly thereafter, or when abnormal liver enzyme levels were detected, were examined by histopathology. Liver steatosis (>5% steatotic hepatocytes) was evident in 13 of 43 cases (30%), and steatohepatitis in 3 (7.0%). The average time to steatosis after LT was 2.74 ± 1.55years. The PNPLA3 rs738409 GG genotype, a steatosis risk factor, was identified in 13 recipients and 10 donors. Steatosis prevalence did not differ according to recipient genotype. However, this condition was significantly more common among patients who received tissue from donors carrying the rs738409 GG genotype compared to those with grafts from donors of the CC or CG genotype (60, 7, and 26%, respectively; P < 0.05). All 3 steatohepatitis cases were associated with the GG donor genotype. The PNPLA3 rs738409 GG donor genotype affects liver steatosis and steatohepatitis risk following living-donor LT.

From genetic single candidate gene studies to complex genomics of GvHD

Graft-versus-host disease (GvHD) is a serious complication affecting the recipients of allogeneic haematopoietic stem cells. In this present review we attempt to summarize the current knowledge on the effect of the donor and recipient genotypes on GvHD, starting from human leucocyte antigen (HLA) matching for an optimal donor selection, typing of non-classical HLA and minor histocompatibility antigens through the polymorphic variations in genes coding for non-HLA proteins contributing to the development of GvHD and response to treatment. The results of recent Candidate Gene Studies (CGS) and Genome-Wide Association Studies (GWAS) are presented and discussed.

Joint Assessment of Donor and Recipient hTERT Gene Polymorphism Provides Additional Information for Early Kidney Transplantation Outcomes.

BackgroundThere are several genes and genetic loci affecting telomere length, including hTERT gene and BICD1 gene as well as polymorphisms within chromosome 18. It has been demonstrated that the age of the donor is a negative factor associated with long-term kidney allograft function, and that post-transplant complications accelerate transplanted organ aging, thus contributing to estimated glomerular filtration rate (eGFR) decreases. The aim of this study was a joint assessment of donors’ and recipients’ hTERT and BICD1 genes as well as chromosome 18 polymorphisms with regard to early kidney transplantation outcomes.Material/MethodsThe study enrolled 74 pairs of Polish Caucasian kidney allograft cadaveric donors (60% male, mean age 45.99±14.62) and recipients (50.0% male, mean age 48.89±13.50). The transplantation procedure (Tx) was performed between 2001 and 2012. All samples were genotyped in duplicate using Real-Time PCR.ResultsThis study showed that rs2735940 hTERT CX-TT donor-recipient genotype pair was associated with almost five times higher odds (OR=4.82; 95% CI: 1.32–18; p=0.016) of delayed graft function (DGF), and that rs2735940 hTERT, rs2630578 BICD1, and rs7235755 chromosome 18 polymorphisms combined pairs were not associated with acute rejection (AR).ConclusionsIn conclusion, both the donor’s and the recipient’s rs2735940 hTERT gene polymorphism was associated with early graft function after transplantation. The odds of DGF were almost five times higher for a combination of CX (CT or CC) donor genotype and TT recipient genotype. Joint assessment of donor-recipient genotype pairs provides more information for prediction of early kidney transplantation outcomes.

Tacrolimus population pharmacokinetics according to CYP3A5 genotype and clinical factors in Chinese adult kidney transplant recipients.

