Platelet CD34 Expression and a Congenital Collar Bone Malformation Associated with a Partial CBFB Deletion in a Case with a Bleeding Disorder

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34.

A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively).

To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases.

Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings.

Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development.