Validation and clinical application of transactivation assays for RUNX1 variant classification.

Area of Science:

• Genetics and Genomics
• Hematology
• Molecular Biology

Background:

• Familial platelet disorder with associated myeloid malignancies (RUNX1-FPD) arises from pathogenic germline variants in the RUNX1 gene.
• Accurate classification of RUNX1 variants is critical for patient diagnosis and family risk assessment.
• Current variant classification guidelines may require reevaluation, particularly for variants of uncertain significance (VUS).

Purpose of the Study:

• To evaluate the applicability of transactivation assays for investigating RUNX1 variants across different protein regions.
• To independently validate transactivation assays for variant classification according to established guidelines.
• To assess the clinical utility of functional assays in diagnosing RUNX1-FPD and guiding patient care.

Main Methods:

• Studied 11 RUNX1 variants using transactivation assays.
• Validated assays against ClinGen Myeloid Malignancies Variant Curation Expert Panel guidelines.
• Applied functional assays to assess variants in familial cases with suspected RUNX1-FPD.

Main Results:

• Two variants of uncertain significance (VUS) were reclassified as likely pathogenic.
• Four VUS demonstrated functionality, but reclassification to likely benign proved challenging.
• Assays confirmed RUNX1-FPD in 3 families; no functional defect was found for 3 variants in non-specific families.
• New assays are needed for C-terminal RUNX1 variants.

Conclusions:

• Transactivation assays are essential tools for RUNX1 variant classification and diagnosing RUNX1-FPD.
• Current classification guidelines may need revision, especially concerning VUS and C-terminal variants.
• Functional assay application facilitates personalized medicine by translating genetic findings into clinical action.