Tacrolimus is characterized by a narrow therapeutic index and a considerable inter- and intraindividual pharmacokinetic variability. The aim of our study was to develop a population pharmacokinetic model of tacrolimus in adult kidney transplant of Chinese patients, identify factors especially CYP3A5*3 genetic polymorphism that explain variability, and determine dosage regimens. Pharmacogenomic data obtained from 83 Chinese kidney transplant patients treated with tacrolimus were determined using polymerase chain reaction-restriction fragment length polymorphism analysis. Trough blood concentration data were collected from all of the patients during the 12months of post-transplantation days and were analysed using the nonlinear mixed-effects modelling program. After building the final model, 1000 bootstraps were performed to validate the final model. A one-compartment model with first-order absorption and elimination adequately described the pharmacokinetics of tacrolimus. In this study, we observed that POD, HCT and CYP3A5*3 genotype were determinant factors in CL/F and POD related with V/F of tacrolimus significantly. The final model with the clearance covariates was presented as: Cl/F=THETA(1)*EXP(THETA(4)*(83/POD))*(39.1/HCT)**THETA(5)*EXP(THETA(6)*CYP3A5), and the final model with the volume covariates was presented as: Vd/F=THETA(2)*POD**THETA(3). The Ka was fixed to 4.5h-1 . The HCT, CYP3A5*3 genetic polymorphism and POD contributed to the interindividual variability of oral tacrolimus in Chinese adult renal transplant patients.

HLA Genotypes in Turkish Hematopoietic Cell Recipients and Likelihood of Finding a Matched Donor Through Family Searches.

Allogeneic hematopoietic stem cell transplant is a life-saving treatment, but donor numbers in Turkey do not meet the increasing demand for this procedure. Here, our objectives were (1) to assess the frequency of HLA-matched related donors in the Turkish population and (2) to identify the HLA antigens and haplotypes that are most frequent in Turkey. The HLA genotypes of 841 consecutive recipients and 3071 family members were retrospectively reviewed. Matched related donors were identified for 368/841 recipients (44%). Extended family donor searches were performed for 111/181 pediatric recipients (61%), with nonsibling matched related donors found for 23 patients (21%). Matched related donors were found for a significantly higher proportion of pediatric patients (52%) than adult patients (41%) (odds ratio of 2.5; 95% confidence interval, 1.9-4.1; P = .02). The percentage of pediatric versus adult patients with 3 or more siblings was 13% versus 46% (odds ratio of 5.6; 95% confidence interval, 3.6-8.5; P = .001). The most frequent HLA class I antigens at each locus were HLA-A*02 (20.2%), HLA-B*35 (19.5%), and HLA-C*07 (19.8%). The most frequent HLA class II antigens at each locus were HLA-DRB1*11 (21.6%) and HLA-DQB1*03 (40.2%). The most common 3-locus haplotypes were HLA-A*24 B*35 DRB1*11 (F:0.020) and HLA-A*01 B*08 DRB1*03 (F:0.015). When adult and pediatric groups were combined, the most common locus haplotypes were found in 43/345 sibling donors (12%) and in 7/23 nonsibling pediatric donors (30%) (odds ratio of 2.7; 95% confidence interval, 1.2-6.4; P = .02). The results indicate that, in Turkey, it can be beneficial to revise donor search algorithms to include an extended family donor search before an unrelated donor search. This type of search can be effective because of the HLA haplotype diversity in Turkey, the frequency of consanguinity, and the country's limited donor pool.

Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato

BackgroundLate blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance.ResultsPopulations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order.ConclusionThis study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype.

Donor CYP3A5 genotype influences tacrolimus disposition on the first day after paediatric liver transplantation.

The aim of the present study was to investigate the influence of the cytochrome P450 (CYP) 3A4/5 genotype in paediatric liver transplant recipients and donors, and the contribution of age and gender to tacrolimus disposition on the first day after transplantation. The contribution of the CYP3A4/5 genotype in paediatric liver transplant recipients and donors to the tacrolimus blood trough concentrations (C0 ) and the tacrolimus concentration/weight-adjusted dose ratio on day 1 was evaluated in 67 liver-transplanted children: 33 boys and 34 girls, mean age 4.5years. Donor CYP3A5 genotype appears to be significantly associated with tacrolimus disposition on the first day after liver transplantation (P < 0.0002). Other physiological factors, such as recipient age and donor gender may also play a role and lead to significant differences in tacrolimus C0 and tacrolimus concentration/weight-adjusted dose ratio on day 1. However, according to the general linear model, only recipient age appears to be independently associated with tacrolimus disposition on the first day after liver transplantation (P < 0.03). Indeed, there was a faster tacrolimus metabolism in children under 6years of age (P < 0.02). Donor CYP3A5 genotype, recipient age and, to a lesser extent, donor gender appear to be associated with tacrolimus disposition on day 1 after transplant. This suggests that increasing the starting tacrolimus doses in paediatric patients under 6years of age who receive a graft from a male extensive metabolizer may enhance the possibility of their tacrolimus levels reaching the therapeutic range sooner.

