Program Overview

National Institute of Allergy and Infectious Diseases

Grant Number: 1U19AI063603-01

Genomics for Kidney Transplantation Project Description (Dr. Daniel Salomon, Principal Investigator)

The theme of the Transplant Genomics Collaborative Group Program Project is to test the application of developing technologies for gene expression profiling (Project 1), proteomics (Project 2) and complex trait genetics (Project 3) to advance our understanding of kidney transplantation in the context of both its clinical problems and the basic biology of transplantation. Our view is that this is a systems biology approach to kidney transplantation. These efforts are supported by a group of collaborators with expertise in all three scientific areas as well as expertise in clinical kidney transplantation and bioinformatics and statistics. The kind of data we will generate will be of two kinds: diagnostic and discovery. The diagnostic component will consist of potentially complex gene and protein expression signatures that will have statistically valid correlations to specific clinical events like acute rejection, chronic allograft nephropathy and long term well-functioning transplants with no rejection. The discovery component will consist of identifying within these complex genomic signatures, specific gene candidates that correlate with transplant events and outcomes. Our hypotheses are: 1) that gene and protein expression signatures can be identified in PBL and kidney transplant biopsies that correlate with biopsy-proven acute rejection and chronic allograft nephropathy, 2) that gene and protein expression profiles provide insights into the molecular pathways involved in both the host immune response and the donor organ's response to transplantation, and 3) that the adequacy of immunosuppression, as defined by the absence of rejection-related gene and protein expression, can be determined by the gene and protein expression profiles. We also propose to examine the possibility that race and sex will influence at least a subset of gene transcripts and proteins expressed post transplant and correlate with outcomes. The first objective of this application will be to integrate data on gene expression profiling in parallel with data generated by proteomics (Project 2) and complex trait genetics (Project 3) to advance a more comprehensive understanding of the molecular basis of clinical kidney transplantation. The second is to establish the requisite proof of principle to design a prospective clinical trial to test the hypothesis that PBL profiles can be used to monitor the efficacy of immunosuppression in 'real-time'.

Project 1: Differential Gene Expression (Dr. Daniel Salomon, Project Leader; Steve Head, Director, TSRI DNA Array Core Facility)

Despite recent reductions in the incidence of acute rejection, chronic allograft nephropathy and immunosuppressive drug side effects are still major causes of graft loss and patient morbidity. In this context, it is essential to advance our understanding of the immune response and the responses of the transplanted organ to both immune and non-immune injury mechanisms. Analysis of gene expression is one means to identify molecular pathways involved in transplantation biology. The objective of Project 1 is to use high-density microarray technology to measure differential gene expression in kidney biopsies and peripheral blood of patients with well-defined phenotypes: acute rejection, chronic allograft nephropathy and patients biopsied by protocol at 12 to 24 months with well-functioning kidney transplants. Our preliminary data based on studies of almost 100 subjects demonstrates characteristic gene expression profiles in biopsies and peripheral blood lymphocytes that correlate with transplant immunosuppression as well as with acute rejection. Using statistical analysis of individual gene expression signals followed by hierarchical clustering and class prediction analysis we can demonstrate significant differences in the gene profiles of biopsies and PBL that define these clinical groups. Our first Specific Aim is to determine the gene expression profiles in transplant biopsies and PBL of patients in 3 well-established clinical classes. Successful completion of this Aim will create a comprehensive, statistically robust data set for gene expression profiles of both transplant biopsies and PBL including many presently unknown genes that can be correlated with clinical events following kidney transplantation. Our second Specific Aim is to establish the patterns for individual gene expression correlated with clinical events and outcomes. Successful completion of this Aim will define candidate gene sets that can be integrated with the parallel studies of proteomics (Project 2) and complex trait genetics (Project 3). We will use in silica tools to identify gene function and connections that reveal novel pathways in transplant biology particularly the response of the donor kidney to transplantation in the context of tissue injury, repair and chronic allograft nephropathy.

