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Functional validation of variants of unknown significance (VUS) in metabolic and pseudohypoxia genes involved in hereditary cancer syndromes

Summary of the Project

Due to application of high-throughput sequencing techniques in diagnostics and clinical research, we identify more and more patients with variants of unknown significance (VUS) in known cancer
associated genes. The impact of these variants on tumorigenesis can only be determined using
functional tests that are specific for the gene in question. Correct classification of germline VUS is of utmost importance for diagnostics of patients and their families with hereditary cancers, allowing for individualized surveillance strategies and treatment decisions, ultimately leading to prevention of cancer in these families. We recently demonstrated that metabolic screening using mass spectrometry based measurements of tumor metabolites facilitates functional characterization of variants in Krebs cycle genes and can be utilized to reclassify VUS in such genes to improve clinical decision-making. This collaborative project aims to establish and utilize in vitro systems to reliable determine the pathogenicity of VUS in known cancer genes involved in metabolism and pseudohypoxia by combining state-of-the-art genetic engineering and analytical approaches.

Scientific Goals

  • Establish isogenic cell models using CRISPR/Cas9 technology to evaluate the functionality of variants of unknown significance in SDHB, FH, and VHL.
  • Set up and validate our in vitro pipeline using metabolite screening and pseudohypoxia activity assays.
  • Functional in vitro testing of selected VUS from our available patient cohorts with our pipeline.
  • Identify targetable phenotypes in our isogenic knockout cell lines using omics.
  • The long-term goal of the presented project is to improve genetic diagnostics of patient with hereditary tumor syndromes by classifying VUS as either benign or pathogenic.

@ Susan Richter

Richter S, Gieldon L, Pang Y, Peitzsch M, Huynh T, Leton R, Viana B, Ercolino T, Mangelis A, Rapizzi E, Menschikowski M, Aust D, Kroiss M, Beuschlein F, Gudziol V, Timmers H, Lenders J, Mannelli M, Cascon A, Pacak K, Robledo M, Eisenhofer G, Klink B. Metabolome-guided genomics to identify pathogenic variants in isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocytoma and paraganglioma. Genet. Med. 2018 Jul;[Epub ahead of print].

Heining C, Horak P, Uhrig S, Codo PL, Klink B, Hutter B, Frohlich M, Bonekamp D, Richter D, Steiger K, Penzel R, Endris V, Ehrenberg KR, Frank S, Kleinheinz K, Toprak UH, Schlesner M, Mandal R, Schulz L, Lambertz H, Fetscher S, Bitzer M, Malek NP, Horger M, Giese NA, Strobel O, Hackert T, Springfeld C, Feuerbach L, Bergmann F, Schrock E, von Kalle C, Weichert W, Scholl C, Ball CR, Stenzinger A, Brors B, Frohling S, Glimm H (2018) NRG1 Fusions in KRAS Wild-Type Pancreatic Cancer. Cancer discovery 2018, [Epub ahead of print].

Rouhi A, Miller C, Grasedieck S, Reinhart S, Stolze B, Dohner H, Kuchenbauer F, Bullinger L, Frohling S, Scholl C (2017) Prospective identification of resistance mechanisms to HSP90 inhibition in KRAS mutant cancer cells. Oncotarget 8:7678-7690

Rump A, Benet-Pages A, Schubert S, Kuhlmann JD, Janavicius R, Machackova E, Foretova L, Kleibl Z, Lhota F, Zemankova P, Betcheva-Krajcir E, Mackenroth L, Hackmann K, Lehmann J, Nissen A, DiDonato N, Opitz R, Thiele H, Kast K, Wimberger P, Holinski- Feder E, Emmert S, Schrock E, Klink B (2016) Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer. PLoS genetics 12:e1006248

Richter S, Peitzsch M, Rapizzi E, Lenders JW, Qin N, de Cubas AA, Schiavi F, Rao JU, Beuschlein F, Quinkler M, Timmers HJ, Opocher G, Mannelli M, Pacak K, Robledo M, Eisenhofer G. Krebs Cycle Metabolite Profiling for Identification and Stratification of Pheochromocytomas/Paragangliomas due to Succinate Dehydrogenase Deficiency. J Clin Endocrinol Metab. 2014;99,3903-3911.


Dr. Susan Richter
Institute for Clinical Chemistry and Laboratory Medicine, University Hospital
Phone: +49 351 458 18068
Email: Susan.Richter(at)uniklinikum-dresden.de

Dr. Barbara Klink
Institute for Clinical Genetics, Carl Gustav Carus Faculty of Medicine at TU Dresden
Phone: +49 351 458 2894
Email: barbara.klink(at)tu-dresden.de