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From the Institute for Transfusion Medicine at the University of Ulm Medical Director Prof. Dr. med. Hubert Schrezenmeier

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1 From the Institute for Transfusion Medicine at the University of Ulm Medical Director Prof. Dr. med. Hubert Schrezenmeier Relevance of smica and MICA polymorphisms as biomarkers in inflammatory diseases: Example hemophagocytic lymphohistiocytosis Dissertation for obtaining a doctorate in medicine from the Medical Faculty of Ulm University presented by Sarah Crispina Gansky from Marbach am Neckar 2014

2 Acting Dean: Prof. Dr. Thomas Wirth 1st reporter: Prof. Dr. Hubert Schrezenmeier 2nd reporter: Prof. Dr. Marion Schneider Promotion Day: January 14, 2016 II

3 Table of Contents Table of Contents ... III List of Abbreviations ... V 1 Introduction Hemophagocytic Lymphohistiocytosis Diagnostic Criteria Pathogenesis The Macrophage Activation Syndrome Therapy Biomarker The MICA Antigen System Structure and Function Polymorphisms Issues and Objectives Material and Methods Structure of the Study Patient Collective and Control Groups Course of the Study Material Chemicals Devices and laboratory materials Software Methods Luminex Bead Array DNA sequencing Assignment of MICA polymorphisms ... 31 III

4 2.3.4 Statistical evaluation results Biomarkers of HLH smica Correlations of the biomarkers MICA genotype Distribution of MICA alleles MICA polymorphisms Multivariate analysis Discussion Methodology Study structure and statistics Experimental investigations Relevance of smica as biomarker Relevance of MICA polymorphisms Distribution of MICA genotypes Disease association of MICA polymorphisms Conclusion and outlook Summary Bibliography ... 91 Appendix Acknowledgments Curriculum Vitae IV

5 List of Abbreviations A Adenine ADAM a disintegrin and metalloproteinase ALL acute lymphoblastic leukemia Aqua dest. aqua destillata ASP ammonium sulfate buffer AST aspartate aminotransferase BSA bovine serum albumin or respectively C cytosine CD cluster of differentiation DNA deoxyribonucleic acid dntp deoxynucleotide triphosphate ddntp dideoxynucleotide triphosphate E exon EBV Epstein-Barrethyl-Virus EDC 1-ethyl amine [3-ethylamine] hydrochloride EDTA ethylene diamine tetraacetic acid ELISA enzyme-linked immunosorbent assay et al. and other companies. Company g mean acceleration due to gravity G guanine HCC hepatocellular carcinoma HIV human immunodeficiency virus HLA human leucocyte antigen HLH hemophagocytic lymphohistiocytosis HPLC high performance liquid chromatography I intron Ig G immunoglobulin G IL-1 interleukin 1 IL-1ß V interleukin 1

6 IL-2 interleukin 2 IvIG intravenous immunoglobulin KD l LADA LE LDH LL-37 dissociation constant liters late onset autoimmunity diabetes in the adult low electroendosmosis lactate dehydrogenase cathelicidin LL-37 m² square meter MAS macrophage activation syndrome MCMC Markov chain Monte Carlo mg milligram MHC major histocomplexity class I-related chain A min minute ml milliliter mm millimolar MM Mastermix NGS next generation sequencing NHS N-hydroxysulfosuccinimide NKG2D natural killer group 2 member D NK cell natural killer cell No. Number PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PCR polymerase chain reaction PE Phycoerythrin pg picogram ph potential hydrogen SAPE Streptavidin Phycoerythrin scd25 soluble CD25, soluble interleukin-2 receptor SDS sodium dodecyl sulphate VI

7 sjia systemic juvenile idiopathic arthritis smica soluble MICA T thymine TBE tris-borate-EDTA TNF-α tumor necrosis factor alpha tris tris (hydroxymethyl) aminomethane U revolutions U Unit WHO world health organization CNS central nervous system C degree Celcius% percent µg micrograms VII

8 1 Introduction 1 Introduction In order to classify an inflammatory disease and then treat it, specific biomarkers are important. This work shows the connection between the biomarker MICA and the disease hemophagocytic lymphohistiocytosis. 1.1 Hemophagocytic lymphohistiocytosis Hemophagocytic lymphohistiocytosis is a life-threatening disease. A strong - but at the same time uncontrolled - activation of the immune system leads to the symptoms of fever, splenomegaly, cytopenia and the characteristic histological appearance of hemophagocytosis [40,46]. In the development of the disease, a distinction can be made between a primary / genetically determined form and a secondary / acquired form [46]. The disease usually begins in childhood, with more and more cases being described with the onset of the disease in adulthood [79,103]. The incidence of primary HLH in Europe is 1.2 children per 1 million children per year [39]. Without treatment, hemophagocytic lymphohistiocytosis quickly leads to death, so rapid diagnosis and treatment are desirable [46] Diagnostic criteria The diagnosis of HLH is made up of characteristic but unspecific symptoms. In both the primary and the secondary form, the onset of the disease is triggered by a - mostly viral - infection [90]. The Epstein-Barr virus was most frequently described in connection with HLH [29,51,92]. The main symptoms of HLH are fever, splenomegaly, and cytopenia. Hyperglyceridemia, hypofibrinogenemia with subsequent coagulopathy, liver dysfunction, as well as increased ferritin and increased serum transaminases occur frequently [1,40,53]. Neurological symptoms associated with CSF hyperproteinemia and pleocytosis are also often described [15,16,42]. Less often, HLH is associated with lymphadenopathy, rash, jaundice, and edema. Characteristic laboratory values ​​are decreased NK cell activity and hypercytokinemia with increased scd25 values ​​[40,44]. Hemophagocytosis can occur in the bone marrow, spleen, lymph nodes, liver and in the 1st

