Validation and clinical application of transactivation assays for RUNX1 variant classification

Heterozygous pathogenic germline variants of RUNX1 cause familial platelet disorder with associated myeloid malignancies (RUNX1-familial platelet disorder [RUNX1-FPD], FPDMM, FPD/AML).^1,2  Patients typically show mild to moderate thrombocytopenia or even platelet counts in the low-normal range as well as qualitative platelet defects.^2-7  Affected individuals face an ∼40% risk of developing hematologic malignancies.⁸  Germline variants of RUNX1 are classified following American College of Medical Genetics/Association for Molecular Pathology guidelines⁹  specified by recommendations of the ClinGen Myeloid Malignancies Variant Curation Expert Panel (MM-VCEP).⁸  In general, classification is based on criteria such as population, computational, segregation, and functional data. RUNX1 missense variants must frequently be classified as variants of uncertain significance (VUS) not allowing clinical conclusions because only variants considered as (likely) pathogenic confirm diagnoses. As demonstrated,¹⁰  functional analyses of VUS can contribute to their reclassification to (likely) pathogenic. Independent of patients’ phenotype and family history, transactivation assays (TAs) address RUNX1’s major function and are, therefore, suitable for first-line screening.¹⁰  In the present study, we evaluate applicability of TAs¹⁰  for different regions of RUNX1 and applied them to 11 RUNX1 variants for independent validation of their clinical utility.

We analyzed the ability of variants to activate transcription of RUNX1 target genes CSF1R, ETV1, and MYL9 by applying luciferase reporter assays in nephrogenic HEK293T and hematopoietic HEL cells.¹⁰  As controls, wild-type (WT) RUNX1b, the pathogenic variant Arg139Gln, and the benign variant Leu29Ser were included. The ability to activate transcription was scored into categories according to the MM-VCEP recommendations: (1) <20% (PS3 moderate); (2) 20% to 80%; (3) >80% to 115% (BS3 supporting); and (4) >115% (PS3 supporting) of WT activity.⁸  Since there are no specific instructions on combining results of multiple TAs, we followed general recommendations^9,11  and concluded the applicability of the functional BS3/PS3 criterion (for details, please refer to Figure 1C). If available in the literature, we included data from secondary assays to further adapt the strength of BS3/PS3 criteria (supplemental Table 3; Figure 1C). This led to a final functional class independent of patients’ phenotype and family history. When both BS3 and PS3 or neither could be applied, variants were classified as variants of uncertain function. Finally, variants were classified following MM-VCEP recommendations including all available data.⁸  Variant nomenclature refers to transcript variant 2 (NM_001001890.3) encoding for RUNX1b (for RUNX1c, please refer to supplemental Table 1).

First, we aimed to validate if TAs are suitable to detect pathogenic RUNX1 variants affecting different regions of the protein. We chose a set of 7 RUNX1 (likely) pathogenic variants leading to premature termination codons. We studied them in TAs using CSF1R, ETV1, and MYL9 reporters (Figure 1A). Using ETV1 and MYL9 reporters, premature termination codon variants up to codon 308 displayed less than 20% of WT activity. The CSF1R reporter was suitable to detect pathogenic variants up to codon 235 because for Gln235*, enhanced transactivation of the CSF1R reporter was seen. In conclusion, present TAs can be applied to variants N-terminal of codon 308 (rETV1, rMYL9) or codon 235 (rCSF1R) covering most of the missense variants in RUNX1-FPD.^3,12  Of note, of 31 reported missense germline RUNX1 variants classified as VUS in the RUNX1 Database, 6 are located C-terminal of codon 308.¹² 

In the present study, we focused on 11 RUNX1 variants affecting codons before residue 308 (Figure 1B). All variants have been identified as heterozygous germline variants in patients with thrombocytopenia, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or as secondary finding. We observed transactivation defects for Ser67Arg, Arg80Ser, Val91Glyfs*11, Arg205Trp, and partly for Gly143Arg (Figure 1B-C). Following present classification standards,^8,9  we reclassified Ser67Arg and Arg205Trp from VUS to likely pathogenic. Our data functionally confirmed the (likely) pathogenic classification of Arg80Ser and Val91Glyfs*11. Additionally, TAs demonstrated functionality of Asp96His, Pro218Ser, Arg223His, and Gln259Lys, but the lack of further benign criteria does not allow reclassification to (likely) benign.