Project 2: Proteomics of Kidney Transplantation (Dr. John Yates, Project Leader)

Kidney transplantation has extended and improved the quality of life for many patients with end stage renal disease. Most transplants involve genetically non-identical donor to recipient combinations. As a consequence the immune response is a major impediment to successful graft survival, necessitating lifelong treatment with potent immunosuppressive drugs. The donor organ and its complex responses to transplantation is also a major player in all the events determining the clinical events post transplant. Despite recent reductions in the incidence of acute rejection, chronic rejection and immunosuppressive drug side effects are still major causes of graft loss and patient morbidity. In this context, it is essential to advance our understanding of the immune response and the responses of the transplanted organ to both immune and non-immune injury mechanisms. We will use quantitative shotgun proteomics based on liquid chromatography tandem mass spectrometery to identify protein expression changes in transplanted kidneys and peripheral blood lymphocytes. Proteins will be identified and their expression levels measured relative to samples from patients with well-functioning transplants and no history of rejection. The objectives will be to identify regulatory processes activated or suppressed as a result of the immune response and identify cellular pathways associated with immunity, cell injury and tissue repair. Proteomics data will be integrated with gene expression measurements to gain a more complete view of the rejection process for both protein and gene transcript expression as well as identify and rank 500 gene candidates for complex trait genetics by SNP analysis in Project 3.

Project 3: Genetics of Kidney Transplantation (Dr. Pui-Yan Kwok, UCSF, Project Leader; Dr. Janet Warrington, Affymetrix)

In this project,we propose to study kidney transplant patients with three possible outcomes (acute rejection, chronic allograft nephropathy, and long-term normal functioning transplants). Our objective is to identify the genetic factors associated with these clinically distinct outcomes. Our first thesis is that understanding the genetic and molecular factors associated with the success or failure in renal transplantation will lead to personalized immunosuppressive therapy that increases the success rate of transplantation while decreasing drug-related morbidity and mortality. Our second thesis is that by understanding the genetic elements of donor organ responses to transplantation that determine clinical outcomes, we can identify a whole new approach to discovering new pathways and drug targets to enhance transplantation success and safety. For each of the three clinically distinct transplant phenotypes, we will identify genetic variations in the 500 gene candidates identified in Projects 1 and 2 and look for genetic association of single nucleotide polymorphism (SNP) markers in these genes. A unique feature of our proposal is that differentially expressed genes in the donor organ will also be pursued with donor DNA. In collaboration with Affymetrix, we will take advantage of two powerful genetic tools to increase the speed of discovery while keeping the cost low. We will also leverage the knowledge gained from the International Haplotype Map Project and take advantage of a pooling strategy for SNP genotyping.

The specific aims of this project are:

1. Screen gene candidates implicated by their differential gene or proteome expression profiles between cases and controls for genetic association by comprehensive DNA resequencing with high-density oligonucleotide microarrays.
2. Screen gene candidates using "haplotype-tag SNPs" (htSNPs) from the HapMap Project for genetic association using a pooled SNP genotyping approach followed by individual SNP genotyping of associated SNPs.
3. Conduct whole-genome screening using a set of high-density SNP microarrays for genetic association.
4. Validate the genetic association by testing a second cohort of donors and recipients obtained prospectively and archived during the first four years of the program project.

Clinical Transplant Center Core A (Dr. Christopher Marsh, Co-Director; Dr. Stuart Flechner, Co-Director)

The Clinical Transplant Center Core will be comprised of 10 major academic transplant centers in the United States doing approximately 1360 adult kidney transplants per year in total. Its activities will be integrated with the three Scientific Projects through the Clinical Database, the Program Project website, yearly scientific meetings and active interim interactions between the 10 Clinical Investigators and the Principal Investigators. Our focused objectives for this Core are to provide the Projects with the requisite patient materials and correlating clinical data to test the hypotheses that: 1) gene expression and proteomic signatures in PBL and kidney transplant biopsies can be identified that correlate with biopsy-proven acute rejection and chronic allograft nephropathy, 2) gene and protein expression profiles will provide insights into the molecular pathways involved in both the host immune response and the donor organ's response to transplantation, 3) the adequacy of immunosuppression as defined by the absence of rejection-related gene and protein expression can be determined at any given time by gene and protein expression profiles, and 4) targeted SNP genotyping and candidate gene sequence studies will identify connections between the genetics of the donor and the recipients that correlate with transplant events and outcomes as well as with gene and protein expression signatures. Arching over all these hypotheses, we propose to examine the possibility that race (Caucasian, African American and Hispanic) and/or sex will genetically influence at least a subset of genes and proteins expressed post transplant and correlate with clinical outcomes. A critical point is that the correct clinical designations of the patient subgroups and successful completion of enrollment and specimen acquisitions, the two primary tasks for this Core, will determine the potential of success for all the scientists in the entire Program Project.