9 1 Introduction CSF occur [40]. However, the number of patients who already have hemophagocytosis at the time of diagnosis in the initial bone marrow biopsy is low [35]. Table 1 summarizes the currently valid diagnostic criteria for hemophagocytic lymphohistiocytosis of the Histiocyte Society. Table 1: Current diagnostic criteria of the Histiocyte Society for HLH from 2004 [40,49]. HLH = hemophagocytic lymphohistiocytosis, scd25 = soluble CD25 (soluble interleukin-2 receptor), NK cell = natural killer cell, LDH = lactate dehydrogenase. Diagnostic criteria of HLH 1. Familial disease / known genetic defect 2. Clinical and laboratory criteria (5/8 criteria) Fever Splenomegaly Cytopenia 2 cell rows Hemoglobin <90 g / l (less than 4 weeks <120 g / l) Neutrophils <1.0 x 10 9 / l thrombocytopenia <100 x 10 9 / l hypertriglyceridaemia and / or hypofibrinogenemia triglycerides on an empty stomach 3 mmol / l fibrinogen <1.5 g / l ferritin 500 µg / l scd U / ml reduced or absent NK cell activity haemophagocytosis in Bone marrow, liquor or lymph nodes The diagnosis is supported by neurological symptoms with moderate pleocytosis and / or increased protein, increased transaminases and bilirubin, LDH> 1000 U / l Pathogenesis Characteristics of primary / congenital HLH is a known genetic defect and usually an early manifestation of the Illness in the first years of life. Secondary / acquired HLH occurs in association with infections, autoimmune diseases, or malignancies in older children and adults [80]. HLH is a syndrome with abnormal and excessive activation of the T cells of the immune system, which leads to a damaging activation of macrophages and other cells of the innate immune system [90]. Pathogen-induced stimulation of the T cells leads to the release of proinflammatory cytokines [2]. The known genetic defects that lead to primary / congenital HLH hinder the cytotoxicity of T and NK cells [53]. Secondary / acquired HLH suggests that multifactorial causes influence the onset of the disease. A 2

10 1 Introduction of the exact pathomechanism has not been researched [46], but here too the disease arises from defective T and NK cell cytotoxicity [80]. This perforin-dependent cytotoxicity can be impaired at various points in the course of the release of the cytolytic granules into the target cell [53]. Normally perforin penetrates the membrane of the target cell and releases granzyme into the cytosol, which leads to apoptosis of the target cell [83]. This process is disrupted at HLH. The activated T and NK cells of the immune system are thus impaired in their function of eliminating infected cells. The stimulation of the pathogen persists, proinflammatory cytokines continue to be released to stimulate the immune system and finally the excessive activation of the immune system leads to the characteristic symptoms of HLH (fever, splenomegaly, cytopenia) [2,40] The macrophage activation syndrome The macrophage activation syndrome is a special form of secondary / acquired HLH considered [17,34,47,63]. It occurs as a complication in the context of a pre-existing autoimmune disease or autoinflammation, often with underlying rheumatic diseases [47,88,119]. It is characterized by excessive activation of tissue macrophages [63]. The MAS was first described in patients with systemic juvenile idiopathic arthritis and occurs most frequently in them [34,88]. The prevalence here is around 10% [17]. Other diseases related to MAS are systemic lupus erythematosus, rheumatoid arthritis, sarcoid, Kawasaki syndrome, chronic infantile neurocutaneous articular syndrome, dermatomyositis, polyarteritis nodosa, and adult Still's disease [34,119]. The diagnostic criteria of the MAS are summarized in Table 2. 3

11 1 Introduction Table 2: Preliminary diagnostic guidelines for macrophage activation syndrome as a complication of systemic juvenile idiopathic arthritis from 2012 [88]. MAS = macrophage activation syndrome, AST = aspartate aminotransferase, CNS = central nervous system, sjia = systemic juvenile idiopathic arthritis. Preliminary diagnostic guidelines for macrophage activation syndrome as a complication of systemic juvenile idiopathic arthritis Laboratory criteria 1. Decreased platelet count (262 x 10 9 / l) 2. Increased AST level (> 59 U / l) 3. Decreased white blood cell count (4.0 x 10 9 / l) 4. Hypofibrinogenemia (2.5 g / l) Clinical criteria 1. CNS dysfunction (irritability, clouding of consciousness, lethargy, headache, seizures, coma) 2. Hemorrhage (purpura, ecchymosis, mucosal bleeding) 3. Hepatomegaly (> 3 cm below the costal arch) Histopathological criterion Detection of hemophagocytosis by macrophages in the bone marrow aspirate Diagnosis rule The diagnosis MAS requires the presence of 2 or more laboratory criteria or 2, 3 or more clinical criteria and / or laboratory criteria. A bone marrow aspirate for the detection of hemophagocytosis is only required in questionable cases. Recommendations The above criteria are only useful in patients with active sjia. The threshold values ​​of the laboratory criteria are given as examples. Similar clinical symptoms occur in HLH and the special form MAS. Presumably, similar immunological changes lead to the clinical pictures [34]. The differentiating criterion is that the MAS always occurs as a complication of an autoimmune disease [47] Therapy goals of HLH are initially an immunosuppression and, in addition, the treatment of the offending infection. In the case of primary HLH and therapy-refractory secondary HLH, a stem cell transplant should also be sought as therapy. The transplant can replace the defective immune system with healthy cells from the donor [90]. The Histiocyte Society currently recommends treatment of the 4th

12 1 Introduction to HLH patients according to the HLH 2004 protocol [37,40]. Table 3 shows the course of therapy based on this protocol. Table 3: Overview of the course of HLH therapy according to the HLH 2004 protocol [37]. HLH = hemophagocytic lymphohistiocytosis. Genetically proven or familial disease HLH patient: 8 weeks of chemotherapy as initial therapy, persistent, non-familial and non-genetic disease recovery from non-familial and non-genetic disease Continuing further therapy until stem cell transplantation The drugs used are listed in the following table. End therapy Reactivation: Continue further therapy until stem cell transplantation Table 4: Overview of the medication used in the HLH 2004 protocol [37]. HLH = hemophagocytic lymphohistiocytosis, IgIG = intravenous immunoglobulin. Initial therapy dexamethasone (10 mg / m² - 1.25 mg / m²) daily etoposide (150 mg / m²) for two weeks twice a week, then once a week cyclosporine A target value 200 mg / l Further therapy dexamethasone (10 mg / m²) as pulse therapy every two weeks etoposide (150 mg / m²) every two weeks cyclosporine A target value 200 mg / l supportive: cotrimoxazole, oral antimycotic (1st-9th week), IvIG (0.5 g / kg) for four weeks, Stomach protection (1st to 9th week) Intrathecal administration of methotrexate is possible if the CNS is involved [40]. Patients who have been shown to have EBV-associated HLH benefit from rituximab as an add-on therapy [48]. In the case of a therapy-refractory course of HLH, plasmapheresis can also be used [47]. Alemtuzumab (CD52 antibody), infliximab (TNF-α antibody), daclizumab (CD25 antibody), anakinra (IL-1 receptor antagonist) and other immunomodulators and chemotherapeutic agents could also be used successfully [29,73,74] . In MAS patients, high-dose corticosteroid therapy is usually very effective, possibly in combination with cyclosporine A [48,119]. The HLH-2004 protocol and other immunomodulators and chemotherapeutic agents can be used for therapy-resistant courses [29,87,119]. 5

