IMPLEMENTATION OF FUNCTIONAL ASSAYS TO CHARACTERIZE AND DETERMINE THE CLINICAL IMPACT OF RUNX1 VARIANTS OF UNCERTAIN SIGNIFICANCE IN SPORADIC DISEASES AS WELL AS IN THE CONTEXT OF FPDMM
Author(s): ,
Melanie Decker
Affiliations:
Department of Human Genetics,Hannover Medical School,Hannover,Germany
,
Tim Lammens
Affiliations:
Department of Pediatric Hematology-Oncology,Ghent University Hospital,Ghent,Belgium
,
Alina Ferster
Affiliations:
Hematology-Oncology,Queen Fabiola Children's University,Brussels,Belgium
,
Lucy Godley
Affiliations:
Section of Hematology/Oncology, Department of Medicine, The University of Chicago Comprehensive Cancer Center,The University of Chicago,Chicago,United States;Department of Human Genetics,The University of Chicago,Chicago,United States
,
Miriam Erlacher
Affiliations:
Division of Pediatric Hematology-Oncology, Department of Pediatric and Adolescent Medicine,University of Freiburg,Freiburg,Germany
,
Ayami Yoshimi
Affiliations:
Division of Pediatric Hematology-Oncology, Department of Pediatric and Adolescent Medicine,University of Freiburg,Freiburg,Germany
,
Charlotte M. Niemeyer
Affiliations:
Division of Pediatric Hematology-Oncology, Department of Pediatric and Adolescent Medicine,University of Freiburg,Freiburg,Germany
,
Doris Steinemann
Affiliations:
Department of Human Genetics,Hannover Medical School,Hannover,Germany
,
Brigitte Schlegelberger
Affiliations:
Department of Human Genetics,Hannover Medical School,Hannover,Germany
,
Thomas Illig
Affiliations:
Department of Human Genetics,Hannover Medical School,Hannover,Germany;Hannover Unified Biobank,Hannover Medical School,Hannover,Germany
Tim Ripperger
Affiliations:
Department of Human Genetics,Hannover Medical School,Hannover,Germany
EHA Library. Ripperger T. Jun 15, 2019; 266943; PS1326
Tim Ripperger
Tim Ripperger
Contributions
×
Abstract

Abstract: PS1326

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Runt-related transcription factor 1 (RUNX1) encodes for a transcription factor subunit. RUNX1 binds core binding factor β (CBFβ) and together they function as a transcriptional regulator. While dimerization with CBFb augments DNA-binding affinity of RUNX1, its subsequent phosphorylation enhances its capacity to activate transcription. Regarding hematopoiesis, RUNX1 is known as a master regulator of transcription. In hematological malignancies, somatic genetic alterations of RUNX1 are frequently observed. Pathogenic germline variants of RUNX1 cause familial platelet disorder with associated myeloid malignancies (FPDMM). FPDMM is an autosomal dominant inherited disease associated with mild to moderate thrombocytopenia, platelet aggregation defects, and an increased risk to develop hematological malignancies, mainly MDS and AML. Functional consequences of RUNX1 missense variants detected in diagnostic analyses are often unclear.

Aims
Develop a first set of assays, in order to functionally characterize RUNX1 variants of uncertain significance (VUS) and determine their clinical impact. As positive controls, known pathogenic RUNX1 variants were investigated in parallel.

Methods
The capability of RUNX1 variants to bind to CBFβ was analyzed by a flow cytometry-based fluorescence energy transfer (FRET) assay. Expression and phosphorylation of RUNX1 variants were investigated by means of western blotting. Finally, the ability to activate transcription was assessed using four independent luciferase reporter constructs. All assays were performed in HEK293T cells.

Results
In comparison to RUNX1 wild type, we analyzed nine missense VUS and six known pathogenic variants of RUNX1. We observed significant impairment in all functional assays for known pathogenic variants. For six out of nine RUNX1 VUS, we gained evidence for their functional relevance. In the FRET assay, one RUNX1 VUS showed no dimerization and reduced dimerization ability was detected for two other VUS. By mobility shift in western blots, four VUS showed markedly decreased amounts of phosphorylation compared to wild type protein. Impaired phosphorylation was detectable for two additional VUS. Results were confirmed using a specific antibody against phosphorylated Ser249 of RUNX1. In contrast to RUNX1 wildtype, three VUS displayed no activation of all four luciferase reporters. Decreased activation capability of individual reporters was seen for three other VUS. In addition, variants lacking transcriptional activation ability, including the known pathogenic variants, tended to accumulate in HEK293T cells indicating their reduced degradation.

Conclusion
In a proof of principle approach, we succeed to establish functional assays addressing dimerization, phosphorylation, and transcriptional activation of RUNX1. Functional data of individual variants are in line with the hierarchy of molecular events of RUNX1 (i.e., 1. dimerization, 2. phosphorylation, and 3. transcriptional activation). Our assays can be used to test both somatic and germline variants and support the classification of VUS as pathogenic if significant functional impairment is observed. This allows testing of healthy family members to exclude carriers as potential HSCT donors and implement rational screening measures. In the future, we will study additional VUS and establish further assays to characterize RUNX1 variants in more detail.

Session topic: 9. Myelodysplastic syndromes - Biology & Translational Research

Keyword(s): Genetic, Mutation analysis

By clicking “Accept Terms & all Cookies” or by continuing to browse, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies