Inherited thrombocytopenias (ITs) are disorders characterized by a reduced platelet count, caused by variants in at least 40 genes. Mutations in CYCS cause a rare autosomal-dominant IT, named Thrombocytopenia 4 (THC4). The CYCS gene encodes cytochrome C (CytC), a small heme protein localized in mitochondria. It functions as an electron carrier in the respiratory chain as well as one of the main actors of the intrinsic pathway of apoptosis.

Although THC4 is the only disease associated with CYCS mutations, its clinical and molecular features are still poorly characterized. Moreover, pathogenesis of THC4 is still unclear. In vitro studies demonstrated that CYCS mutations exert a proapoptotic activity and enhanced peroxidase activity, as well as affect the mitochondrial respiratory function in yeast.

The role of apoptosis in platelet biogenesis is a highly controversial issue. Some studies suggested that megakaryocytes must undergo apoptosis to promote platelet release. In fact, mice carrying mutations impairing apoptosis exhibited thrombocytopenia and pharmacological inhibition of caspases blocked platelet production, supporting this hypothesis. Conversely, investigations on mice genetically modified for the BCL-2 family proteins, initiators of apoptosis, concluded that megakaryocytes must restrain apoptosis in order to generate platelets.

Since thrombocytopenia is the only phenotypic manifestation of CYCS mutations, the investigators believe that THC4 represents a unique model for deciphering the role of Cytc and apoptosis in megakaryopoiesis and platelet biogenesis.

Preliminary data:

The applicant unit is a reference center for ITs. The database includes 375 families; among these, 7 presented with mutations in CYCS gene, accounting for 26 THC4 patients.

Patients presented mild thrombocytopenia, normal mean platelet volume and morphology, without hematological or extra-hematological defects.

The investigators isolated platelets and hematopoietic progenitors from peripheral blood of 9 patients. Platelets were analyzed under baseline conditions; a part was treated with an inducer of apoptosis (ABT-737).

Surprisingly, the expression of cytC was found reduced in platelets of all analyzed patients. Platelets showed no signs of early death. Instead, when stimulated with ABT-737, they showed inability to undergo apoptosis, suggesting that CYCS mutations confer resistance to apoptosis.

Hematopoietic progenitors from 3 patients were differentiated in megakaryocytes. Maturation and differentiation were comparable between THC4 and healthy controls. Instead, their capability to form proplatelets was profoundly defective.

These preliminary data suggest that CytC deficit could be associated with inability to form proplatelets in THC4 megakaryocytes, and that this defect underlies thrombocytopenia, supporting the crucial role of cytC in platelet production.

Inclusion Criteria:

• Molecular diagnosis of THC4, through the identification of a pathogenic variant of the CYCS gene
• Acquisition of written informed consent

All subjects with suspected Inherited Thrombocytopenia (IT) observed at the SC Medicina generale 1, Fondazione IRCCS Policlinico San Matteo