13 1 Introduction Long-term observations of patients who were treated according to the previous HLH-94 protocol, which differed only slightly in content, are already available. These show that% of the patients respond to the therapy [113]. About 20% of HLH patients die within six months of being diagnosed, even before a stem cell transplant. A total of 54% of the patients under therapy are long-term survivors [113] Biomarkers The diagnostic criteria of the Histiocyte Society list known HLH biomarkers [40]. These biomarkers are characteristic, but cannot be clearly assigned to hemophagocytic lymphohistiocytosis. Autoimmune diseases or autoinflammatory syndromes, which are the underlying diseases of MAS, also show characteristic biomarkers. Clear biomarkers are necessary for a quick diagnosis and the start of therapy without delay. The biomarker scd25 The alpha chain of the heterodimeric soluble IL-2 receptor is referred to as scd25 [95]. Increased scd25 values ​​are observed in diseases associated with the activation of T lymphocytes [93]. Pathological scd25 values ​​have been described in patients with hepatocellular carcinoma, acute leukemic leukemia and sepsis [12,67,77]. Scd25 values ​​above 2400 U / ml are used as markers in HLH diagnostics [49,100]. The serum level of scd25 in patients with untreated HLH is increased and decreases in parallel with effective therapy [57]. The scd25 values ​​of HLH patients can thus be viewed as markers for disease activity and prognosis [44,57]. Since the scd25 values ​​are influenced by various diseases, in addition to a pathological scd25 value, further positive clinical or laboratory criteria are necessary for the diagnosis of HLH [40,93] The Ferritin Biomarker Healthy people have low ferritin values. Ferritin is increased as an acute phase protein in connection with the activation of the immune system [58]. Ferritin values> 500 µg / l are a diagnostic criterion of the HLH [40]. Measurements of the ferritin level 6

14 1 Introduction gels in the blood are inexpensive and can be carried out quickly [1]. A comparison of different patient groups with ferritin values> 500 µg / l shows that ferritin levels> 10000 µg / l occur in connection with HLH [1]. An unfavorable course of HLH with early death correlates with an increase in ferritin values ​​to> 2000 µg / l at the start of therapy and in the first two weeks of therapy [112]. A reduction in ferritin levels during therapy is in turn associated with low mortality [66]. Since ferritin, as an acute phase protein, is always increased when the immune system is activated, it can only be used as a diagnostic criterion in combination with other HLH criteria [40,58] The biomarker IL-1ß The biomarker IL-1ß is a proinflammatory cytokine. Elevated IL-1ß levels indicate fever and fatigue. In inflammatory diseases, peripheral inflammation is associated with a central release of IL-1ß [61]. Elevated IL-1ß levels are also associated with autoimmune diseases [30]. High S-100 protein levels can be detected in sjia. S-100 proteins lead to increased IL-1ß production [19]. Incubation of PBMCs with serum from sjia patients leads to increased IL-1ß production [81]. Thus, because of their autoimmune disease or autoinflammatory underlying disease, increased IL-1ß levels are to be expected in MAS patients. In contrast, there is no increase in the IL-1ß level in HLH patients with a primary / congenital cause of the disease [38] The biomarker Cathelicidin LL-37 As a protein of the immune system, Cathelicidin LL-37 has immunomodulatory and antimicrobial properties [11 ].In the case of infections and inflammatory reactions, LL-37 is increasingly expressed [9]. An increase due to autoinflammation, which can lead to MAS, is therefore likely. No elevated LL-37 values ​​have been described in connection with HLH. 1.2 The MICA antigen system Bahram et al. described MICA for the first time in 1994 as a non-classical MHC molecule [5]. 91 MICA alleles are now registered by the World Health Organization [91; Release,]. Overall, the MIC family consists of genes 7

15 1 Introduction MICA and MICB, as well as the five pseudogenes MICC, MICD, MICE, MICF and MICG. The genes are located adjacent to the MHC class I gene locus on chromosome 6 [43]. With the exception of the CNS, MICA-RNA can be detected in all tissues [101]. In contrast to classic MHC molecules, MICA does not bind any antigens [32]. In the physiological state, the MICA proteins are only detectable in the gastrointestinal epithelium on the cell surface [31]. MICA is increasingly expressed under cell stress, on tumor cells and on pathogen-infected cells [31,36,41,60]. MICA can be present in the blood as a membrane-bound molecule on the cell surface and as a soluble protein smica [41,101]. In connection with various immune diseases such as ulcerative colitis, diabetes mellitus type I and periodontitis, an increased expression of smica and the presence of special MICA polymorphisms were observed [23,85,123] Structure and function The structure of the MICA protein is similar to that of classic MHC molecules : MICA consists of three extracellular domains -α1, α2 and α3- as well as a transmembrane domain and a cytoplasmic residue [65]. Exon 2 codes for the α1 domain, exon 3 for the α2 domain and exon 4 for the α3 domain. The transmembrane domain is encoded by exon 5 and the cytoplasmic remainder by exon 6 [4]. In contrast to classic MHC molecules, however, MICA has no association with ß2-microglobulin [31]. MICA is a ligand of the activating NKG2D receptor that is expressed on NK cells, γδ-t cells and αβ-cd8 + T cells [6,13]. The NKG2D receptor is a C-type lectin receptor. The binding of MICA to NKG2D takes place via an induced-fit mechanism. This leads to a change in the conformation of the receptor [64,86]. The dissociation constant of the MICA-NKG2D complex is 37 C with K D = 1 µm. This means that MICA has a higher binding affinity for NKG2D than that of most NK and T cell receptors for the classic MHC molecules [10,64,72]. A special feature of MICA is that it has opposing effects on the NKG2D receptor as a membrane-attached surface molecule and as a soluble MICA. Cell-bound MICA has an activating effect, whereas soluble MICA has an inhibiting effect on the NKG2D receptor [6,7,33]. A healthy immune defense is kept in balance by a balanced stimulation of activating and inhibiting NK and T cell receptors. MICA contributes to this through its effect on the NKG2D receptor [116]. 8th

16 1 Introduction Polymorphisms Within the classical and non-classical MHC molecules there is a multitude of polymorphisms. MICA is one of those molecules that have a high number of polymorphisms [5.91; Release,]. In the case of some MICA polymorphisms, a connection with certain diseases can be observed [7,85,109,123]. Table 5 presents the currently known MICA alleles and their corresponding polymorphisms in intron 4, exon 5 and exon 3 [91; Release,]. Table 5: MICA alleles and their corresponding polymorphisms [91; Release,]. MICA = major histocompatibility complex class I-related chain A, T = thymine, A = alanine. *** = currently no information is available on the polymorphisms of these alleles. - = no amino acid at position 129, as there is an early stop codon in the allele. MICA allele 7T / 8T polymorphism (intron 4) alanine polymorphism (exon 5) MICA-129 polymorphism (exon 3) MICA * 001 8T A4 methionine MICA * 002: 01 8T A9 methionine MICA * 002: 02 *** *** Methionine MICA * 002: 03 *** A9 Methionine MICA * 002: 04 *** *** Methionine MICA * 004 7T A6 Valine MICA * 005 *** *** Valine MICA * 006 7T A6 Valine MICA * 007: 01 8T A4 Methionine MICA * 007: 02 *** *** Methionine MICA * 007: 03 *** A4 Methionine MICA * 007: 04 *** *** Methionine MICA * 007: 05 *** ** * Methionine MICA * 007: 06 *** *** Methionine MICA * 008: 01: 01 7T A5.1 Valine MICA * 008: 01: 02 7T A5.1 Valine MICA * 008: 02 *** A5.1 Valine MICA * 008: 03 *** *** Valine MICA * 008: 04 *** A5.1 Valine MICA * 009: 01 7T A6 Valine MICA * 009: 02 7T A6 Valine MICA * 010: 01 7T A5 Valine MICA * 010: 02 7T A5 valine 9

