888 Abstract# A508 Abstract# A509 Abstract# A510

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on. 05/30/2018. 888. Abstract# A508. Dissecting a Subtle Endothelial Signal in Human Kidney Transplants. With Antibody-Mediated Rejection. J. Venner, P.
Abstract# A508

evolves in response to immune system pressure and the factors that determine whether EBV infection leads to disease. We characterized the genomic diversity of EBV by using a publically available dataset containing high throughput sequencing reads from EBV-infected cell lines. Following alignment of reads to the B95-8 reference genome, the GATK SNP calling module was used to determine sites that differed from the reference. This was followed by calculation of the average diversity (as quantified by Shannon entropy) over a sliding window across the genome. Overall, over 3,000 sites were found to be variable, 1400 of which were in protein-coding regions, and 796 were non-synonymous variants, representing a dS/dN ratio of 1.24. This suggests, as expected, that the EBV genome is under strong positive selection. The most diverse annotated genes as measured by SNP density were EBNA3c, LMP1, and EBNA3b, all genes involved in the latent phase of the viral lifecycle. This high level of diversity provides evidence consistent with tetramer-staining studies showing these genes are immunodominant and thus under strong selective pressure. At a larger scale, we identified several hotspots of diversity both within and outside of protein-coding regions that may be used in the future to track or elucidate the EBV family tree both within and between individuals. We are currently proceeding with sequencing transplant patient samples over time to analyze the effect the immune response has on viral diversity and connect variants with clinical outcomes.

Dissecting a Subtle Endothelial Signal in Human Kidney Transplants With Antibody-Mediated Rejection. J. Venner, P. Halloran. University of Alberta, Edmonton, Canada.

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We aimed to describe the molecular changes most associated with ABMR in comparison to other events – IFNG effects, inflammasome activation, innate immunity, and complement inhibitors. In a set of 403 human kidney transplant indication biopsies, we used microarrays to compare ABMR to all other biopsies, including T cell-mediated rejection, acute kidney injury, polyomavirus nephropathy, and glomerulonephritis. Transcripts previously identified by a molecular classifier for ABMR – eg DARC, CDH13, FGFBP2 (Sellares et al. Am J Transplant 13(4):2013) – had the greatest association with ABMR (Figure 1, p-values as low as 10-14). Other transcripts with moderate associations with ABMR (p-values >10-8) included: inflammasomerelated CASP1 and IL1B (which is activated by CASP1) and complement inhibitor transcripts CD55 (DAF) and CD59 (MAC-IP). Toll-like receptors 7/8/9; inflammasome-related IL18, CASP8 and CD95 (Fas); CD46 (complement regulatory protein); and the parenchyma (kidney solute carrier expression) changed little, unlike in TCMR.

Abstract# A510 Genetic Variations in a Sestrin2/Sestrin3/mTOR Axis and Development of New-Onset Diabetes After Transplantation. D. Vu,1,2,3,4 T. Shah,2,3,4,5 H. Manzoor,4 I. Hutchinson,1,5 N. Lemp,1,6 Y. Qazi,5 J. Cicciarelli,1,6 R. Naraghi,2,4 D. Min.2,3 1Mendez National Institute of Transplantation, Los Angeles; 2Saint Vincent Medical Center, Los Angeles; 3 Western Unveristy of Health Sciences, Pomona; 4Transplant Research Institute, Los Angeles; 5USC Keck School of Medicine, Los Angeles; 6 Viracor-IBT Laboratories, Los Angeles. Background: Genetic variations in Sestrin2/Sestrin3/ mTOR axis may cause obesity-associated metabolic syndrome, including lipid accumulation and insulin resistance, and then increase the individual’s risk of diabetes. Accordingly, we explored the association between single nucleotide polymorphisms (SNPs) of these genes and new-onset diabetes after transplantation (NODAT) in our Hispanic kidney transplant recipients. Methods: We genotyped 8 potential functional polymorphisms in Sestrin2, Sestrin3 and mTOR genes using the Taqman method in a study of 129 Hispanic RTRs with no evidence of pre-existing diabetes who developed NODAT and 186 controls with no history of diabetes. The Cox proportional hazard model was used to examine risk factor for NODAT. Nongenetic and genetic characteristics were included in the multivariate risk model. Results: We observed significant associations between NODAT and mTOR rs2295080 TT (OR=1.6, 95% CI =1.14-2.82, p=0.01), Sestrin2 rs580800 AA (OR=0.42, 95% CI =0.27-0.67, p=0.0002), and Sestrin3 rs684856 AA (OR=0.45, 95% CI=0.27-0.75, p=0.001). There was an interaction between the mTOR rs2295080 and Sestrin3 rs684856 and risk of NODAT (p interaction = 0.046). Of the non-genetic factors, use of tacrolimus, older age, and acute rejection were associated with increased risk for NODAT. Conclusion: Polymorphism in the Sestrin2/Sestrin3/ mTOR gene may confer certain protection or predisposition for NODAT

