A Novel Cycs Missense Mutation in the α-Helix of the Cycs C-Terminal Domain Causes Non-Syndromic Thrombocytopenia

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. The CYCS gene (MIM123970) encodes cytochrome c, somatic, a small heme protein localized in the inner membrane of mitochondria. Cytochrome c is an electron carrier in the mitochondrial respiratory chain as well as a key initiator of apoptosis (Cell Death Differ. 2018;25(1):46-55). To date, only three families with CYCS missense variants have been reported: a large New Zealander family (p.Gly42Ser), an Italian family (p.Tyr49His), and a white/mixed British family (p.Ala52Val) (Nat Genet. 2008;40(4):387-9, Biochim Biophys Acta. 2014;1842(2):269-74, Haematologica. 2016;101(10):1170-1179). These patients were affected by an autosomal dominant phenotype characterized by low platelet count without extra-hematological findings. Moreover, these patients' peripheral blood cell morphologies, including platelet size, were all normal.

In this study, we identified a novel heterozygous mutation in CYCS using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α-helix of the cytochrome c (CYCS) C-terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. By the deletion mutation, the Ala102 position is considered to be replaced with the neighboring Thr103, which would impair the hydrophobic core and destabilize protein folding. All three reported missense mutations are clustered within the highly conserved Ω-loop (Biochim Biophys Acta. 2014;1842(2):269-74, Sci Rep. 2016, 27;6:30447, Biophys Chem. 2017;230:117-126). Previous structural analyses also showed that two mutations in the Ω-loop (p.Gly42Ser and p.Tyr49His) of CYCS disrupted hydrogen bonds (Sci Rep. 2016, 27;6:30447, Biophys Chem. 2017;230:117-126). Although in silico analysis suggested the significant structural impact of p.Lys101del, it is arguable that a one amino acid in-frame deletion outside the Ω-loop would have a mild or neutral effect. To verify the pathogenicity of this variant in the less characterized C-terminal region, we used the fission yeast Schizosaccharomyces pombe as a model system (Biosci Biotechnol Biochem. 2013;77(7):1548-55). Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in-frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. Similar deleterious effects in terms of mitochondrial respiratory function were reported for two other pathogenic mutations, p.Gly42Ser and p.Tyr49His in CYCS (Biochim Biophys Acta. 2014;1842(2):269-74). The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. Hence, we suggest that loss-of function variants in CYCS cause non-syndromic thrombocytopenia.

In conclusion, our findings provide further support for CYCS mutations being causative for pure isolated thrombocytopenia. In a clinical setting, differential diagnosis of thrombocytopenia is important for evaluating the risk of developing hematological malignancies. Genetic testing complemented with the relevant functional studies should be considered part of appropriate patient care.

The study protocol was approved by Institutional Review Boards of Yokohama City University School of Medicine and the Gunma University Graduate School of Medicine. Written informed consent was obtained from all participants.