17 1 Introduction MICA * 011 8T A6 Methionine MICA * 012: 01 8T A4 Methionine MICA * 012: 02 *** *** Methionine MICA * 012: 03 *** *** Methionine MICA * 013 *** *** Valine MICA * 014 *** *** Methionine MICA * 015 8T A9 Methionine MICA * 016 7T A5 Valine MICA * 017 8T A9 Methionine MICA * 018: 01 8T A4 Methionine MICA * 018: 02 *** A4 Methionine MICA * 019 7T A5 Valine MICA * 020 *** A10 Methionine MICA * 022 *** *** Valine MICA * 023 *** A5.1 Methionine MICA * 024 *** *** Valine MICA * 025 *** *** Methionine MICA * 026 *** A6 Methionine MICA * 027 7T A5 Valine MICA * 028 *** A5.1 Valine MICA * 029 8T A4 Methionine MICA * 030 8T A6 Methionine MICA * 031 *** *** Methionine MICA * 032 *** *** Methionine MICA * 033 7T A5 Valine MICA * 034 *** *** Methionine MICA * 035 *** *** Methionine MICA * 036 *** *** Methionine MICA * 037 *** * ** Methionine MICA * 038 *** *** Methionine MICA * 039 *** *** Methionine MICA * 040 *** *** Methionine MICA * 041 8T A9 Methionine MICA * 042 *** *** Methionine MICA * 043 *** A4 methionine MICA * 044 *** *** valine MICA * 045 *** A4 methionine MICA * 046 8T A9 methionine MICA * 04 7 *** A6 methionine MICA * 048 *** A5 valine 10

18 1 Introduction MICA * 049 *** A6 Valine MICA * 050 *** A7 Methionine MICA * 051 8T A4 Methionine MICA * 052 *** A9 Methionine MICA * 053 *** A5.1 Valine MICA * 054 *** A5 Valine MICA * 055 *** A8 Methionine MICA * 056 *** A5 Valine MICA * 057 *** *** Valine MICA * 058 *** A5.1 Valine MICA * 059 *** *** Methionine MICA * 060 *** *** Methionine MICA * 061 *** *** Methionine MICA * 062 *** *** Valine MICA * 063N *** *** - MICA * 064N *** *** Valine MICA * 065 *** *** Valine MICA * 066 *** *** Valine MICA * 067 *** *** Valine MICA * 068 *** *** Methionine MICA * 069 *** *** Valine MICA * 070 *** *** Valine MICA * 071 *** *** Methionine MICA * 072 *** *** Methionine MICA * 073 *** *** Valine MICA * 074 *** *** Valine MICA * 075 *** *** Methionine T / 8T polymorphism (intron 4) Intron 4 of the MICA gene can exist in two variants. There is either a seven-fold or an eight-fold repetition of the base thymine at the 3 - end of the intron. This is known as the 7T or 8T polymorphism [24] alanine polymorphism (exon 5) In exon 5, the short tandem repeat (GCT) n codes for a variable number of repetitions of the amino acid alanine in the transmembrane domain of the MICA protein. There are alleles with the polymorphisms A3, A4, A5, A6, A8, A9 and A10 in which 11

19 1 Introduction corresponding to the designation, there are 3, 4, 5, 6, 8, 9 or 10 alanine repetitions in each case [25,75,82,94]. In addition, the polymorphism A5.1 can occur as a special case. Here, a guanine insertion after the second GCT triplet leads to a reading frame shift and this creates an early stop codon [75]. The changed allele has a shortened cytoplasmic residue and is not expressed on the basolateral cell membrane, as is otherwise usual for MICA proteins, but apically [108]. The A5.1 polymorphism occurs, among other things, with the allele MICA * 008, which has the peculiarity that soluble MICA is released in exosomes [3] MICA-129 polymorphism (exon 3) As an amino acid at position 129 of the α2- domain of the MICA protein can be either methionine or valine. Depending on the amino acid expressed, the MICA alleles have different binding affinities for NK and T cells. Valine shows a weak affinity, methionine shows a strong affinity for the NKG2D receptor. This polymorphism may have an effect on the functional properties of the MICA protein [106]. 12th

20 1 Introduction 1.3 Issues and objectives Hemophagocytic lymphohistiocytosis is an inflammatory disease which, without rapid diagnosis and treatment, leads to death in childhood [46]. No clear biomarker is available to characterize HLH [40]. The aim of this work is to analyze the relevance of smica and MICA polymorphisms as biomarkers of HLH. The following questions are examined: - Do HLH patients have increased levels of smica in the blood? - Do HLH patients have peculiarities in the MICA genotype? - Are special MICA polymorphisms associated with increased smica values ​​in HLH patients? The analyzes should support a quick diagnosis of the HLH with simple test methods. The assignment of special MICA polymorphisms to the HLH disease helps to find indications of an increased susceptibility to disease. Based on this, test options for early detection of HLH are conceivable. As a biomarker of HLH, smica can also be considered as a disease progression parameter. In the future, studies on the disease-influencing effect of smica are obvious. These can provide information on the pathogenesis of HLH as well as new therapeutic approaches. 13th

