DOI: 10.1182/blood-2023-186060 ISSN: 0006-4971

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

Konstantina Giavi, Stavros Glentis, Anthi Bouchla, Anastasia Apostolidou, Nikolaos M. Marinakis, Antonis Kattamis, Eleni Katsantoni, Vasiliki Pappa
  • Cell Biology
  • Hematology
  • Immunology
  • Biochemistry

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 heterozygous in all family members tested. To investigate the functional role of the novel variant and its potential link to the thrombocytopenic phenotype FACS has been performed to isolate immature and mature megakaryocytes (positive for CD42a, CD42b, CD31 and medium positive for CD71) followed by analysis of apoptosis in Patient 2, Patient 3 and healthy controls. Cells undergoing early apoptosis have stained positive for Annexin V, while late apoptotic cells have been characterized by positive staining for both Annexin V and 7-AAD. Our preliminary results show increased numbers of apoptotic cells in the patients compared to controls (Figure 1B). We are currently validating this finding by inserting the CYCS gene variant in the megakaryocytic cell line DAMI, using the CRISPR-Cas9 system.

Conclusions: A novel missense variant in the CYCS gene has been identified in a Greek family with thrombocytopenia, where the phenotype is mild with isolated thrombocytopenia, no hemorrhagic diathesis and no any other organ dysfunction. FACS analysis of two variant carriers has revealed increased apoptosis of immature and mature megakaryocytes compared to controls. This study will delineate the molecular mechanisms underlying thrombocytopenia in the family and it is expected that the findings will be translated in novel therapeutic management strategies for patients with thrombocytopenic related pathologies.

Figure 1. A novel missense variant in the CYCS gene increases apoptosis in megakaryocytes. A. Segregation analysis of the family: Thrombocytopenia phenotype is marked with pink and carriers of the variant with dot. B. FACS analysis of megakaryocytes stained with Annexin V/7-AAD: Percentages of total apoptotic cells (early and late) are shown on the chart. Values represent mean ± SE, N=2 for patients and N=8 for controls (three FACS experiments have been performed for patient 2, two for patient 3 and one for each control).

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