Introduction In inherited thrombocytopenic disorders (IT) associated with germline genetic variants in the transcription factors RUNX1 (RUNX1-RT) and ETV6 (ETV6-RT) or ankyrin 26 (ANKRD26-RT), bleeding is usually not clinically relevant. However, patients have a high risk (10-45%) of developing hematologic malignancies (HM). Genetic diagnosis at an early stage of life is essential for these patients and their families, as the underlying genotype determines the natural history of the disease and, consequently, its prognosis and clinical management. High-Throughput Sequencing (HTS) has greatly facilitated diagnosing and providing personalized medicine for these patients. Objective The aim of this study was to evaluate the frequency, clinical characteristics, platelet phenotype and molecular alterations of patients with IT predisposing to HM, within the series of IT patients recruited by the Spanish Group of Inherited Platelet Disorders (GEAPC). Methods Since 2008, we have enrolled 296 patients (174 families) suspected of having any type of IT. They were selected according to their clinical and biological background. Bleeding (bleeding score (BS); ISTH-BAT), complete blood count and smear, and in many patients platelet phenotyping (platelet aggregation (LTA), cytometry, microscopy, etc.) were performed. Sanger sequencing of candidate genes (until 2014) or HTS was used for genetic diagnosis of patients. Candidate genetic variants were classified in accordance with the ACMG/AMP criteria, also taking into account the adapted specifications for RUNX1. Results Genetic diagnosis was obtained in 208 patients (76.3%) (135 families), identifying 26 different types of IT. Thirty patients (17 families) had RUNX1-RT (14.4%), 16 (5 families) had ANKRD26-RT (7.7%) and 7 cases (4 families) had ETV6-RT (3.4%). In patients with RUNX1-RT, the median age at identification of cytopenia and at genetic diagnosis was 17 (0-56) and 27 (1-71) years, respectively. Mild-to-moderate thrombocytopenia (95x109 pl/L (15-142)) and mild bleeding (BS=2(0-12)) was observed in 89% of these patients. Of the 13 patients evaluated, 83.3% had a reduced LTA at 2µg/ml collagen and 80% had low levels of glycoprotein Ia (collagen receptor). We identified 15 different variants in RUNX1 (8 novel); 47% missense, 30% nonsense, 12% large deletions, 6% splicing and 6% frameshift. Sixty-five percent of the variants were classified as pathogenic (PV), 6% as likely pathogenic (LPV), and 29% of uncertain significance (VUS). In 7 cases the variant was de novo. Ten patients (33.3%) developed HM (median age 49 years) (Table 1). Of these, 53% had at least one family member with HM (AML, 53.5%; MDS, 10.7%; CML, 7.1%; AML-post-MDS, 3.6%) or solid tumor (breast cancer, 14.2% [50% BRCA2+]; prostate, 7.1%; stomach, 3.6%). In 16 cases with ANKRD26-RT, thrombocytopenia was identified at age 16 (0-60 years), while the genetic diagnosis was made at age 36 (0-79 years). In all cases the thrombocytopenia was moderate [median: 53.5x109 pl/L (8-119)], mostly asymptomatic (BS=1.5(0-12)) and not associated with platelet dysfunction (5 cases studied). We detected 3 different variants in the 5'UTR of ANKRD26 (1 LPV, 2 VUS). One patient had a de novo variant. Twenty-five percent developed neoplasia (3 HM cases; 1 breast cancer) at 42 years of age (median). Two of 5 families had a history of HM (MDS or AML) (Table 1). The ETV6-RT patients were genetically diagnosed at the age of 30 (3-59) years, while thrombocytopenia, moderate [97 x 109 cells/L (8-119)] and not associated with a bleeding diathesis (BS=0 (0-4)), was detected at the age of 12 (2-33) years. In one case, a defect in LTA, activation and platelet dense granule number was detected in platelet function studies. In this group we identified 4 different ETV6 missense variants (2 PV and 2 LPV/VUS). One patient developed B-ALL at the age of 12 years (Table 1). Conclusions Inherited thrombocytopenia that predisposes to HM is the most common form of IT in this Spanish cohort (25.5% of cases [19% of families]). We found an excessive delay (10-20 years) in the genetic diagnosis of patients with respect to the age of identification of the cytopenia. The high rate of development of HM of 28% reinforces the critical importance of early genetic diagnosis of these IT in order to apply optimal surveillance and clinical management aimed at early detection of their evolution to neoplasm.

Inherited platelet function disorders (IPFDs) are rare diseases caused by defects in platelet surface receptors, enzymes, granules, or signaling proteins. In humans, GPVI and P2Y12 deficiency cause autosomal recessive bleeding disorders, while TBXAS1 deficiency is related to Ghosal hematodiaphyseal dysplasa, a rare autosomal recessive disorder characterized by increased long bone density and platelet dysfunction without bleeding. To date, at least 20 patients have been identified with molecular defects in P2RY12, 12 cases with molecular defects in GP6, and 34 cases with molecular defects in TBXAS1. Here, we report a novel nonsense and missense variants in P2RY12, a novel nonsense variant in GP6, and a novel missense variant in TBXAS1. These variants selectively affect the platelet reactivity to ADP and collagen/CRP, predisposing to bleeding. P2RY12 c.835 G>A [p.Val279Met] variant did not affect receptor expression whereas P2RY12 c.44delG [p.Ser15Ilefs*33] lead to decreased levels of the receptor in one of the patients. This was confirmed both by RT-qPCR and immunoblotting analysis. Decreased expression of both GPVI and FcRγ-chain was detected in patients carrying GPVI nonsense variant in heterozygosis. The deleterious effect of these variants was also confirmed in a transfected cell line model. TBXAS1 variant triggered decreased TxA₂ production using a cell line model. These variants expand the genetic landscape of P2RY12, GPVI and TBXAS1 inherited deficiency.