We analyzed the top 100 ABMR-associated transcripts (defined by the class comparison p-value) for overrepresentation in IPA Canonical Pathways: top pathways (p-values around 10-15) were associated with antigen presentation and contained HLA molecules (eg HLA-A/B/C, -DQ); and when the transcripts used in these pathways were purged and we and re-analyzed the remaining ABMR-associated transcripts – Step 2, Figure 2 – top pathways now were leukocyte Adhesion and Diapedesis (p-values around 10-9). Subsequent removal of these transcripts (eg CDH5, SELE, CX3CL1) left relatively weak associations (p>10-4) such as Nitric Oxide, Angiopoietin, and NK Cell Signaling – Step 3, Figure 2. ABMR-associated transcripts in these pathways included endothelial expressed GNG11, VEGFC, CAV1, TEK (angiopoeitin receptor) and NK expressed FCGR3A and CD94. Thus the transcripts most strongly associated with the ABMR phenotype reveal the endothelial injury, IFNG effects, and NK transcripts, but the more moderate association also suggest some of the molecular events in the microcirculation response to injury, including nitric oxide, angiopoietin, VEGF, caveolin, and complement inhibitors CD55 and CD59. DISCLOSURES: Halloran, P.: Speaker’s Bureau, Astellas, Novartis, OneLamba.

Abstract# A511 Effect of the New Functional CYP3A4*22, CYP3A5 and ABCB1 Polymorphisms On the Tacrolimus Concentrations in Hispanic Kidney Transplant Recipients. D. Vu,1,2,3,4 H. Qwak,5 P. Sakharkar,1 J. Kim,3 T. Shah,1,2,4 I. Hutchinson,1 R. Naraghi,2,4 D. Min.1,2 1Western Univesity of Health Sciences, Pomona; 2Saint Vincent Medical Center, Los Angeles; 3Mendez National Institute of Transplantation, Los Angeles; 4 Transplant Research Institute, Los Angeles; 5Ewah Women’s University, Seoul, Korea, Republic of. Background: Tacrolimus is a substrate of metabolic enzyme cytochrome P450 3A, and a cell membrane transporter, ABCB1 gene product, P-glycoprotein (P-gp). This study aims to investigate the effect of the new functional CYP3A4*22, CYP3A5 and ABCB1 polymorphisms on Tac trough concentrations in renal transplant recipients (RTRs). Methods: We investigated the impact of the CYP3A4*22 (intron 6 C>T), CYP3A5*3 6986 G>A, ABCB1 C1236>T and ABCB1 C3435>T polymorphisms on Tac pharmacokinetics in 229 Hispanic RTRs at months 1, 3, and 6 post transplantation. Trough blood levels ([Tac](0) in ng/ml), dose-adjusted [Tac](0) (ng/ml per mg/ kg bodyweight) as well as doses (mg/kg bodyweight) required to achieve target concentrations were compared among patients according to allelic status for CYP3A4*22, CYP3A5 and ABCB1. Results: Frequencies of variant alleles among the RTRs were CYP3A5*3, 82.3%; ABCB1 1236T, 54% and ABCB1 3435T, 56.2%. The CYP3A4*22 variant allele was observed in only 17 (6.6%) patients. The overall mean daily-dose requirement to reach the same predose Tac blood concentration was 29% lower for carriers of the CYP3A4*22 T variant allele than for CC patients (95%CI, -43% to 18%; p=0.02). When CYP3A4/CYP3A5 genotypes were combined, the difference was even more

