Page 29 - Haematologica April 2020

RUNX1 variant curation
Lymphoid Tissues’ incorporated the classification of myeloid neoplasia with germline predisposition in their 2016 revised edition.4,5
In parallel, clinical laboratories are increasingly offering broad next-generation sequencing-based tests for patients with myeloid neoplasia for somatic testing, and will read- ily detect germline variants, if present in a patient. While there is increased clinical awareness of the potential for these germline variants to contribute to a patient’s dis- ease, there are often insufficient data in the literature to definitively classify whether a detected variant is con- tributing to the patient’s phenotype.6,7 For example, famil- ial platelet disorder with predisposition to AML (FPD/AML) is an autosomal dominant disorder in which germline mutations in RUNX1 result in thrombocytope- nia, platelet functional and/or ultrastructural defects, and/or susceptibility to hematologic malignancies com- monly including MDS, AML, and other malignancies8-11 (Table 1). ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) is a database repository of clinically actionable genomic vari- ants12,13 that currently lists 325 germline RUNX1 variants deposited by clinical laboratories. More than half of these variants are currently reported as being of uncertain sig- nificance.
Worldwide, most clinical laboratories follow the 2015 American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines for sequence variant interpretation.14 In this framework, germline variants are classified using a five-tier system: benign (BEN), likely benign (LBEN), vari- ant of uncertain significance (VUS), likely pathogenic (LPATH) and pathogenic (PATH). During sequence vari- ant interpretation, laboratories systematically review the supporting criteria of a genomic variant, such as: minor allele frequencies (MAF), computational predictions, functional experiments and segregation with disease in order to determine the five-tier classification.14-16
Although the ACMG/AMP guidelines provide a com- prehensive framework for sequence variant interpreta- tion, the high rate of VUS and curation discrepancies con- tinue to be an impediment to accurate clinical annotation and interpretation of genomic variants.6,7 To encourage genomic and phenotypic data sharing, and engage experts in consensus-driven variant interpretation, the Clinical Genome Resource (ClinGen) convened Variant Curation Expert Panels (VCEP) to develop gene- and disease-specif- ic modifications of the original guidelines and provide expert-reviewed variant classification for depositing into ClinVar (Online Supplementary Figure S1).17 In 2018, the American Society of Hematology (ASH) sponsored a ClinGen Myeloid Malignancy Variant Curation Expert
Panel (MM-VCEP), composed of 34 international mem- bers, who started working on gene- and disease-specific rules for RUNX1 as the first of several genes conferring predisposition to myeloid malignancies (Online Supplementary Figure S1A). After designing, modifying and testing the preliminary RUNX1 rules on 52 pilot variants, which improved classification in 33% VUS or variants with conflicting interpretations (CONF), MM-VCEP- specified ACMG/AMP rules were approved by the ClinGen oversight committee and efforts to curate vari- ants to ClinVar using the Variant Curation Interface have commenced (Online Supplementary Figure S1B).18 This pilot effort resulted in one variant being upgraded to PATH, two variants being upgraded to LPATH, and three vari- ants being downgraded to LBEN. ClinGen’s website con- tains the MM-VCEP variant classification recommenda- tions and any subsequent modifications to these codes over time (https://www.clinicalgenome.org/affiliation/10034/).
Herein, we demonstrate the application of RUNX1-spe- cific rules (Table 2) to classify nine representative RUNX1 variants in six examples (Table 3) while reviewing pheno- typic criteria for FPD/AML and summarizing molecular and functional roles of RUNX1.
Example 1. Early nonsense variants, (p.Arg204Ter) (PATH with PVS1, PM2, PS4_supporting, and PP1)
A 50-year old female with new pancytopenia was referred to a hematology service. A bone marrow biopsy showed hypocellularity with severe trilineage dysplasia and 12% blasts, diagnostic of MDS with excess blasts (MDS- EB-2). Further investigation showed pathogenic variants in RUNX1 (NM_001754:c.610C>T, (p.Arg204Ter)), BCOR and ASXL1 with a normal karyotype. The medical histo- ry was positive for thrombocytopenia (baseline 70- 120x109/L) and a propensity to excessive bleeding after tooth extractions. The family history was positive for two sons with persistent thrombocytopenia (baseline 50- 100x109/L) not otherwise explained and a granddaughter with thrombocytopenia and MDS with monosomy 7 (Figure 1). During the initial assessment, an increase in lactate dehydrogenase and the peripheral blast count were noted. A second marrow biopsy confirmed transfor- mation into AML with 40% blasts. The patient under- went induction chemotherapy without achieving remis- sion and clofarabine bridging for unrelated stem cell transplantation. During conditioning, the patient devel- oped sepsis with Gram-negative bacteria and died shortly afterwards. Since she had a remarkable personal and fam- ily history pointing towards a germline predisposition syndrome, a skin biopsy was performed at the time of the diagnosis of MDS, and DNA testing from cultured skin
Table 1. Clinical phenotypes of RUNX1 familial platelet disorder and hereditary malignancies.
Clinical and laboratory features
Hematologic malignancy
Thrombocytopenia
Platelet functional and/or
ultrastructural defects
Details
Commonly AML or MDS; less frequently T-ALL; and rarely mixed MPN/MDS
such as CMML, as well as B-ALL, and hairy cell leukemia
Mild to moderate thrombocytopenia with normal platelet size, in the absence
of other causes
Includes impaired platelet aggregation (particularly in response to collagen
and epinephrine) and platelet alpha or dense granule secretion defects
Life-time risk
~44%
Most patients
Not known
Adapted from Table 2 from Luo and Feurstein, et al.18 AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; MPN: myeloproliferative neoplasms; MDS: myelodys- plastic syndrome, CMML: chronic myelomonocytic leukemia.
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