Novel RUNX1 Mutation in a Family with an Uncharacterized Secretion Defect

Background: Platelet function disorders are a common cause of a bleeding problem. While many rare and severe forms of platelet function disorders are well studied, many common platelet function defects are uncharacterized. In a family with uncharacterized platelet secretion defects, we identified a single base pair insertion in the gene RUNX1 by exome sequencing. We report on the clinical and laboratory phenotype of the defect in this family.

Methods: Bleeding histories of affected and unaffected family members, obtained using a standardized questionnaire, were scored using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). Laboratory data evaluated included blood counts, platelet aggregation responses, dense granule ATP release findings (lumiaggregometry) and platelet dense granule counts, evauated by whole mount preparations and electron microscopy. Affection status was based on the recorded opinion of the subject's hematologist, which concurred with diagnostic laboratory findings. Exome sequencing was performed on the index case, followed by evaluation of all family members for the candidate mutation by polymerase chain reaction (PCR) amplification and Sanger sequencing.

Results: Family members investigated (median age: 25.5, range 1-69] included 5 males (affected: n=4, unaffected: n=1) and 3 females (affected: n=2, unaffected: n=1). ISTH BAT bleeding scores for affected members were elevated (median: 10.5, range 4-20), unlike unaffected family members (n=2) and healthy controls (n=40) (ranges: 0-1 and 0-2). Platelet counts were low in 2/6 affected individuals (10⁹ platelets/L : affecteds: median: 164, range: 125-169). While unaffected family members had unremarkable platelet function findings, the affected individuals had absent or reduced dense granule secretion with all agonists tested (including thrombin, collagen, epinephrine, U46619 and arachidonic acid), and in aggregation tests, they had absent secondary aggregation with epinephrine, reduced maximal aggregation with collagen (1.25 and 5.0 μg/mL) and thromboxane analogue U46619, variable responses to arachidonic acid (reduced in 4/5 affecteds), and normal responses to ADP and ristocetin. Only some (3/6) affected family members had dense granule deficiency (lower reference interval limit: 4.9/platelet; affecteds: median: 5.2, range: 4-6). A single base pair insertion (A) in exon 6 of the RUNX1 gene (NM_001754.4:c.583dup) was identified in the index case using exome sequencing. The mutation results in a reading frame shift at codon Ile195, that is expected to end in a premature termination codon 17 positions downstream. Sanger sequencing further confirmed this mutation was present in 6/6 affected family members and it was absent in both unaffected family members. Further evaluation of the family history indicated that only one family member (deceased aunt of the index case) had developed leukemia or myelodysplasia although her mutation status is unknown.

Conclusion: We identified a novel, frameshift RUNX1 mutation in a family with an inherited platelet function disorder associated with increased bleeding symptoms. This variant has not been previously reported in exome sequencing of over 60 000 individuals as documented by the ExAC consortium. The variable platelet count and dense granule numbers among affected individuals in this family emphasize the importance of phenotyping multiple family members. RUNX1 defects should be considered as a potential cause of inherited abnormalities in dense granule secretion, even in individuals without dense granule deficiency or a striking family history of leukemia/myelodysplasia.