Abstract# A509 High-Throughput Sequencing of EBV Genomes Reveals Evidence for Strong Positive Selection Pressure From the Host Immune System. S. Schaffert, S. Krams, C. Esquivel, O. Martinez. Surgery/Division of Abdominal Transplantation, Stanford University School of Medicine, Stanford, CA. Around 90% of the adult population worldwide is Epstein-Barr Virus (EBV) seropositive and in most cases infection is controlled by host immunity. However, EBV is also linked to a number of malignancies including post-transplant lymphoproliferative disorder. Despite the prevalence of this virus, little is known about its genome-wide diversity, which could provide key insights into how the virus

888 © The Authors. Compilation © The American Society of Transplant Surgeons, The Transplantation Society and the American Society of Transplantation

striking as the so-defined CYP3A poor metabolizer group presented dose-adjusted concentration 1.4- and 3.9-fold higher for Tac than the intermediate metabolizer and extensive metabolizer groups, respectively. Renal function, assessed by calculation of SCr (mg/dL) was not statistically significant between CYP3A4*22 T allele carriers at 1,3,6 months of follow-up compared with wild-type patients. Higher Tac trough blood concentrations were observed in the homozygous CYP3A5*3 allele and homozygous ABCB1 C1236>T TT genotype. Conclusions: The CYP3A4*22 (intron 6 C>T) polymorphism is associated with a significant alter Tac metabolism. Analysis of CYP3A4*22 intron 6 C>T along with CYP3A5*3 and ABCB1 just before transplantation may help to identifying patients at risk of Tac overexposure.

serologic status D+/R-, acute rejection and thymoglobulin induction. The allelic as well as genotypic frequencies of NF-kB SNPs did not significantly differ between CMV infection and non-CMV group. Conclusion: These results indicated that the NF-kB activation pathway might be associated with the CMV infection.

Abstract# A514 Peritubular Capillary Vessels and Hypoxia/Angiogenesis Genes in Kidney Biopsies With Transplant Glomerulopathy. A. Konvalinka,1 R. John,2 P. Boutros,3 J. Kim,1 S. Yang,1 J. Scholey,1 A. Herzenberg,2 H. Reich.1 1Medicine, Division of Nephrology, Toronto General Hospital, University of Toronto, Toronto, ON, Canada; 2Pathology, Toronto General Hospital, University of Toronto, Toronto, ON, Canada; 3Informatics and Biocomputing Platform, Ontario Institute for Cancer Research, Toronto, ON, Canada.

Abstract# A512 Application of Second Generation Sequencing to the Organ Transplantation Arena. B. Keating,1 Y. Li,1 K. Olthoff,2 J. Christie,2 J. Wang,3 A. Shaked.2 1Center for Applied Genomics, The Children’s Hospital of Philadelphia, Philadelphia, PA; 2Department of Surgery (Division of Transplantation), University of Pennsylvania, Philadelphia, PA; 3Genomics, BGI, Shenzhen, Yantian District, China.