21 2 Material and Methods 2 Material and Methods Various materials and methods were used to analyze the relevance of smica and MICA polymorphisms as biomarkers. 2.1 Structure of the study A group of HLH patients was examined and compared with other collectives. Data from healthy blood donors were used as controls. In addition, a group of MAS patients was considered as a comparison group. All collectives were tested independently of one another. The samples from the various groups had been collected over a longer period from 1999 to 2011. Patient collective and control groups 56 HLH patients were selected as a patient group, from whom DNA and blood samples were available. Based on the known scd25 values, only HLH patients were included in the study who had an active disease at the time the sample was taken. In addition, some of the patients already knew the values ​​of ferritin, IL-1ß and LL-37. The EBV status of some patients was also known. The patient samples had been collected over several years and were now analyzed together. The diagnosis was made according to the current HLH criteria at the time of diagnosis. DNA and blood samples were available from 87 MAS patients as a comparison group. Values ​​for scd25, ferritin, IL-1ß, LL-37 and the EBV status were also known from some patients. Doctor's letters were available to clearly differentiate between HLH and the special form MAS, as well as the µ-score for a large number of patients. This special score is used to clearly differentiate between HLH and MAS using the parameters scd25, IL-1ß, CD86 + / CD2 + and ferritin [99,120]. It was not considered whether in the group of HLH patients a primary / congenital or secondary / acquired form of the disease was present in the development of the disease. An uncontrolled activation of the immune system leads to the course of HLH in both forms, so the initial underlying cause of HLH and the factors triggering the disease are not relevant for the present study. The diagnostic 14

22 2 Material and Methods The criteria of the HLH of the Histiocyte Society do not differentiate between primary / congenital and secondary / acquired form, but rather cover both causes equally. DNA and blood samples from two groups of healthy blood donors were used as controls. DNA samples were sequenced from 112 test persons and the smica values ​​in the blood of 89 test persons were determined. Table 6 shows the composition of the individual groups in terms of gender and age. Control group A consists of 89 test persons whose smica values ​​were analyzed. The gender distribution is available for this control group. To analyze the MICA genotypes, control group B was used with 112 test persons whose gender distribution is unknown. In terms of age, it is known from healthy blood donors in groups A and B that the subjects were of legal age at the time the sample was taken. In three of the HLH patients, no information was available on the age at which the sample was taken. For all patients younger than 12 months, the age was given as 1 year. Table 6: Demographic data of the four comparison groups: HLH patients, MAS patients, control group A for smica analysis and control group B for MICA genotype analysis. The samples of the patients and control groups come from the years 1999 to 2011 and were analyzed at the University of Ulm. HLH = hemophagocytic lymphohistiocytosis, MAS = macrophage activation syndrome, n = number, smica = soluble major histocompatibility complex class I-related chain A. HLH patients MAS patients group A control groups number n group B male 32 57.1% 50 57.5% 50 56.2% unknown female 24 42.9% 37 42.5% 39 43.8% unknown median [years] 3 5 unknown unknown minimum [years] maximum [years] Course of the study The examinations were carried out after approval of the ethics committee of the University of Ulm (ethics application number 34/12 of the ethics committee of the University of Ulm), so that the ethical principles for medical research on humans set out in the Declaration of Helsinki were observed. 15th

23 2 Material and methods Corresponding declarations of consent for the use of their DNA and blood samples for research purposes were available from both the blood donors and the patients or their legal guardians. In addition to the values ​​already available, a measurement of the smica level in the blood and DNA sequencing to determine the MICA genotype and analysis of the MICA polymorphisms were carried out in the patients and healthy test persons. The data were supplemented with relationships that were already known from the literature. The measured values ​​were statistically evaluated in order to present and interpret the results. 2.2 Material Chemicals, equipment, laboratory materials and software were required to carry out the methods. The material used is listed here Chemicals Table 7: List of the chemicals used Agencourt Ampure Beads from Beckmann Coulter, order number APN PBS, 1% BSA, pH 7.4 (Sigma P3688, 138 mm NaCl, 2.7 mm assay buffer KCl, 1% BSA) 200 ml PBS + 1 g BSA Bio-Plex Bead Activation Buffer, 85 ml, S 60, L Rev B Bead Activation Buffer Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA Bio-Plex Bead Wash Buffer, 4ml, L Rev B, Bio -Rad Bead Wash Buffer Laboratories, 2000 Alfred Nobel Drive, Hercules CA BigDyeTerminatorV1.1 (company ABI article number) BigDye-Buffer 5xSequencingBuffer (company ABI article number) pab anti-mouse Ig G [Biotin], NB720-B Lot 677, Biotin Antibody NOVUS Biologicals Art.Nr, Size 500g, Lot Nr C, Biozym Biozym LE-Agarose Scientific GmbH, D Hess.Oldendorf Bio-Plex Blocking Buffer, 10ml, L Rev B Blocking Buffer Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA Tris 900mM (C4H11NO3) Boric Acid 900mM (H3BO3) Borate Buffer (TBE) 10% EDTA 25mM Titriplex III (C10H14N2NA2O8xH2O) aqua dest. Coupling Reaction Tubes, Bio-Plex Coupling Reaction Tubes, L Rev C, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA

24 2 Material and Methods Detection Antibody Part, R&D Systems Europe, Ltd. Detection antibodies 19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK Ultrapur dntp-set (Amersham, 100mM, order number) dntp-mix (Amersham) Product No, 5g EDC Thermo Scientific, Pierce Biotechnology 3747 N. Meridian Road, Rockford IL USA Ethanol 96% (Merck, Article No.) Ethanol 70% Aqua dest. Ethanol 96% from Merck, order number, 07 Ethidium Bromide, CX 12948, 10ml, A9EB012, InnoTrain Ethidiumbromid Diagnostik GmbH, D Kronberg / Taunus Capture Antibody Part, R&D Systems Europe, Ltd. 19 catcher antibodies Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK LiChrosolv water company Merck, Art.No LiquiChip System Fluid 10x concentrate LiquiChip System Fluid fully demineralized water Tris powder EDTA disodium salt SDS sodium lauryl sulfate Loading-Buffer Bromophenol blue Sucrose distilled water. Dulbecco s PBS, 500ml, order no. PL-12-L, ccpro GmbH, PBS (ccpro GmbH) Bio-Plex PBS Buffer, 135ml, S 60, L Rev B PBS Buffer (Bio Rad) Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA PCR- Ammonium sulfate buffer 10x (ASP 10x) PCR master mix (MM) PE-conjugated-streptavidin Tris (hydroxymethyl) aminomethane, Merck, 1kg, pH 8.8, order number) Ammonium sulfate (Merck, Calbiochem, 100g, order number) HPLC water (Merck Company, 1.0l, Order No) Tween 20 (Merck, 500ml, Order No) HPLC water (Merck, 1.0l, Order No) 10x ammonium sulfate buffer 25mM MgCl2 (Merck) Glycerin 99 , 5% (Merck, 1.0l, order number) dntp-mix 100mM (Amersham) PE-conjugated streptavidin (lyophilized) distilled water. Polystyrene Beads COOH Bead 011, Bio-Plex COOH Beads # SAPE-Solution SAPE Buffer PE-Conjugated-Streptavidin Bio-Plex Staining Buffer, 105ml, S 60, L Rev C, Bio-Staining Buffer Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA Standard Standard Part, R&D Systems Europe, Ltd. 19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK 17

