A Novel Variant of the CYCS Gene Alters Apoptosis of Megakaryocytes in a Family with Thrombocytopenia

Introduction: In humans, the CYCS gene encodes Cytochrome c, a protein important for the respiratory electron transport chain in mitochondria and for apoptosis. The clinical features of known CYCS pathogenic variants have been reported to be linked with thrombocytopenia. Herein, we present a novel variant of CYCS gene found in a Greek family with thrombocytopenia and its effects in apoptosis. Patients: A 1 year-old patient (Patient 1) with moderate thrombocytopenia (platelets=42×10 3/μl, MCV=76fL) and reported family history of thrombocytopenia has been referred to “Aghia Sophia” Children's Hospital for Whole Exome Sequencing. A 28 year-old female (Patient 2) and her 52 year-old mother (Patient 3) have been referred to the Hematology Unit of Attikon University General Hospital due to chronic grade III thrombocytopenia with no evidence of hemorrhagic diathesis. A thorough hematological examination of these two adult patients, including peripheral blood - bone marrow morphology, functional platelet assays, marrow cytogenetics and NGS, has detected no abnormalities. A trial of gamma globulin and steroids has been performed with no platelet response. Family history revealed more family members with thrombocytopenia on the side of the mother. In total five family members with moderate thrombocytopenia and four members with normal platelet counts have been tested with Sanger sequencing. Methods: Whole Exome Sequencing has been performed on Patient 1 and Sanger sequencing/Segregation analysis in nine members of the family. Florescence Activated Cell Sorting (FACS) has been performed in bone marrow mononuclear cells of Patient 2, Patient 3 and eight healthy controls. The antibody panel used included PE anti-human CD42a, PE/Cyanine7 anti-human CD42b, APC/Cyanine7 anti-human CD31 and APC anti-human CD71 antibodies. The stained cells (immature and mature megakaryocytes) have been sorted on a FACS Aria IIu (BD Biosciences) and subsequently stained with Annexin V FITC/7-AAD, to allow the detection of early-stage and late-stage cell apoptosis. Results: A novel missense variant of unknown significance (VUS) in the CYCS gene ( CYCS(NM_018947.6):c.292T>C (p.Tyr98His)) has been identified using whole exome sequencing in Patient 1. Segregation analysis of the variant in the family showed complete genotype-phenotype correlation (five family members with moderate thrombocytopenia, four members with normal platelet counts) (Figure 1A). The variant has been found to be heterozyg