BACKGROUND: Transplant glomerulopathy (TG) is an important cause of late renal allograft loss. TG has been linked to the presence of alloantibodies, chronic rejection, and persistent inflammation, however the mechanisms responsible for TG are incompletely understood. Peritubular capillary (PTC) loss and hypoxia contribute to pathogenesis of chronic rejection in experimental lung transplantation. In these models HIF1A gene upregulation in response to hypoxia contributes to microvascular repair. A role for PTC loss and hypoxia-related gene expression has not been examined in TG. While persistent inflammation is implicated in TG-mediated graft injury it is not known whether this reflects downstream effects of PTC loss. To study the role of PTC loss in the pathogenesis of TG we quantified PTC density, and evaluated expression of hypoxia-associated and inflammatory pathways in kidney biopsies of patients with TG compared to acute antibody-mediated rejection (AMR) and interstitial fibrosis/tubular atrophy not otherwise specified (IFTA NOS). METHODS: We utilized custom Taqman Low Density Arrays (TLDA) designed to measure expression of 48 genes in pathways of interest in archived kidney biopsies for cause. Formalin-fixed paraffin embedded sections were stained with antibody to CD31, an endothelial cell marker for detection of microvessel density. PTC leukocytes were immunophenotyped in TG cases. RESULTS: Among 19 cases of TG, 16 of IFTA NOS, and 10 of AMR, we found significant differences in the expression of VCAM1, CCL5, CCL2 and IFNg in TG compared to AMR and IFTA NOS. CD68 (monocyte marker) was most elevated in AMR, and the lowest in IFTA NOS. Hypoxia/angiogenesis genes (HIF1A, VEGFA and EDN1) were increased in AMR with no significant difference between TG and IFTA NOS. Peritubular capillary density was comparable in TG and other groups. The majority of PTC leukocytes in TG were macrophages and T cells. CONCLUSIONS: Inflammation is a prominent finding in TG biopsies, and macrophages and T cells appear to be important mediators. We found no evidence of prominent PTC drop out or hypoxia responses in TG. Neither PTC drop out nor hypoxia appear important in established TG.

Over the last two decades, over 300,000 solid organ transplantations have been performed in the USA alone. While there has been significant progress with immunosuppression therapies there is still significant risk of graft organ rejection, even when appropriate HLA matching has been performed. Transplant rejections from maledonor to male-recipient and females to females are much lower than male to female. Cross-ethnic rejection rates are also higher, suggesting that additional genetic factors are at play in the rejection process. Possible sources of genetic variation underpinning rejection are homozygous deletion copy number variants (CNVs) spanning whole gene or exon regions and Loss of Function (LoF) variants ablating two copies of a given gene, resulting in incompatibility across the proteomes of donor and recipient. It has been shown through large-scale whole genome sequencing studies that the average human has 20 genes disrupted (i.e. with LoF) in two copies. We have analyzed CNV data from over 68,000 individuals in CHOP subjected to genome-wide association studies (GWAS), mostly using 610,000 SNPs arrays, as well as published GWAS and second generation sequencing (SGS) datasets. We designed 6.5MegaBase (Mb) of targeted capture DNA for over 300 hdCNVs, and non-exonic PGx content, and spiked it into the 64Mb EZ v3.0 whole exome capture product (Roche). In total, DNA from 562 tissue/blood samples from transplant donors and recipients (lung, liver, heart and kidney) within the Clinical Trials in Organ Transplantation 3 study (CTOT3) were subjected to the 70.5Mb capture tool followed by SGS on HiSeq2000 (Illumina). We compare hdCNV and LoF carriages across the entire donor pool compare with the entire recipient pool in order to assess inherent differences between these two populations and demonstrate over 3 dozen putative candidate allogenic targets.

Abstract# A513 Impact of Inhibitor kappa B-alpha (IkB-α) Promoter Polymorphisms On the Risk of Cytomegalovirus Infection Among Hispanic Renal Transplant Recipients. D. Vu,1,2,3,4 T. Shah,2,3,4 Y. Chiang,5 I. Hutchinson,1 R. Naraghi,2,4 D. Min.2,3 1Mendez National Institute of Transplantation, Los Angeles; 2Saint Vincent Medical Center, Los Angeles; 3Western University of Health Sciences, Pomona; 4Transplant Research Institute, Los Angeles; 5 School of Pharmacy, Taipei Medical University, Taipei, Taiwan.