25 2 Material and Methods Storage Buffer Streptavidin-HRP Sulfo-NHS Taq DNA Polymerase Wash Buffer Bio-Plex Storage Buffer, 25ml, S 60, L Rev B, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA Part R&D Systems Europe, Ltd. 19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK N-Hydroxysulfosuccinimide, 500mg, g / mole, CAS, Prod # 24510, Thermo Scientific, 3747 N.Meridian Rd., Rockford, IL USA Taq Polymerase 5U / ml, QIAGEN GmbH, Hilden PBS 0.05%, TWEEN-20, ph7.4 Sigma P3563, 138 mm NaCl, 2.7 mm KCl, 0.05% TWEEN 500 ml PBS µl TWEEN devices and Laboratory materials Table 8: List of equipment and laboratory materials used 96-well microtiter plates without barcode 96-well microtiter plates with barcode 96-well microtiter plates for Luminex Assay Alpha Innotech photo unit electrophoresis chamber Eppendorf Combitips Plus Eppendorf pipette tips (yellow) Filter for vacuum pump Rubber mats Hamilton Microlab Star Laboratory Balance Luminex 100 Device LiquiChip Workstation Microwave Device Mains Connection GPS 200/400 ThermoFast 96, Non Skirted, Cat # AB-0600, Thermo Scientific MicroAmp Optical 96-well Reaction Plate with Barcode, REF, Applied Biosystems MultiScreen Filter Plates for High Throughput Separations, MultiScreen -HV Clear Plates, Cat.Num.MAHVN4510, Millipore Corporation, Billerica, MA USA Biozym Scientific GmbH, D Hess.Oldendorf SGU-030T-02, CBS Scientific Company, INC. Box 856, Del Mar, CA 92014, USA 0.2ml B146534I, Eppendorf AG, Hamburg, Germany 0.5ml G, Eppendorf AG, Hamburg, Germany Reload 0.1-10 µl and Reload µl, Eppendorf AG, Hamburg Millex -FG 50, 0.2µm Rated Hydrophobic Filter, Catalog Number: SLFG05010 Millipore Corporation Billerica, MA USA Plate Septa 96-Well, No., Applied Biosystems GmbH, Foster City, USA Serial No. (room 24), Hamilton AG, Via Crusch 8, CH-7402 Bonaduz Ohaus Navigator, serial no. S / N, Ohaus GmbH Germany, Ockerweg 3a, Gießen Serial Number LX, Revision A, Controller Version Luminex Analyzer LX Luminex XY Platform LXY Luminex Pump SD Qiagen GmbH, Qiagen Straße 1, D Hilden, Germany exquisit ED8525.2S, serial no , EGS GmbH, Sandstr. 13, Lippstadt, serial no. DD006208, Pharmacia LKB Biotechnologie, S Uppsala, Sweden 18

26 2 Material and methods Pipette tips 1-10 µl, µl, µl Reaction vessels 1.5 ml Shaker Automatic sequencing TECAN Genesis RSP 150 Thermocycler Vacuum Pump Centrifuge Biozym Safe Seal tips Premium sterile Order No, Eppendorf AG, Hamburg Vortexer VM300, serial number, NeoLab Migge , Laborbedarf-Vertriebs-GmbH, Richerstr. 7, Heidelberg Vortexer, serial number, VWR International GmbH, James-Franck-Ring 9, Ulm / Donau Shaker for Luminex incubation IKA MTS 2/4 digital,, IKA-Werke GmbH & Co KG, D Staufen 3730 DNA Analyzer, serial no., Applied Biosystem, Applera Corporation Business, Applera Deutschland GmbH, Darmstadt, serial no. 4315, company TECAN Deutschland GmbH, Theodor-Storm-Straße 11, Crailsheim Thermocycler Gene Amp PCR System 9700, serial no. 805S, Applied Biosystems Applera, Deutschland GmbH, Frankfurter Str. 129B, Darmstadt Thermocycler Peq Star, PEQLAB Biotechnologie GmbH, Carl-Thiersch-Str. 2B, Erlangen Bio Rad vacuum pump, 262BR3218, flow rate 900 cu ft / min Heraeus Multifuge 3S +, serial number, Heraeus Instrument GmbH, Kendro Laboratory Products GmbH Hanau laboratory centrifuge SIGMA 2-6, serial number, Sigma laboratory centrifuge, An der Unteren Söse 50, D Osterode Software Table 9: List of the software used Alphamanager for electrophoresis gel documentation Alphamanager, AlphaImmotech, Biozym Scientific GmbH, Hessisch Oldendorf, Germany Hamilton Microlab Star ABC program for creating work protocols for Hamilton Microlab Star, Version 1.2. 8, in-house program IBM SPSS Statistics IBM SPSS Statistics Version 20, IBM Deutschland GmbH Ehningen Luminex Bioplex Manager Microsoft Excel Version (32-bit), part of Microsoft Office Professional 2010, Microsoft Corporation Microsoft Word Version (32-bit), part of Microsoft Office Professional 2010, Microsoft Corporation Sequence Pilot Sequence Pilot Version Build 508, JSI medical systems GmbH Tecan Gemini V ersion

27 2 Material and methods 2.3 Methods The following methods were used for the analyzes. Luminex Bead Array A Luminex Bead Array was used to detect the smica concentrations in the blood samples of the patients and the control group. The smica to be detected is first bound to polystyrene beads via a capture antibody. A detection antibody coupled with an enzyme is added later. It binds smica to a different epitope than the catcher antibody, resulting in a sandwich-like structure: the catcher antibody binds smica, which binds the detection antibody with the enzyme. Figure 1: Detection of the smica concentration in the blood using a Luminex Bead Array: coupling of smica to polystyrene beads using capture antibodies and coupling of smica to the enzyme using detection antibodies. F = capture antibody, D = detection antibody, smica = soluble major histocompatibility complex class I-related chain A. The smica concentration is detected by identifying the beads and measuring chemiluminescence. After adding the substrate PE-conjugated-streptavidin, this is converted by the enzyme. The smica values ​​can thus be determined on the basis of the fluorescence. The measurement is carried out according to the principle of flow cytometry. The analyzer uses two lasers for measurement: a red laser identifies the beads and a green laser measures their fluorescence. This principle is suitable for performing measurements with high sensitivity. Coupling of the catcher antibodies to beads To prepare the Luminex bead array, the polystyrene beads were coupled with the catcher antibodies in order to later create the sandwich-shaped structure illustrated above when carrying out the Luminex bead array. The beads were first mixed for 30 seconds using the vortexer. 100 μl of the beads were poured into a reaction vessel, which corresponds to an amount of 1.25 * 10 5 beads 20

