Identification of novel tumor predisposition families and underlying genetic defects

7 REVIEW OF THE LITERATURE . 9 1. Tumorigenesis . 9 2. Human genome . 11 3. Tumor genome . 13 3.1 Driver mutations and genes . 15 4. Inherited susceptibility to tumors . 16 4.1 Rare high risk variants . 17 4.2 Rare moderate risk variants . 18 4.3 Common low risk variants . 18 5. Infectious causes of cancer . 19 6. Identification of rare high risk tumor susceptibility genes . 22 6.1 Linkage analysis . 22 6.2 High-throughput sequencing . 23 7. Tumor susceptibility conditions scrutinized in this study . 24 7.1 Kaposi sarcoma. 24 7.2 Nodular lymphocyte predominant Hodgkin lymphoma . 25 7.3 Meningioma . 26 AIMS OF THE STUDY . 28 MATERIALS AND METHODS. 29 1. Ethical considerations . 29 2. Study subjects and samples . 29 2.1 Patients in the Finnish Cancer Registry (I) . 29 2.2 Kaposi sarcoma patients (II) . 29 2.3 Hodgkin lymphoma patients (III) . 30 2.4 Meningioma patients (IV) . 31 2.5 Control samples (II, III, and IV) . 32 2.6 DNA and RNA extractions (II, III and IV) and cDNA synthesis (II and III) . 32 3. Clustering of the patients in the Finnish Cancer Registry (I) . 33 3.1 Clustering procedure (I) . 33 3.2 Cluster score calculation (I) . 33 4. Genetic analyses . 33 4.1 Genotyping (II, III and IV) . 33 4.2 Transcriptome analysis (III and IV) . 34 4.3 DNA copy number analysis (IV) . 34 4.4 Linkage analysis (II, III, and IV) . 34 4.5 Exome and genome sequencing (II, III, and IV) . 35 4.6 PCR and Sanger sequencing (II, III, and IV) . 37 4.7 Loss of heterozygosity analysis (IV) . 37 5. Protein analyses . 37 5.1 Serological testing of human herpes virus 8 (I and II) . 37 5.2 In silico predictions and modelling (II, III and IV) . 37 5.3 Flow cytometry (II) . 37 5.4 Cloning and site-directed mutagenesis (IV) . 38 5.5 Cell culturing and production of ShhN conditioned media (IV) . 39 5.6 Luciferase assay (IV) . 39 5.7 Immunoprecipitation and western blot (IV) . 39 5.8 Immunocytochemistry (IV) . 39 RESULTS . 40 1. Cancer registry based familial clustering of cancers in Finland . 40 1.1 Frequent familial clustering of Kaposi sarcoma in Finland . 40 2. STAT4 as a candidate susceptibility gene for Kaposi sarcoma . 42 2.1 Prioritization of the candidate variants . 43 2.2 STAT4 activation is not affected by p.Thr446Ile mutation. 44 2.3 IFN- production is decreased in the p.Thr446Ile variant carriers . 44 2.4 IL-4 and IL-17 responses in the p.Thr446Ile carriers . 44 2.5 Screening of STAT4 and associated genes . 44 3. NPAT may predispose to nodular lymphocyte predominant Hodgkin lymphoma . 45 3.1 Analysis of large chromosomal alterations and EBV-infection . 45 3.2 Identification of NPAT as a candidate susceptibility gene . 45 3.3 Expression of NPAT in the c.2437-2438delAG carriers. 46 3.4 In-frame deletion of p.Ser725 in Hodgkin lymphoma . 47 4. Loss-of-function mutation in SUFU in meningioma predisposition . 47 4.1 NF2 is not associated with the Finnish meningioma family . 47 4.2 Identification of meningioma susceptibility genes . 48 4.3 In silico modelling of the p.Arg123Cys SUFU mutation . 50 4.4 Mutated SUFU leads to dysregulated Hedgehog signaling . 51 4.5 Screening of SUFU in additional meningioma samples . 52 DISCUSSION . 54 1. Familial aggregation of tumors in Finland . 54 1.1 Identification of potential tumor susceptibility families. 56 2. Kaposi sarcoma in the Finnish population . 57 2.1. STAT4 and Kaposi sarcoma . 58 3. NPAT in Hodgkin lymphoma predisposition . 60 4. SUFU, Hedgehog signaling, and meningioma . 62 CONCLUDING REMARKS AND FUTURE PROSPECTS . 65 ACKNOWLEDGEMENTS . 68 WEB-BASED RESOURCES . 70 REFERENCES . 71