Administrative Core B (Dr. Daniel Salomon, Co-Director; Dr. Caroline Lanigan, Co-Director)

The objectiveof the Administrative Core is to coordinate all the interactions of the Program Project's various components internally and externally. In practice the Administrative Core will be the working center of the Program Project bridging the many staff of the Clinical Centers with the staff of the scientific cores and managing all the regulatory and administrative duties of a large, multi-project endeavor. Its primary goals will be to:

1. Manage the administrative duties so efficiently that the Principal Investigator can concentrate on developing the science proposed,
2. Support the three scientific projects and their staff to optimize their productivity,
3. Create the constructive working relationships at the administrative level that are so critical for a Program Project to function successfully,
4. Support the Clinical Center Investigators and the Study Coordinators so that the critical task of patient enrollment, specimen acquisition, shipping and follow-up is facilitated and the work of the Study Coordinators at the Centers is efficient and productive,
5. Manage the complex sample and data generation from the Centers, the Clinical Database, the movement of materials from Scripps to UC, San Francisco for Project 3, support the two scientific projects at Scripps so that the integrity of the data set created is insured to enable the critical task of finding the correlations between genomic profiles and clinical events and outcomes in kidney transplantation,
6. Insure the data is made accessible to the public at the Program Project web site to facilitate outside analysis and advance the entire Field's efforts to understand the biology of transplantation.

Bioinformatics and Statistics Core C (Dr. Steve Horvath, Director)

The objective of this program project is to integrate three different genomic disciplines to advance our working understanding of clinical kidney transplantation. Patients with acute rejection and chronic allograft nephropathy will be compared to patients without rejection and long term, well-functioning transplants. Specifically, we propose to use both kidney transplant biopsies and peripheral blood lymphocytes. Gene expression profiling will be done using Affymetrix oligonucleotide-based microarrays (Project 1). Proteomics will involve the use of liquid chromatography coupled with linear ion trap mass spectrometry (Project 2). Gene candidates identified by data generated with these two technologies will comprise a set of 500 genes for which we will perform complex trait SNP genetic analysis (Project 3). The Bioinformatics and Statistics Core will provide the bioinformatics and statistical support for all projects. All data will flow to the Core for both advanced analysis and archiving. At the first level this support will include monitoring experimental designs for statistical integrity, organizing complex data sets generated in each Project so that they are more accessible to the Principal Investigators for interpretation and discovery and perform data mining using bioinformatic tools. Clinical data entered into the web site at Scripps will also be integrated with these efforts in a real-time fashion. At the second level the Core will supervise the selection and statistical validation of the 500 gene candidate set based on gene expression and proteomic data intended for complex trait genetics in Project 3. We will use supervised and unsupervised data mining methods such as clustering and class prediction tools. We will also identify differentially expressed genes and proteins in the different kidney transplant group comparisons. At the third level the Core will integrate data generated in all three Projects so that connections, pathways and mechanisms can be recognized, defined and validated including possibly novel relationships between patient and donor genetics.

Program Overview/Executive Summary

The Clinical Centers will provide samples representing three well-defined classes of patients:

• Group 1: Biopsy-proven Acute Rejection (AR; n=100)
• Group 2: Biopsy-proven Chronic Allograft Nephropathy (CAN; n=100)
• Group 3: Protocol biopsies between 12 and 24 months post transplantation with normal, stable transplant function (TX; n=100)

All the patients in Group 1 and Group 2 will have additional PBL sampled at 6 and 12 months following the entry biopsies and undergo a second biopsy by protocol 12 months later.

Group 3 patients are the control group. They will be 12 to 24 months post transplant with no history of rejection. A key classifier will be a biopsy showing no evidence of AR, CAN or other nephropathies. These patients will have serum creatinines < 1.5 mg/dl for women, < 1.6 mg/dl for men that are stable for at least three months based on three or more measurements, calculated or measured creatinine clearances of > 45 ml/minute and well controlled blood pressures ( < 130/80). We will exclude patients with diabetes (Type I or II) and any patients that were biopsied at any time post transplant for acute graft dysfunction regardless of the biopsy histology.

Within each class we will enroll 50 Caucasians, 25 African-Americans and 25 Hispanics and approximately equal numbers of males and females in all groups.