Abstract# A515

Background: Cytomegalovirus (CMV) is one of the most common cause of viral infection, causing morbidity and mortality among kidney transplant recipients (RTRs). The dimeric NF-kB 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 and immune responses. Inhibitors of NF-kB, known as inhibitor kappa B-alpha (IkB-α), block NF-kB transcriptional activity by forming stable IkB-α:NFkB complexes. The aim was to investigate the association of gene polymorphisms in the NF-kB and IkB pathway with CMV infection in RTRs. Methods: IkB-α promoter polymorphisms at position -881 (A/G), -826 (C/T), and 297 (C/T) were studied in 247 RTRs (52 RTRs with CMV infection and 195 without CMV infection), using DNA-based polymerase chain reaction with sequence-specific primers and restriction. In the case of NF-kB genotypes, the following single nucleotide polymorphisms (SNPs) were included: NF-kB1 (rs3774959, rs3774932, rs3774937, rs230526, rs230519), NF-kB2 (rs1056890, rs7897947, rs12769316) and NF-kB inducing kinase (NIK) (rs9908330, rs7222094). Results: Median time to CMV infection was 6 months with a mean peak CMV viral load of 7925 copies per ml. Patients with donor-positive/recipient-negative [D+/R-] serostatus were found to be associated with a high risk of CMV infection (p=0.001). A statistically significant correlation was found between IkB-α -297T allele polymorphism and the risk of CMV. Three common haplotypes were found, of which haplotype 2 (GTT) was significantly associated with an increased risk for CMV infected group as compared to non-CMV group (p=0.01). The association was independently significant in multiple logistic regression (p=0.02) along with

The International HapMap Projectlaid down the foundation to assess the diversity of genetic variation amongst the major human populations, and led to the development of Genome-wide genotyping arrays, typically using 300K to over a million SNPs. These have been a tremendous success, unveiling several thousand associations of SNPs with diseases and traits across the human genome. We designed a low-cost genome-wide SNP array specifically tailored for the transplantation community. It comprised 782,200 markers including: 1. exonic, and Loss of Function (LoF) content (n= 274K variants) from a recent updated of all available exome/LoF content from >40,000 individuals; 2. Genome-wide markers (n=463K SNPs) with all available European ancestry population datasets from the 1000 genome project with supplementary content from ~90K African ancestry panels SNPs to boost GWAS coverage in this ancestry. Furthermore we have added significant content (~55K SNPs) for UTRs; 3. Pharmacogenomic markers (7.5K SNPs) from PharmaADME. org, PharmaGKB, and from literature searching for serious adverse events and PGx related to immunosuppression therapies; 4. Copy number Variants (16.4K probes) from the Wellcome Trust and UKbiobank (n=2.4K) and analysis of 68,000 GWAS samples from CHOP; 5. Expression QTL markers (17.4 K SNPs) from NCBI/NIH GTEx eQTL database and 6. Deeper and more updated HLA, KIR, MHC, CKD, Cardiovascular & metabolic content for NODAT & Cardiac allograft vasculopathy (CAV) as well as a collation of SNPs from >600 PubMed manuscripts. We have run this array on over 21,000 individuals from a range of studies, including DeKAF, CTOTs and large European cohorts and we present the initial findings for LoF, CNV and SNPs across this dataset.

Concept, Design and Implementation of a Sedicated Genome-Wide Transplant SNP Array With > 780,000 Markers. B. Keating,1 N. Nair,1 M. Holmes,1 W. Oetting,2 P. Jacobson,2 A. Israni,3 A. Shaked.1 1Surgery (Division of Transplantation), Univ of Pennsylvania, Philadelphia, PA; 2 College of Pharmacy, University of Minnesota, Minneapolis, MN; 3 Epidemiology, University of Minnesota, Minneapolis, MN.

889 © The Authors. Compilation © The American Society of Transplant Surgeons, The Transplantation Society and the American Society of Transplantation