For 6 of the 11 protein variants included in our study, clinical data were available to assess the clinical utility of our functional investigations (Figure 2). Families A-C were characterized by RUNX1-FPD-typical phenotypes including AML, MDS, thrombocytopenia, and platelet defects. Our functional analyses of Ser67Arg, Arg80Ser, and Arg205Trp,¹³  identified in these families, revealed functional defects and led to or confirmed their likely pathogenic classification. Molecularly confirmed RUNX1-FPD allowed targeted management of index patients, predictive genetic testing, and, subsequently, surveillance of carriers.¹⁴  Surveillance permits early detection of MDS and, thus, may improve prognosis because stem cell transplantation can be timely performed.¹⁵  In family D, 2 carriers of the variant Asp96His were identified, 1 of them presenting with platelet defects. Owen et al¹⁶  have reported a classical RUNX1-FPD family also carrying this variant. It was recently reclassified to VUS following expert guidelines⁸  and present TAs displayed functionality. In pedigree E, Pro218Ser was identified in twins presenting with thrombocytopenia^17,18  and, later, also in their father having normal platelet counts and function. This raised suspicion regarding the causality of the variant. In line with this, no experimental evidence for a functional impairment was observed. The variant Arg223His was reported to us in 2 independent families. In family F, it was identified by whole exome sequencing as a secondary finding in a girl with a complex phenotype. It was additionally reported in the index patient in family G having thrombocytopenia and MDS, but no RUNX1-FPD-specific family history. No functional impairment was observed by our TAs because all reporters showed at least 70% of WT activity. In conclusion, our functional data and the resulting variant classification confirmed RUNX1-FPD suspicion in families A-C, which was consistent with patients’ phenotypes and family histories. In families D-F, no FPD-suspicious family history was reported; from our perspective, this is a rare occasion in known RUNX1-FPD families. In line with this, we demonstrated functionality of the reported variants. Implementation of easy-to-apply TAs for missense VUS N-terminal of codon 308 is recommended and allows functional classification independent of patients’ phenotype. This improves translation of genetic data into personalized clinical care by supporting RUNX1-FPD diagnoses or denying functional impact of variants investigated.

In summary, we validated the clinical utility of our previously reported TAs¹⁰  by studying additional RUNX1 variants and discussing them in their clinical context. Limitations of TAs regarding variants C-terminal of the codon 308 have to be considered, but, in the context of RUNX1-FPD, this applies only to a minority of observed RUNX1 missense variants.^3,12  Implementation of adequately performed TAs in the MM-VCEP classification scheme can be key to reclassify VUS observed in RUNX1, which is essential for patient care because only (likely) pathogenic variants molecularly confirm diagnoses. Following the present guidelines, the majority of VUS without functional impairments cannot be reclassified to (likely) benign, even if secondary assays demonstrate functionality.¹⁰  This limitation illustrates the need to consider a careful reevaluation of the present MM-VCEP guidelines. TAs support translation of genetic data into personalized clinical care, regarding both index patients and relatives at risk and, if applicable, should be integrated as first-line screening when VUS are identified. Future attempts must focus on the establishment of functional assays covering the C-terminal part of the protein and consider high-throughput assays that allow fast and accurate classification of RUNX1 variants. Because RUNX1-FPD may be underdiagnosed among patients with AML^12,13,19  and germline testing of all patients with hematologic malignancies might be considered in the future,²⁰  variant classification will become even more important to support clinical decision-making.

The authors thank the RUNX1 research program and ERN GENTURIS for referral of clinicians regarding functional investigation of RUNX1 variants and are most grateful for getting access to the Amaxa Nucleofector IIb of the research group of the Institute of Clinical Chemistry at Hannover Medical School.

The project was funded by the European Hematology Association John Goldman Clinical Research Grant 2017 (T.R.) and the Research Network MyPred (01GM1911B) funded by the German Federal Ministry of Education and Research (M.D., A.F., T.I., and T.R.).

Contribution: T.R. designed the study; M.D. and T.R. designed and established experiments, which were performed, analyzed, and visualized by M.D. and T.R.; M.D., A.F., and T.R. performed the variant classification; Gene variants and clinical data were provided by A.B., J.C., H.H.-V., I.H., M.N., A.V., A.A., A.D., M.P.T.E., M.H.G.P.R., T.H.A.T., and N.D.; and M.D. and T.R. wrote the manuscript critically revised by all coauthors.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Tim Ripperger, Department of Human Genetics, Hannover Medical School (MHH), OE 6300, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; e-mail: ripperger.tim@mh-hannover.de.