Comparative Temporal Transcriptome Profiling of Wheat near Isogenic Line Carrying Lr57 under Compatible and Incompatible Interactions.

Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general.

Clinical impact of the CYP3A5 6986A>G allelic variant on kidney transplantation outcomes.

Meta-analyses and large cohort studies provide confusing results on the association of the CYP3A5 6986A>G allelic variant and adverse outcomes in kidney transplant recipients under tacrolimus-based immunosuppressive regimen. A residual effect of CYP3A5 recipient genotype is unexpected if kidney transplant recipients have similar exposure of tacrolimus. We have undertaken a population-based, observational study, to investigate all the consecutive patients who received a kidney transplant at the Necker hospital between 2005 and 2015, who were treated with tacrolimus and for whom the CYP3A5 genotype was available. We analyzed 577 patients followed for up to 5 years. We found a significant association of CYP3A5 genotypes with tacrolimus daily dose as well as with tacrolimus dose-adjusted concentrations. We however found no association of CYP3A5 genotypes with histology scores on biopsies, measured renal function, biopsy-proven acute rejection episodes and graft survival.

REG3A Polymorphism Is Associated with the Incidence of Extensive Chronic Gvhd after Allogeneic BMT

[Introduction] Regenerating islet-derived protein 3 alpha (REG3A), a protein produced by Paneth cells, has been reported as a biomarker of lower gastrointestinal graft-versus-host disease (GVHD). The single nucleotide polymorphism (SNP) rs7588571 is located at the upstream of REG3A gene within 2kb, suggesting that the expression of REG3A might be affected by the SNP. In this study, we analyzed the association between the rs7588571 genotype in recipients and transplant outcomes.[Patients and methods] The study population included 118 adult Japanese patients who had undergone T cell replete allogeneic bone marrow transplantation from an HLA-identical sibling donor with GVHD prophylaxis consisting of short-term methotrexate and cyclosporine between 1987 and 2006. Sixty-eight (58%) male and 50 (42%) female patients, with a median age of 35 years (range 15-56), were included. Eighty-eight (75%) patients were classified as having standard risk disease defined as acute myeloid leukemia in first or second complete remission, acute lymphoblastic leukemia without Philadelphia chromosome in first complete remission, chronic myeloid leukemia in chronic phase, myelodysplastic syndrome without excess of blasts, aplastic anemia and paroxysmal nocturnal hemoglobinuria, while other patients were classified as having high risk disease. Conditioning regimen was myeloablative in 113 (96%) patients and reduced-intensity in other patients. Genomic DNA was extracted from patient’s peripheral blood or bone marrow by standard methods. Genotyping was performed using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, California, USA) on ABI 7300 Real-Time PCR systems (Applied Biosystems). Multiple logistic regression model was used to identify risk factors associated with acute and chronic GVHD, and multivariate Cox model was created for overall survival (OS), non-relapse mortality (NRM), and relapse. Variables with a P-value < 0.1 in the univariate analysis were entered into the stepwise selection method. All tests were two-sided, and P <0.05 was considered significant. This study was approved by the ethics committees at the Nagoya University Hospital and the Japanese Red Cross Nagoya Daiichi Hospital.[Results] The frequencies of the GG, AG, and AA genotypes were 41%, 43%, and 16%, respectively, which were compatible with those reported in the HapMap-JPT database. There was no significant association between the distribution pattern of the rs7588571 genotypes and each patient characteristic. Grade II-IV acute GVHD developed in 12% of all patients, 14% in those with GG genotype, 14% with AG genotype and 11% with AA genotype, respectively. In univariate analysis, the higher patient age (≥35) was the only significant factor for higher incidence of grade II-IV acute GVHD (OR: 5.4, 95% CI: 1.3-32, P=0.011). Extensive chronic GVHD developed in 38% of all patients, 27% in those with GG genotype, 45% with AG genotype and 50% with AA genotype, respectively. The incidence of extensive chronic GVHD in non-GG genotype (46%) was significantly higher than that in GG genotype (27%) (P=0.05). In multivariate analysis, non-GG genotype (OR: 2.5, 95% CI: 1.0-5.8, P=0.04), female to male transplant (2.8, 1.2-7.0, 0.023), and high risk disease (2.8, 1.1-7.2, 0.038) were significant factors for higher incidence of extensive chronic GVHD. The relapse rates at 4-year were 26% in non-GG and 17% in GG genotype (HR: 1.16, 95% CI: 0.49-2.7, P=0.73). High risk disease was the only significant factor for higher relapse rate. The OS and NRM at 4-year were 57% and 20% in non-GG, and 65% and 17% in GG genotype, respectively. The rs7588571 genotypes affected neither OS nor NRM.[Discussion] REG3A polymorphism was associated with the incidence of extensive chronic GVHD. Given its capacity to control the tissue regeneration and intestinal microbiota, REG3A may not only reflect the intestinal damage caused by GVHD but also have a certain role in the pathogenesis of GVHD. Our findings need to be confirmed in larger cohort, and further investigation is required in order to clarify the biological effect of rs7588571 genotypes and the exact role of REG3A in the pathophysiology of GVHD. DisclosuresKiyoi:MSD K.K.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Alexion Pharmaceuticals: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Chugai Pharmaceutical Co. LTD.: Research Funding.

