Session: 509. Bone Marrow Failure and Cancer Predisposition Syndromes: Congenital: Poster III
Hematology Disease Topics & Pathways:
clinical trials, Research, Acute Myeloid Malignancies, AML, adult, Fundamental Science, Bone Marrow Failure Syndromes, Inherited Marrow Failure Syndromes, elderly, Clinical Research, Genetic Disorders, pediatric, Diseases, young adult , Myeloid Malignancies, Biological Processes, Study Population, Human, pathogenesis
To functionally characterize each variant, we used well-established in vitro functional assays to assess the damaging effect of each RUNX1 VUS, including transactivation reporter assays, electrophoretic mobility shift assays, co-immunoprecipitation, immunofluorescence, and western blot with cell fractionation. All assays include RUNX1 wildtype and known benign and pathogenic controls, as well as co-expression with its binding partner CBFβ. The VUS Ser94Arg located in RHD of RUNX1 has showed impaired CSF1R transactivation (< 20% of wildtype), reduced subcellular co-localization of RUNX1 with CBFβ, and reduced heterodimerization with CBFβ. These results together with population frequency data, in silico prediction, phenotype and segregation data support the reclassification of this variant into the likely pathogenic category. On the other hand, the VUS Asp123His shows no damaging effect in vitro, and could be reclassified to likely benign. Using these assays, we have confirmed or reclassified several RUNX1 variants using the criteria set by the ACGM Myeloid Malignancy VCEP (Luo et al, Blood Adv. 2019; 3:2962). Together, these results highlight the importance of using in vitro functional assays for RUNX1 variant classification.
Moreover, we sought to determine the potential of using platelet transcriptomics for RUNX1 variant classification and to gain a deeper understanding of FPDMM pathogenesis. Clariom™ D microarrays identified 218 differentially expressed genes (DEG; absolute fold change ≥ 2; q-value < 0.05) between FPDMM patients and healthy controls, with 90 genes (41%) being upregulated and 128 genes (59%) being downregulated in patients. Hierarchical clustering showed that the selected FPDMM patients with pathogenic and likely pathogenic RUNX1 variants clustered together and separately from healthy controls, with well-established pathogenic variants, such as Arg201Ter and Arg201Gln, at the opposite end of the dendrogram, and likely pathogenic variants such as Tyr403Cysfs153 closer to healthy controls. Our results show that FPDMM patients exhibit reduced expression of important genes related to thrombocytopenia and platelet dysfunction. Furthermore, several top upregulated genes identified in FPDMM patient’s platelets are associated with poorer outcomes in AML patients, according to the TCGA dataset.
Overall, our study shows the importance of performing functional assays to aid RUNX1 variant classification and to facilitate the study of FPDMM pathogenesis. We are currently analyzing the platelet proteome data of FPDMM patients by LC-MS/MS, which will be reported at the annual meeting.
Disclosures: No relevant conflicts of interest to declare.