Germline Variants of RUNX-1 in Myeloid Malignancy
Authors: Nielsen Alek d , Alharbi Sayer , Hirsch Cassandra M. , Przychodzen Bartlomiej P , Sekeres Mikkael A. , Saunthararajah Yogen , Carraway Hetty E. , Maciejewski Jaroslaw P 
Published: November 2019

Abstract
Mutation of the master transcription factor RUNX1 (NM_001754) has a well characterized role in the pathogenesis of myeloid neoplasms. RUNX1 is located at 21q22 and is composed of a c-terminal transactivation domain (TAD) (269-480) and an n-terminal RUNT domain (amino acid 76-209) that binds DNA and mediates heterodimerization with CBF-β. If the RUNT domain is disrupted a RUNX1 mutant will behave in a dominant negative fashion. Mutations leaving the RUNT domain intact result in haploinsufficiency compounded by CBF-β binding site competition with the wild-type allele. Germline (GL) RUNX1 mutations cause the autosomal dominant predisposition syndrome familial platelet disorder with propensity to myeloid malignancy (FPDMM). In FPDMM, RUNX1 mutations, including RUNT domain missense mutations or nonsense mutations throughout the gene, confer mild chronic platelet disorders and a lifetime risk of MDS/AML. As with other GL predisposition syndromes a secondary event is considered necessary for progression, typically an additional mutation in the wild type RUNX1 allele. Onset of MDS/AML occurs at a median age of 33 years but the latency can be variable and ease of detection in younger cases likely distorts summary statistics. Inherited mutations in adults may be difficult to distinguish and have not been systematically explored. We hypothesize that a fraction of patients with otherwise typical RUNX1-positive MDS or related disorders are in fact carriers of a RUNX1 GL mutation.
DNA obtained from a cohort of 1451 patients with myeloid neoplasia was analyzed using a multiamplicon deep next-generation sequencing (NGS) panel including all ORFs of RUNX1. A total of 124 patients (8.5%) were found to carry 117 unique RUNX1 mutations. Of these, 59 hits were missense and 58 nonsense; and in 94 (76%) of cases the RUNT domain was disrupted. Eighteen (15.3% RUNX1 positive patients) were previously described in FPDMM. We applied various bio analytic criteria to designate somatic status in 57 patients. The remaining 64 RUNX1 mutations were further investigated, when possible, by paired capillary sequencing of CD3- mononuclear DNA and in-vitro expanded CD3+ T-Cell DNA. We found 5/31 (16%) of these cases carried mutation in both myeloid and lymphoid lineages and were designated GL. This included 2 post-RUNT truncations (p.Y281*, p.S410*) and two RUNT domain missense mutations (p.R80C, p.S141L) all of which are expected to produce a dominant negative phenotype. A 5th GL mutation of uncertain significance in the TAD (p.M310I) was also discovered. Excluding this TAD missense mutation, each of these have been described as somatic factors in myeloid disease. The truncation at amino acid 410 is further downstream than all reported FPDMM variants to date. Chart review revealed an anamnestic presence of thrombocytopenia in 80% of proposed FPD patients. Suggestive family history was found in both truncated cases, with hematologic malignancy presenting in a 1st degree relative before 30 years of age. Median age at diagnosis of MDS/AML in GL cases was 46 years (range: 18-68 years) compared to 65 years in the cases designated somatic (range: 37-87 years). Two cases (p.S410* and p.M310I) had cytogenetic abnormalities on the 21st chromosome (trisomy 21 and t(8;21) respectively) and had no other molecular abnormalities detected by our NGS panel. A mutation in GPR98 of unknown significance was found with p.Y281*. Lastly the p.S141L mutant had cooperating mutations in chromatin modifiers ASXL1 and BCOR, and in RNA splicing gene LUC7L2. This study of an exemplary GL leukemia gene suggests that systematic searches for known and potential GL predisposition genes in otherwise typical adult cohorts may reveal a GL role in the evolution of myeloid neoplasms. Future work to determine genetic predisposition to leukemia in adults is essential.

Disclosures
Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.