28 2 Material and Methods. After four minutes of centrifugation at g, the supernatant was removed. The batch in the reaction vessel was resuspended with 100 μl of bead washing buffer and the mixture was vortexed for 30 seconds. After four minutes of centrifugation at g, the supernatant was again discarded. This was followed by the addition of 80 μl of bead activation buffer. The batch was vortexed for 30 seconds. Next, 10 μl of S-NHS solution (50 mg / ml) was added and mixed using the vortexer. The S-NHS solution consisted of 1 ml of bead activation buffer mixed with 50 mg of S-NHS powder. Then 10 μl of EDAC solution (50 mg / ml) was added to the batch and mixed with the vortexer. The EDAC solution consisted of 1 ml of bead activation buffer and 50 mg of EDAC. The reaction vessel with the light-sensitive beads was wrapped with aluminum foil to protect it from light and incubated with shaking at room temperature for 20 minutes. Then 150 μl of PBS were added and the mixture was vortexed for 10 seconds. After four minutes of centrifugation, the supernatant was removed. These steps were repeated: 150 μl of PBS were added, followed by mixing using the vortexer for 10 seconds. After a further 4 minutes of centrifugation, the supernatant was discarded. Another 100 μl of PBS was added. The batch was mixed with the vortexer for 30 seconds. The capture antibody was then added. 25 μl of capture antibody were pipetted into the reaction vessel, which corresponds to an absolute amount of 8 μg of capture antibody. The total volume was made up to 500 µl by adding 375 µl PBS. The batch was mixed well with the vortexer. This was followed by an incubation time of two hours, during which it was incubated with shaking and protected from light at room temperature. The mixture was then centrifuged for four minutes at g and the supernatant was removed. The beads were resuspended with 500 µl PBS. The mixture was centrifuged for a further four minutes at g, then the supernatant was discarded. 250 µl of the blocking buffer were added. The batch was mixed for 15 seconds with the aid of the vortexer. An incubation time of 30 minutes followed. During this, it was incubated with protection from light and shaking at room temperature. The mixture was then centrifuged for four minutes at g and the supernatant was removed. The approach was supplemented with 500 µl storage buffer and 21

29 2 Material and methods Mixed with the vortexer for 20 seconds. It was then centrifuged for six minutes at g and the supernatant discarded. Finally, 150 μl storage buffer was added to the batch. These catcher antibodies coupled to beads were stored in a light-protected place at 2-8 ° C. until they were used. Validation of the bead coupling To check the coupling of the catcher antibodies to the beads, a validation was carried out as a test run. This validation served to confirm the correct coupling of the capture antibodies to the beads. A test run that simulated smica-positive sample material and a negative control that was smica-negative were used in this test run. Four coupling reaction tubes each were labeled and prepared as reaction vessels for the test batch and negative control. The beads coupled with the capture antibody were vortexed for 15 seconds. 4 μl of beads were then pipetted into each reaction vessel, this corresponds to an amount of about 5000 beads. 200 μl of the biotin antibody with a concentration of 1 μg / ml were added to each of the four reaction vessels of the test mixture. The batches of the negative control were each supplemented with 200 μl of assay buffer. All reaction vessels were incubated for 30 minutes at room temperature on the vortex shaker, protected from light. In preparation for the measurement, a MultiScreen filter plate was blocked with 200 μl of assay buffer per well. After an incubation time of ten minutes at room temperature, the plate was suctioned off. 50 μl test mixture or negative control and 50 μl streptavidin with a concentration of 6 μg / ml were pipetted onto the filter plate. 22nd

30 2 Material and Methods A B C D E F G H T T T T N N N N Figure 2: Detection of the smica concentration in the blood using Luminex Bead Array: Scheme of the bead validation. smica = soluble major histocompatibility complex class I-related chain A, T = test approach, N = negative control. This was followed by an incubation period of ten minutes. During this time, the filter plate stood on the vortex shaker at room temperature, protected from light. The filter plate was then washed three times with washing buffer. For this purpose, 200 μl washing buffer was added to each well and sucked off again with each passage. The wells were then filled with 100 µl LiquiChip each and the measurement started with the Luminex 100 device analyzer. The validation of the bead coupling was successful if a signal was detectable during the test and the negative controls did not show any measurable values. A successful validation was a prerequisite for the use of the beads for the Luminex Bead Array to detect the smica concentrations in the blood samples. Luminex Bead Array The Luminex Bead Array was used to measure the smica concentrations of the blood samples from patients and control persons. When carrying out the Luminex bead array, a MultiScreen filter plate was first blocked for two minutes with 200 μl assay buffer per well and then sucked off again. The following mixture was then pipetted into each well: 25 μl assay buffer as matrix, 25 μl blank sample / standard / blood sample and 25 μl of the capture antibody coupled to beads. Each approach was represented twice. The blank sample (B), the defined standard samples (S1-S8) and the blood samples to be examined (1-39) were applied according to the following scheme: 23

31 2 Material and methods A B S4 S B B S4 S C S1 S D S1 S E S2 S F S2 S G S3 ​​S H S3 S Figure 3: Detection of the smica concentration in the blood using the Luminex Bead Array: scheme of the Luminex Bead Array. smica = soluble major histocompatibility complex class I-related chain A. B = blank. S1 = standard 1, S2 = standard 2 to S8 = standard 8. 1 = patient sample number 1, 2 = patient sample number 2 to 39 = patient sample number 39. Assay buffer was used as a blank. The concentrations of the standard samples were calculated using the dilution factor x = 3. Table 10: Detection of the smica concentration in the blood using the Luminex Bead Array: Luminex Bead Array standard concentrations. smica = soluble major histocompatibility complex class I-related chain A, S1 = Standard 1, S2 = Standard 2 to S8 = Standard 8. Standard S, 0 S2 4666.7 S3 1555.6 S4 518.5 S5 172.8 S6 57 , 6 S7 19.2 S8 6.4 concentration [pg / ml] The blood samples were centrifuged for two minutes at 5000 rpm before pipetting. In order to mark smica, the capture antibodies coupled to polystyrene beads were added. The batch was incubated for 60 minutes. During this time, the batch stood on the vortex shaker at room temperature, protected from light. Three washes followed after the incubation. In each run, 200 μl washing buffer were added per well and aspirated again. Then 25 μl of the detection antibody was added per well. An incubation period of 24 followed