Impact of genomic risk factors on survival after haematopoietic stem cell transplantation for patients with acute leukaemia.

The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N=458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2 –/–mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2 –/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising theNational Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across thelinear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Efficacy and Safety of Sofosbuvir-Based Anti-viral Therapies for the Treatment of Recurrent Hepatitis C Post Liver Transplantation: A “Real-Life” Single-Center Experience

Introduction: Recent introduction of Interferon free regimens with Direct Acting Antiviral agents have yielded excellent results in clinical trials for the treatment of HCV. However, data in the real world setting in post liver transplant patients is limited and focused primarily on HCV infections with genotype 1. We report the efficacy and safety of various Sofosbuvir-based antiviral therapies for the treatment of recurrent hepatitis C infection with different genotypes in liver transplant recipients. Methods: This is a Retrospective analysis of patients with recurrent HCV infection after liver transplantation treated with Sofosbuvir-based antiviral therapy. Primary endpoint was achievement of sustained virological response 12 weeks after the end of treatment (SVR-12). Results: 80 patients, transplanted between 2003 and 2016, were treated. Mean age was 58.4 years; and 62.5% of patients were male. Treatment combinations included Sofosbuvir (SOF), Simeprevir (SIM), Ribavirin (RBV) and Ledipasvir (LDV). Mean time from LT to treatment was 42.1 months. Duration of therapy was 12 weeks for 43.24% of the patients and 24 weeks for the remainder. Treatment was discontinued on two patients due to severe anemia on the SOB + RBV regimen. 4 patients were lost to follow up. Overall, SVR-12 was achieved in 91.9% of the patients who completed treatment. SVR-12 rates per treatment regimen were - SOF + SIM (97.06% n=37), SOF + RBV (80.95% n=24), SOF + RBV + SIM (100% n=1) and SOF + LDV (94.12% n=17). SVR-12 rates per genotype (GT) were - GT1a (95%), GT1b (88.24%), GT1a/1b (100%), GT2 (85.71%), GT3 (87.5%), GT4 (100%), GT1a/2 (100%) and GT1b/2 (100%).Figure 1Figure 2Figure 3Conclusion: To our knowledge, our study is one of the few studies that have comprehensively studied the safety and efficacy of different Sofosbuvir based treatment regimens for HCV infections in LT recipients with different genotypes in a “real-life” setting. Our findings for GT1 were comparable with the COSMOS clinical trials for non-LT patients. SVR-12 rates were lowest for patients on SOF+RIB regimen (81%) and highest for patients on SOF+SIM (+/-RIB) regimen (97-100%). SVR-12 rates were highest for HCV GT4 (100%) and lowest for GT2 (86%). In conclusion, Sofosbuvir-based anti-viral therapies are safe, effective and well tolerated for the treatment of HCV recurrence post transplantation. In our experience, the SOF + RBV regimen had a higher rate of treatment failure and increased risk of side effects requiring treatment discontinuation.

