A Novel CBFA2 Mutation in a Family with Familial Platelet Disorder with Associated Myeloid Malignancy and Low Platelet MPL Expression

HELLER PAULA G; GANDHI MANISH J; GLEMBOTSKY ANA C; MARTA ROSANA F; CUMMINGS CARRIE; DRACHMAN JONATHAN G; MOLINAS FELISA C

San Diego, Estados Unidos

Congreso; 45th annual meeting of the American Society of Hematology; 2003

American Society of Hematolgy

Hereditary heterozygous mutations in the hematopoietic transcription factor CBFA2 have been identified in patients with familial platelet disorder with associated myeloid malignancy (FPDMM), which is characterized by autosomal dominant thrombocytopenia and propensity to develop myeloid malignancies. Haploinsufficiency or mutation of CBFA2 in humans impairs megakaryopoiesis although the mechanisms underlying this effect have not been clarified. Thrombopoietin (Tpo) binds to the MPL receptor on the surface of platelets and megakaryocytes and has a major role in megakaryopoiesis. To determine whether abnormalities in this regulatory pathway contribute to the pathogenesis of thrombocytopenia in this disorder, we studied Tpo levels and platelet MPL expression in a 3-generation family with FPDMM. The propositus (I:1), who had a history of thrombocytopenia, developed chronic myelomonocytic leukemia and died secondary to SNC bleeding. Her two daughters (II:1, II:2) and a granddaughter (III:1) have thrombocytopenia, platelet counts 62x109/l, 140x109/l and 90x109/l respectively, moderate bleeding diathesis, normal MPV, normal membrane glycoprotein Ib-IX and IIb-IIIa expression, dense granule deficiency and markedly abnormal platelet aggregation. Plasma Tpo levels, measured by ELISA (R D), were 200 pg/ml, 47 pg/ml and 98 pg/ml in patients II:1, II:2 and III:1 respectively, normal controls <15-78 pg/ml. Platelet MPL surface expression was assessed by flow cytometry with a polyclonal anti-MPL antibody (Kirin Brewery, Tokyo, Japan) and a ratio between fluorescence intensity of MPL respect to an isotype control was calculated, normal controls 1.62 0.23. Patients II:1 and III:1 revealed absent staining while it was decreased in patient II:2, ratio 1.25. Immunoblot analysis of platelet MPL content was performed by probing with anti-MPL antibody and reprobing with anti- 3 integrin antibody and a ratio between both bands was expressed as percentage of normal controls. Low levels of MPL were found in the 3 patients: II:1, 17%; II:2, 50%; III:1, 16%. Stimulation of platelets from patients II:1 and III:1 with Tpo failed to induce significant tyrosine phosphorylation of proteins, by immunoblotting with anti-phosphotyrosine antibody. The entire coding region and splice-junction sites of the MPL and CBFA2 genes were sequenced from the genomic DNA of affected individuals. We found that the MPL gene was entirely normal. However, a single basepair deletion was found in exon 6 of CBFA2 that causes a frameshift and premature chain termination (P245fs X252). This mutation, located just downstream of the DNA-binding runt domain, has not been previously identified and was present in all thrombocytopenic individuals. Studies are underway to investigate whether this mutation alters DNA-binding activity of the truncated protein. Of particular interest, two consensus DNA-binding sites for CBFA2 are present on the MPL promoter at positions -124 and -711. To our knowledge, this is the first description of a mutation in CBFA2 causing diminished MPL expression and provides a potential explanation for thrombocytopenia in affected family members.