32 2 Material and Methods of 60 Minutes. The batch was protected from light and stood on the vortex shaker at room temperature. After the incubation, 25 μl PE-conjugated streptavidin was added per well. The mixture was incubated for 30 minutes at room temperature, protected from light. This was followed by three washes. Three times 200 μl washing buffer were added to each well and sucked off again. The wells were finally filled with 125 µl LiquiChip each. Then the measurement started with the Luminex 100 device analyzer. A standard curve was generated for each run using the standard values ​​S1 to S8. On the basis of this, the absolute smica values ​​of the patients and the control group were determined from the measured fluorescence. Figure 4: Detection of the smica concentration in the blood using the Luminex Bead Array: Standard curve of the Luminex Bead Array. The curve shows a sigmoidal course. The higher the smica concentration, the stronger the detected signal. smica = soluble major histocompatibility complex class I-related chain A detection limit of the smica values ​​For very low smica values, a value extrapolated from the standard curve was specified in the Luminex bead array as a measured value. To determine the detection limit of the measurable smica values, the mean value of the smica values ​​extrapolated from the standard curve was determined and its standard deviation was calculated. The detection limit determined in this way was 2.50 pg / ml ± 1.72 pg / ml. 25th

33 2 Material and methods DNA sequencing To determine the MICA genotypes, the DNA samples of the patients and the control group were sequenced. For this purpose, the relevant DNA sections were first copied and then the base pattern of the MICA gene was determined. The relevant DNA regions were amplified by means of PCR. The PCR approach for a DNA sample consisted of the following components: 18 µl master mix, 2 µl sequence-specific primer, 0.2 µl Taq polymerase and 2 µl DNA. The PCR was carried out using a method by Fürst et al. performed as a generic PCR of exons 2, 3 and 4 in combination with two group-specific PCRs of exon 5. The following primers were used: Table 11: MICA sequencing project: PCR primers. MICA = major histocompatibility complex class I-related chain A, I = intron, E = exon, G = guanine, A = adenine, T = thymine, C = cytosine, PCR = polymerase chain reaction. MICA primer 5 3 sequence amplified area MICA * I 1 fwd MICA * I 3 rev MICA E4 F seq MICA-I5 rev 2g MICA I4-7T fwd MICA-I5 rev 2g MICA-I4-8T-fwd MICA-I5 rev 2g GGA AGG TTG GGA CAG CAG AC GAT GTG CCA ACA GGA AAT GC AAG AGA AAC AGC CCT GTT CTT CTC C CCC AAA TTC CCC AAC TTT CAT CC CAG TGT ATA ACA AGT CCC TTT TTT TC CCC AAA TTC CCC AAC TTT CAT CC CAG TGT ATA ACA AGT CCC TTT TTT TTC CCC AAA TTC CCC AAC TTT CAT CC exon 2 and 3 exon 4 group-specific PCR for exon 5 The primers were used in four PCR batches. Approach A consisted of a master mix with primer for exons 2 and 3. Approach B was a master mix with primer for exon 4. Approach C consisted of a master mix and primer for exon 5 / 7T. Approach D consisted of a master mix and primer for exon 5 / 8T. 96-well microtiter plates were used to carry out the PCR. The batch was applied to the plates according to the following scheme: 26

34 Approach A Approach B Approach C Approach D 2 Material and methods A B C D E F G H Figure 5: MICA sequencing project: Scheme of the MICA-PCR. 1 = DNA of patient number 1, 2 = DNA of patient number 2, to 8 = DNA of patient number 8. Approach A contains primers for exons 2 and 3, approach B contains primers for exon 4, approach C contains primers for exon 5 / 7T, batch D contains primers for exon 5 / 8T. MICA = major histocompatibility complex class I-related chain A, DNA = deoxyribonucleic acid, PCR = polymerase chain reaction, T = thymine. After the batch had been pipetted, the microtiter plates were sealed against evaporation with rubber mats and centrifuged at 400 g for one minute. The samples then went through various steps in the thermal cycler: At 96 C, the DNA double strands were denatured and opened. The Taq polymerase was then activated at 94 ° C. in ten cycles. During the cooling to 65 C, the primers attached to their DNA binding sites and a specific synthesis of new matrix took place. This was followed by 30 cycles in which the primers attached to the single strands at 61 ° C. and the Taq polymerase synthesized new DNA strands at its optimum temperature of 72 ° C. The MICA gene region of the DNA samples was amplified in this way. After the reactions, the PCR products were cooled at 4 ° C. 27

35 2 Material and methods Table 12: MICA sequencing project: MICA-PCR process. MICA = major histocompatibility complex class I-related chain A, PCR = polymerase chain reaction. Step Temperature [C] Time [s] Number of cycles Gel electrophoresis followed to check the PCR. A two percent agarose gel was prepared for this purpose. 300 ml 1x borate buffer and 6 g Biozym-LE agarose were mixed and heated in the microwave. As soon as a temperature of 60 ° C. was reached during the subsequent cooling, six drops of ethidium bromide were added.The ethidium bromide is used to stain the nucleic acids. The mixture was poured into a gel electrophoresis chamber. After a cooling time of about 20 minutes, 7 μl of the PCR products were mixed with blue loading buffer and poured into the gel pockets. The electrophoresis was carried out for 15 minutes at 180 volts and 400 milliamps and then evaluated with the aid of a photo unit. Clearly visible bands indicated that amplification of the DNA had taken place. 28

36 2 Material and methods Approach A Approach B Approach C Approach D Figure 6: MICA sequencing project: gel electrophoresis of approaches A, B, C and D for the 8 different DNA samples number 1-8. Approach A contains primers for exons 2 and 3, approach B contains primers for exon 4, approach C contains primers for exon 5 / 7T, approach D contains primers for exon 5 / 8T. The light bands show the location of the MICA gene products that were amplified by the PCR. No MICA gene products are detectable at positions B8 and D7. MICA = major histocompatibility complex class I-related chain A, DNA = deoxyribonucleic acid, PCR = polymerase chain reaction, T = thymine. Before further processing, the PCR products were purified using Agencourt Ampure Beads and ethanol precipitation of the DNA. The Hamilton Microlab Star was used as the pipetting robot. This purification serves to remove excess salts and short DNA fragments from the PCR reaction. 96-well microtiter plates with a barcode were used for the subsequent sequencing. A master mix of 6 µl specific primer, 1.5 µl buffer and 0.5 µl BigDye was provided per well and 2 µl of the purified PCR product was added. The following table shows the primers used: Table 13: MICA sequencing project: sequencing primers. MICA = major histocompatibility complex class I-related chain A ,, E = exon, G = guanine, A = adenine, T = thymine, C = cytosine. MICA Primer E2-fwd-seq E2-rev-seq E3-fwd-seq E3-rev-seq E4-fwd-seq E4-rev-seq E5-rev-seq 5 3 Sequence GGA AGG TTG GGA CAG CAG AC GTG CCG GCT CAC CTC CCC TGC T TTC GGG AAT GGA GAA GTC ACT GC GAT GTG CCA ACA GGA AAT GC AAG AGA AAC AGC CCT GTT CCT CTC C TCC CTG CTG TCC CTA CCC TG CCC AAA TTC CCC AAC TTT CAT CC The primers were used for sequencing applied to the 96-well microtiter plate as shown: 29