An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.

Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.

John Cunningham (JC) virus genotypes in kidney transplant recipients, rheumatoid arthritis patients and healthy individuals in Isfahan, Iran.

In healthy individuals John Cunningham virus is latent without any clinical signs, but in the cases of the use of immunosuppressive drugs in graft recipients, autoimmune diseases and also increasing of age, that the immune system is suppressed it may cause disease in reactivation. Progressive multifocal leukoencephalopathy (PML) is the well-known disease caused by the virus. It has also been associated with nephropathy and tumorogensis. At present, based on vp1 capsid gene 7 genotypes have been detected. Genetic variations of JC virus in different geographical areas and the presence of different subtypes is a useful tool for reconstructing of the genetic information of JC virus and understanding of its evolution. The aim of this study was to investigate different genotypes of the JC virus in the urine of 100 kidney transplant recipients, 43 rheumatoid arthritis patients, and 100 healthy individuals as control group in Isfahan. DNA was extracted by phenol-chloroform method and subjected to a nested PCR using specific primer for vp1 capsid gene designed by Oligo 7 software. Fisher's exact test was used for statistical analyses. Using MEGA 6 software the sequences were aligned using Clustal W tool and phylogenetic trees were constructed by neighbor joining method. Thirty-one positive samples were sequenced. Genotypes 1, 3, and 4 of the virus were detected for the first time in Iran. For the first time genotype 3 was reported as the dominant genotype in Iran. For the first time in the world, genotype 4 was detected in rheumatoid arthritis patients. J. Med. Virol. 89:337-344, 2017. © 2016 Wiley Periodicals, Inc.

Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation

AbstractThe ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1. HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Challenges in Determining Genotypes for Pharmacogenetics in Allogeneic Hematopoietic Cell Transplant Recipients

As part of a pharmacogenetic study, paired blood and oral fluid samples were tested for the IL28B polymorphism (rs12979860) before and after hematopoietic cell transplantation (HCT) to evaluate changes in the genotype and investigate the utility of genotyping in oral fluid in HCT recipients. In 54patients with leukemia >18 years of age, samples were collected approximately 7 days before HCT and 60 days after HCT. IL28B polymorphism testing was performed using real-time PCR with allele-specific probes. Twenty-four patients had the same genotype as their donors. In 30 patients, the genotype was different from that of the donor. In the oral fluid samples, 4 retained the recipient's genotype, and 18 had a genotype that matched that of the donor. In the remaining 8 patients, the results could not be characterized and appeared to be a combination of both, suggesting mixed proportions of donor and recipient cells. The assumption was that the sloughed epithelial cells of the mouth are of recipient origin. However, oral fluid is a mixture that contains varying numbers of cells of the recipient and immunomodulatory cells from the donor. Therefore, the use of oral fluid after HCT for clinical pharmacogenetics purposes needs further investigation.

