Accurate germline RUNX1 variant interpretation and its clinical significance

TO THE EDITOR:

The frequency of germline RUNX1 variants in an unselected acute myeloid leukemia (AML) population is poorly defined and likely underestimated. The recent study by Simon et al¹  is particularly important as a first attempt to define this underlying frequency. Because RUNX1 is part of most next-generation sequencing panels performed on leukemic samples, germline variants are invariably found, highlighted by this and other studies.^1-4  Human and medical geneticists, genetic counselors, molecular pathologists, hematopathologists, and hematologists are particularly likely to encounter patients with germline RUNX1 variants and may benefit from guidance on how to interpret these variants and their clinical implications.

In the Simon et al¹  study, 10.7% (44/430) of AML patients had a somatic or germline RUNX1 variant. Germline variants represented 27.3% (12/44) of RUNX1 variants, suggesting a 2.8% frequency of germline RUNX1 variants in an unselected AML population. However, it was not clearly delineated whether the identified germline variants were all disease causing (ie, pathogenic or likely pathogenic), although the term “mutation” implies pathogenicity. Inconsistent usage of “variant” and “mutation” can lead to miscommunication of scientific findings, as well as clinical testing results: “mutation” refers to pathogenic/likely pathogenic variations that are deleterious and found less frequently in a population or are nongermline changes in a tumor cell (somatic mutations) that are predictive/therapeutic, diagnostic, or prognostic biomarkers (Table 1).⁵ 

Germline variant classification is performed using 5 ranks of pathogenicity: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign. Variants of uncertain significance, as well as likely benign and benign variants, should not be attributed to disease causality (Table 1). Accurate variant classification is critically important for attribution of pathogenicity of the identified variants and their actionability, because the identification of a deleterious germline variant has clinical implications that extend far beyond the treatment of the diagnosed individual.

In response to interlaboratory curation differences, the Clinical Genome Resource (ClinGen) has launched Variant Curation Expert Panels (VCEPs) to develop gene- or disease-specific American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) criteria.⁶  The Myeloid Malignancy (MM)-VCEP was formed in 2018 and published RUNX1-specific ACMG/AMP criteria in 2019.^7,8  Given our familiarity with the RUNX1 variant curation rules, we have reviewed the variants described in the Simon et al¹  study and found that only 7 of the 12 germline variants meet the criteria for pathogenic/likely pathogenic classification (Table 2). Thus, the actual yield of deleterious germline RUNX1 variants is 16% (7/44) of all RUNX1 variants and 1.6% (7/430) of all AML patients. Other than early truncating variants leading to non-sense–mediated decay, most causative RUNX1 variants are dependent on a variety of pathogenic evidence. In the case of RUNX1, this is usually a combination of computational and predictive, functional, population, and segregation data in a Bayesian framework.^7,9 

With regard to RUNX1 variant curation in the Simon et al¹  study as an example, we would like to highlight the following points. (1) Three major RUNX1 isoforms (A, B, and C) are expressed by the use of 2 promoters and alternative splicing. Isoform function, biological relevance, and expression differ in hematopoietic tissue,^10,11  which makes PVS1 not applicable for N-terminal truncating variants affecting only isoform C.^7,12  (2) Different strength levels of pathogenic functional evidence (PS3) are based on decreased or enhanced transactivation activity, with or without a secondary assay showing decreased DNA binding affinity, diminished heterodimerization ability with CBFb, abnormal cellular localization, reduced colony-forming potential, or abnormal function of mutant RUNX1 in vivo. Of note, other functional assays, such as interaction of RUNX1 with MLL, are not valid secondary assays for RUNX1 function.^13,14  (3) The presence of a RUNX1 germline variant in a proband with hereditary myelodysplastic syndrome (MDS)/acute leukemia, even with the typical phenotype including lifelong thrombocytopenia and platelet dysfunction, does not justify a pathogenic classification, but it is always dependent on a combination of additional functional, cosegregation, predictive, or population data. More information regarding the application of RUNX1-specific ACMG/Association for Molecular Pathology criteria in the classification of variants identified in this study is shown in Table 2.

As of 30 June 2020, 591 RUNX1 variants have been reported in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). Many germline disease-causing RUNX1 variants are unique to individuals or families; thus, detailed annotation is not always available for reference when a new RUNX1 variant is identified.¹⁵  Only 21% of RUNX1 variants are clinically significant (pathogenic/likely pathogenic), whereas the majority (79%) are benign/likely benign or variants of uncertain significance, which are not clinically actionable (Figure 1). It is worth mentioning that 50% of RUNX1 variants are variants of uncertain significance that warrant more collaborative efforts for the scientific community to up- or downgrade them based on new evidence, such as observation in multiple probands, segregation with disease, or functional impact of the variant or absence in affected individuals, nonsegregation with disease, or no effects on protein function.

Phenotypic criteria have been proposed by the ClinGen MM-VCEP, and they can be helpful in the determination of RUNX1 variant pathogenicity, because a high penetrance, with regard to thrombocytopenia and/or underlying platelet dysfunction, is typically recognized, and patients display ≥1 of the following features⁷ : mild to moderate thrombocytopenia with normal platelet size and volume in the absence of other causative factors; platelet ultrastructural and/or functional defects; and diagnosis of a hematologic malignancy, most commonly affecting the myeloid lineage (causing AML or MDS) and less frequently involving the lymphoid lineage and manifesting as T-cell acute lymphoblastic leukemia or others.

The following example highlights the importance of variant annotation for management decisions. A 56-year-old female with a diagnosis of MDS and a family history of hematologic malignancies was identified to have a germline RUNX1 c.167T>C (p.Leu56Ser) variant and was counseled that this RUNX1 variant was disease causing. Family members were tested for this variant to determine who lacked the variant and, thus, could be an appropriate stem cell transplant donor for the index patient and who in the family carries the variant and should receive surveillance on research protocols for RUNX1-associated familial platelet disorder with myeloid malignancy. Importantly, upon further review at the time of a second opinion, the RUNX1 c.167T>C (p.Leu56Ser) variant was reclassified to be a benign germline variant and a “red herring” in the evaluation of this family.

Patients with chronic otherwise unexplained thrombocytopenia, platelet ultrastructural and/or functional defects, and/or AML, MDS, or T-cell acute lymphoblastic leukemia should undergo genetic testing whenever there is a positive family history for a RUNX1 phenotype and when the patient has been diagnosed at a young age or a RUNX1 variant has been identified upon molecular testing of the leukemic clone. Germline material for testing should represent tissues that are not contaminated with blood/circulating blasts, such as cultured skin fibroblasts, which are the gold standard. Upon confirmation of a germline disease-causing RUNX1 variant, additional family members can be tested and followed-up long-term, including a baseline bone marrow biopsy with cytogenetic/molecular analysis and additional biopsies at the time of any significant/persistent change in blood counts. Most importantly, a family member with the RUNX1 variant should not be considered as a related stem cell donor, which makes recognition of the underlying germline syndrome paramount.^16-18 

Our clinical example and the variant interpretation by Simon et al¹  highlight how easily variants can be misclassified when criteria are not applied correctly, too much weight is put on the observation of the variant in affected probands, or the criteria are not combined correctly to reach the level of clinical significance (ie, disease causing). The accuracy of RUNX1 variant classification and interpretation is of great importance for treatment and follow-up of affected patients, related donor selection, and counseling of family members. Therefore, we emphasize that MM-VCEP RUNX1-specific rules, as the most accurate standards of germline RUNX1 variant classification, should be applied in clinical and research settings.^7,8