Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

........................................................................................ 9 INTRODUCTION ................................................................................. 11 REVIEW OF THE LITERATURE .............................................................. 12 1 KIDNEY TRANSPLANTATION .............................................................. 12 1.1 General aspects of kidney transplantation ..................................... 12 1.2 Genetic aspects of end stage renal disease .................................... 13 1.3 Human leukocyte antigen ........................................................... 14 1.3.1 Basic features of human leukocyte antigen biology ................ 14 1.3.1.1 Immunogenetics of the human leukocyte antigen system ................................................................. 14 1.3.1.2 Antigen processing and presentation ........................ 17 1.3.1.3 Human leukocyte antigen and T cell selection ............ 18 1.3.1.4 Interaction between specifi c human leukocyte antigen molecules and peptides........................................... 19 1.3.2 Clinical histocompatibility and human leukocyte antigen matching ......................................................................... 19 1.4 Complications in kidney transplantation ........................................ 20 1.4.1 Acute rejection ................................................................. 21 1.4.1.1 Immune systems involved in rejection ...................... 22 1.4.1.2 Allorecognition ...................................................... 23 1.4.1.3 Tolerance .............................................................. 24 1.4.2 Delayed graft function ........................................................ 24 1.4.3 Cytomegalovirus infection .................................................. 25 1.4.4 Vascular complications ....................................................... 25 1.5 Genes associated with acute rejection, delayed graft function, cytomegalovirus and vascular complications ........................................ 26 1.5.1 Cytokine genes ................................................................. 27 1.5.1.1 Tumor necrosis factor ............................................. 29 1.5.1.2 Transforming growth factor beta 1 ........................... 29 1.5.1.3 Interleukin 10 ....................................................... 29 1.5.1.4 Interleukin 6 ......................................................... 30 1.5.1.5 Interferon gamma .................................................. 30 1.5.2 Thrombosis-associated genes.............................................. 31 1.5.2.1 Coagulation factor V ............................................... 31 1.5.2.2 Coagulation factor II .............................................. 31 1.5.2.3 Methylenetetrahydrofolate reductase ........................ 32 1.5.2.4 Protein C, tissue factor pathway inhibitor, coagulation factor XIII ............................................................. 32 1.5.3 Other genes studied in renal transplantation ......................... 34 1.6 Methods and biomarkers studied by these methods for diagnosing acute rejection .......................................................................... 34 1.6.1 Histology .................................................................................. 35 1.6.2 Enzyme-linked immunoabsorbent assay ........................................ 36 1.6.3 Fluorescence activated cell sorting ............................................... 37 1.6.4 Mass spectrometry ............................................................ 37 1.6.5 TaqMan real-time quantitative polymerase chain reaction ....... 38 1.6.6 Microarray ........................................................................ 38 2 AIMS OF THE STUDY ........................................................................ 42 3 PATIENTS ....................................................................................... 43 4 ETHICAL PERMITS ........................................................................... 43 5 METHODS ...................................................................................... 44 5.1 Isolation of DNA (I, II, IV) .......................................................... 44 5.2 Human leukocyte antigen typing (I) ............................................. 44 5.3 Determination of single nucleotide polymorphisms (I, II, IV) ........... 44 5.4 Isolation, quantifi cation and validation of RNA from whole blood (III, V) ..................................................................................... 45 5.5 Real-time quantitative polymerase chain reaction (III, V) ................ 45 5.6 Low-density array (V) ................................................................ 47 5.7 Microarray (V) ........................................................................... 47 5.8 Statistical analyses (I–V) ............................................................ 48 6 RESULTS AND DISCUSSION .............................................................. 49 6.1 Acute rejection (I, IV) ................................................................ 49 6.1.1 Only a few gene polymorphisms were associated with acute rejection (I)...................................................................... 50 6.1.2 Genetic variation in thrombosis-associated or cytokine genes did not associate with AR (IV) ............................................. 52 6.2 Genetic variation in interleukin 10 but not in other cytokine genes predisposed to CMV infection (II) ................................................. 53 6.3 Genetic variation in thrombosis-associated or cytokine gene polymorphism did not predispose to thrombosis (IV) ............................ 56 6.3.1 Thrombosis-associated or cytokine gene polymorphisms did not predispose to infarction (IV) .......................................... 57 6.3.2 Thrombosis-associated or cytokine gene polymorphisms were not associated with graft survival ........................................ 58 6.4 Gene expression markers showed similar time-related variance during hospital stay in control patients (III) ................................... 59 6.5 The expression of two genes was different in rejection patients than in control patients (III) ............................................................... 61 6.6 Rejection patients differed from non-rejection patients with regard to one gene expression marker (III) ................................................. 63 6.7 Candidate gene expression markers did not differ between patients with subclinical rejection and control patients (V) ........................... 64 6.8 Gene expression markers of the low-density array were unable to identify patients with subclinical rejection (III) ............................... 65 6.9 A robust biomarker specifi c for subclinical rejection could not be identifi ed in whole genome microarray analysis (III) ....................... 66 STRATEGIES FOR THE FUTURE ............................................................. 68 CONCLUSIONS ................................................................................... 69 ACKNOWLEDGEMENTS ........................................................................ 70 REFERENCES ..................................................................................... 71 ORIGINAL PUBLICATIONS .................................................................... 95