NF‐E2 mutation as a novel cause for inherited thrombocytopenia

Inherited thrombocytopenias (IT) are genetic diseases that affect platelet production and function, resulting in thrombocytopenia and a tendency for bleeding. Novel genetic mutations responsible for IT are being reported continuously (Almazni et al., 2019; Lentaigne et al., 2019). We present a case of thrombocytopenia with a homozygous frameshift mutation of nuclear factor, erythroid 2 (NF-E2), which has not been reported before. The patient was an eight-week-old Filipino boy born at full term of non-consanguineous parents with no family history of bleeding disorders. The mother had gestational diabetes. The patient developed multiple petechiae on day 2 of life and thrombocytopenia was identified. Physical examination revealed a well-developed baby boy with petechiae and purpura on face, trunk and extremities. There was no other active bleeding. Examination of respiratory, cardiovascular, abdominal and lymphatic systems was unremarkable. Further investigations for viral infections, immunoglobulin levels, lymphocyte subsets, Coombs test and abdominal ultrasound were unrevealing (Table SI). Bone marrow examination revealed cellular marrow with the presence of all cell lines including megakaryocytes. The thrombocytopenia was refractory to platelet transfusion, intravenous immunoglobulin and prednisolone (Fig 1). He was referred to The University of Hong Kong for genetic work-up. Genetic and functional studies on referred patients, data archival in the Asian Primary Immunodeficiency Network (APIN) database (Lee & Lau, 2011), and DNA storage were approved by the Clinical Research Ethics Review Board of the University of Hong Kong and Queen Mary Hospital (Ref. no. UW 08-301) with informed consent obtained from the parents of subjects. DNA from the patient and his mother were sent to Hong Kong together with his peripheral blood and bone marrow slides. A peripheral blood smear revealed thrombocytopenia with normal-sized platelets (Figure S1). A bone marrow smear revealed few normal-looking megakaryocytes without dysplasia, active erythropoiesis and granulopoiesis (Figure S2). Unfortunately the patient was lost to further follow-up in the Philippines. The patient was initially screened for Wiskott–Aldrich syndrome but PCR-Sanger sequencing of WASP was normal (Lee et al., 2009). Therefore, whole-exome sequencing (WES) was performed on the patient and his mother, which revealed the patient had a homozygous frameshift mutation in NF-E2 (c.952delA, p.T318fsX326), which resulted in a truncated transcription factor NF-E2 45 kDa subunit (p45/ NF-E2) (Fig 2). The mother was a carrier of the mutation. The WES was performed as described in Data S1 and Figure S3. Analysis in silico using PROVEAN (Choi et al., 2012) and SIFT Indel (Hu & Ng, 2012) predicted the frameshift mutation in NF-E2 to be deleterious. PROVEAN predicted the mutation as deleterious with a PROVEAN score of 143 071 (scoring less than 2 5 is considered deleterious). SIFT Indel predicted the mutation effect as damaging with a confidence score of 0 858. Mutations in NF-E2 were further searched for in a group of 13 patients in our APIN database with documentation of thrombocytopenia in the first two months of age, or between two and four months of age if without small platelets or organomegaly. None of the 13 patients had a mutation in NF-E2. NF-E2 is a heterodimeric transcription factor formed by p45/NF-E2 and small MAF proteins through the interaction of their basic leucine zipper bZIP domains (Fig 2) (Levin et al., 1999). NF-E2 is essential for promoting megakaryocytic maturation (Motohashi et al., 2010), proplatelet formation and release (Lecine et al., 2000; Tiwari et al., 2003; Chen et al., 2007) (Figure S4). The bZIP domain facilitates binding of NF-E2 to DNA, allowing the p45/NF-E2 subunit to transactivate platelet gene expression (Fig 2). p45/NF-E2 is expressed in erythroid cells, megakaryocytes, granulocytes and mast cells (Levin et al., 1999). Although studies demonstrated that mice without either p45/NF-E2 or small MAF proteins had thrombocytopenia, only those without p45/NF-E2 died from lethal haemorrhage, indicating p45/NF-E2 is the critical component (Shivdasani et al., 1995; Catani et al., 2002). Post-translational modifications of p45/NF-E2 are critical to modulate its functions. Lysine 368 on p45/NF-E2 is the site for sumoylation, the addition of small ubiquitin-like modifier (SUMO), enhancing the DNA binding affinity and transactivation capability of p45/NF-E2, which has been shown to be critical in b-globin expression (Shyu et al., 2005). The frameshift mutation in our patient (c.952delA, p.T318fsX326) resulted in a truncated p45/NF-E2 protein and loss of the sumoylation site, leading to impaired DNA binding, transactivation, megakaryocyte maturation and platelet release. correspondence