1. Ivana Jochmanova, Karel Pacak Genes selection and sample preparation for genome sequencing MGL User Group Meeting August 20th, 2014

2. Outlines 1Pheochromocytoma/paraganglioma (PHEO/PGL) 2Gene selection for sequencing 3Backround and rationale for sample selection 4Sample preparation method

3. Pheochromocytoma/paraganglioma (PHEO/PGL) catecholamine producing tumors arising from sympathetic- or parasympathetic-associated chromaffin tissue. PGLs derived from chromaffin cells of adrenal medulla are called PHEOs.

4. Genetics of PHEO/PHL PHEO/PGL susceptibility genes Major: VHL, RET, NF1, SDHB and D Minor: TMEM127, SDHA and C, SDHAF2, MAX New genes: KIF1Bβ, PHD2, HIF2A, IDH, FH, H-RAS, K- RAS, BAP1 mutations in PHEO/PGL susceptibility genes affect HIF- α signaling pathway Gimenez-Roqueplo et al.: Horm Metab Res 2012; 44:328-33 Zhuang et al.: N Engl J Med 2012; 367:922-30 Jochmanova et al.: Physiol Res 2014; 63:S251-62

5. Invasion and metastasis Cell proliferation/(de)differentiation/ survival Glucose/energy metabolism Genomic instability Epithelial-mesenchymal transition Red blood cell production and iron metabolism HSP90 HRE p300 CPB HIF- α HIF- β Angiogenesis HIF target genes Jochmanova et al.: J Natl Canc Inst 2013; 105:1270-83

6. Summary of the pathways affecting HIF turnover involved in the development of various PGLs. TMEM127 MAX NF1 RET Jochmanova et al.: J Natl Canc Inst 2013; 105:1270-83

7. Selection of genes for sequencing Rationale for gene selection -in our research we are focusing on signaling pathways changed or disrupted in PHEO/PGL, especially HIF signaling pathway -selection of genes was done based on their involvement in the signaling pathways which were found to be altered in cancer, including PHEO/PGL -we also included genes already known to be mutated in patients whose samples were sequenced – this step was important to validate the sequencing data

8. Examples of genes selected for sequencing Genes known to be mutated in PHEO/PGL SDH-A, -B, -C, -D; SDHAF2, FH, VHL, NF1, RET, MAX, TMEM127, EGLN1, EPAS1 Genes involved in Krebs cycle: SDH-A,-B, -C,-D; FH; IDH; … Genes involved in MAPK signaling pathway: MAP2K1, -K2, -K4, MAP3K1 Genes involved in PI3K/AKT signaling pathway: PIK3CA, PIK3CG, PIK3R1, PIK3R2, AKT1, 2; PDK1, TSC1, 2 Genes involved in mTOR signaling pathway: EIF4E Genes involved in HIF signaling pathway: HIF1AN, HIF3A, EPAS1, VHL,…

9. Backround and rationale for sample selection 16 samples were selected: 8 genomic DNA samples (blood) 8 tumor DNA samples 10 samples were from patients with already known mutation associated with PHEO/PGL development (SDHB, SDHC, SDHD (2 samples), MAX, TMEM127, RET, and NF1 – these were selected to “validate” the sequencing accuracy 6 samples were from patients with not yet known mutations, thus to find out if sequencing will reveal any possibly new damaging mutations associated with PHEO/PGL

10. Sample preparation Blood and/or tumor tissue samples were obtained from patients after written consent. Blood sample preparation Isolation of DNA using commercial kit (Qiagen Dneasy Blood & Tissue Kit) Tissue DNA sample preparation Isolation of DNA using commercial kit (Qiagen Dneasy Blood & Tissue Kit)

11. THANK YOU FOR YOUR ATTENTION