Human leukocyte antigen G single-nucleotide polymorphism -201 (CC–CC) donor–recipient genotype matching as a predictor of severe cardiac allograft vasculopathy

In heart transplant recipients, human leukocyte antigen G (HLA-G) has been shown to inhibit endothelial and smooth muscle cells injury in vitro, suggesting protection against cardiac allograft vasculopathy (CAV). Although the expression of HLA-G is regulated by single-nucleotide polymorphisms (SNPs), their association with CAV remains unknown. Therefore, the objective of this study was to determine the association between recipient and donor HLA-G SNPs with CAV. We retrospectively analyzed DNA for HLA-G SNPs of 251 adult heart recipients, 196 of whom had their corresponding donors included. Severe CAV was defined as ISHLT Category 2 or 3. The association between donor-recipient genotypes and diagnosis of severe CAV over time was evaluated with parametric hazard regression models. Recipient age was 48 ± 12 years, whereas donor age was 35 ± 14 years. Median follow-up was 5.0 years (range 1 day to 13.2 years). At 10 years after transplantation, freedom from severe CAV, retransplantation or death was 64%. In multivariable analysis adjusted for donor age, recipient weight and pre-transplant Class II antibodies, the presence of donor-recipient SNP -201 (CC-CC) matching was associated with an increased risk of severe CAV (hazard ratio 11.9; 95% confidence interval 4.3 to 32.9; p < 0.001). Matching of donor-recipient SNP -201 (CC-CC) was an independent risk factor for the diagnosis of severe CAV. HLA-G SNP genotypes may reveal a pathogenic pathway to be explored for diagnostic and therapeutic strategies for CAV.

Effects of IL10-592 locus of AA genotype on the incidence of aGVHD and survival after HLA-matched unrelated allogeneic hematopoietic stem cell transplantation

目的研究供患者IL10基因-592 (rs1800872）单核苷酸多态性位点（SNP）不同基因型对HLA全相合无关供者异基因造血干细胞移植（allo-HSCT）预后的影响。方法104对HLA全相合供患者和100名健康人的DNA样本，均采用Sanger法基因测序技术检测IL10-592位点SNP，并结合临床资料分析不同基因型对allo-HSCT各预后因素的影响。结果当供患者IL10-592位点基因型相同且分别为AA/AA、AC/AC、CC/CC时，移植后Ⅲ~Ⅳ度急性移植物抗宿主病（aGVHD）发生率分别为47.1%、3.7％和0，三组间差异有统计学意义（P=0.002）。当供患者IL10-592位点基因型不同，且患者为AA或供者为AA基因型时，不同基因型组合Ⅲ~Ⅳ度aGVHD发生率差异均有统计学意义（P=0.046，P=0.041）。当患者IL10-592位点为AA、AC、CC基因型时，移植后Ⅲ~Ⅳ度aGVHD发生率分别为27.8%、10.2%、11.1%（P=0.072）；肠道aGVHD的发生率分别为22.2%、5.1%、11.1%（P=0.040）；2年总生存（OS）率分别为48.2%、75.1%、85.7%（P=0.002）；2年无病生存（DFS）率分别为48.5%、66.3%、76.2%（P=0.045）。当供者IL10-592位点为AA、AC、CC基因型时，患者Ⅲ~Ⅳ度aGVHD发生率分别为26.5%、8.9%、0（P=0.024）；肠道aGVHD发生率分别为20.4%、4.4%、0（P=0.026）。多因素分析结果提示患者或供者IL10-592位点AA基因型组移植后有较高Ⅲ~Ⅳ度aGVHD发病风险（OR=3.3，P=0.049；OR=3.9，P=0.043）。而患者或供者IL10-592位点为AA、AC、CC不同基因型时，对慢性GVHD发生率和复发率均无明显影响。结论在HLA-10/10全相合无关供者allo-HSCT中，患者和（或）供者IL10-592位点AA基因型是allo-HSCT后发生较高Ⅲ~Ⅳ度aGVHD和较低OS、DFS率的不利因素。