Pure Red Cell Aplasia - a New Manifestation of CTLA4 Mutation

The cytotoxic T lymphocyte antigen-4 (CTLA4) is an essential negative regulator of immune responses. Its loss causes fatal autoimmunity in mice. Patients with heterozygous mutations in CTLA4 were recently reported to present with an autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features (Schubert, D. et al., Nat. Med. 20: 1410-1416, 2014; Kuehn, H.S. et al., Science 345, 1623-1627, 2014). Here we report a family with a CTLA4 mutation that presents with hematological manifestations not previously associated with CTLA4 loss.

The index case was a girl referred to our hospital with a very severe thrombocytopenia and anemia at the age of 17. Bone marrow examination revealed no megakaryocytes and just a few erythroblasts. In contrast, granulopoiesis was normal and clonal lymphocytes were not detected. No etiology of the lack of thrombopoiesis and erythropoiesis was discovered. Lack of response to high-dose steroid treatment, platelet counts below 10x10⁹/l, short-lasting effect of transfused platelets and a bleeding tendency urged us to start treatment with horse anti-thymocyte globulin (ATG) and cyclosporine after 2 months. A positive clinical response was observed in 2 weeks. Hb and platelet counts were normalized by 4 weeks, and all medication discontinued by 5 months. Two months later, the patient suffered from generalized tonic-clonic seizures. MRI revealed multiple cerebral lesions, lymphadenopathy and multiple infiltrates in lungs and kidneys. A core kidney biopsy revealed no clonal lymphoid infiltrates. A cerebral biopsy suggested crystal-storing histiocytosis. Steroids were administered, and the infiltrates disappeared gradually during the following months. More than two years later the patient suffered from a Salmonella-induced gastroenteritis, and again she became severely anemic. Her bone marrow revealed now lack of erythropoiesis whereas thrombopoiesis and granulopoiesis were normal. She was then diagnosed with pure red cell aplasia (PRCA).

An uncle of the index case had been referred to us with the diagnosis MDS at the age of 47, one year prior to the index patient's last referral and PRCA diagnosis. Dysplasia was not verified, and since the patient completely lacked red cell precursors, the diagnosis was changed to PRCA. In addition, both he, his mother and the index case had been suffering from diarrhea for years without any diagnosis. His mother also had pernicious anemia.

The two familial cases of PRCA, a rare hematological manifestation, prompted us to conduct whole exome sequencing which revealed that the two patients with PRCA were both heterozygous for a 5 base pair-insertion in exon 1 of the CTLA4 gene, leading to a frameshift and premature stop codon causing haploinsufficiency. Familial segregation was tested by Sanger sequencing and showed that also the grandmother and the mother of the index case were carriers of the same mutation.

Functional assays of our index patient, the two family members with autoimmunity (her uncle and grandmother) and her unaffected mother revealed fewer CD19+ B- and naïve T cells than normal. Furthermore, we found dysregulation of regulatory T (Treg) cells, reduced levels of T cell CTLA4, particularly FoxP3+ Treg CTLA4 (mean fluorescence intensity as well as frequency of CTLA4+ T cells and Tregs) and signs of hyperactivity of effector T cells. Our findings were as reported in the above cited publications.

The discovery of a CTLA4 mutation in this family with 3 affected members and one unaffected carrier might have immediate implications for our index patient. As she did not respond to high-dose steroids as her uncle did, her genetic diagnosis opens up to additional treatment opportunities. Actual medication for patients with mutations in CTLA4 and autoimmunity could be mTOR inhibitors like sirolimus and possibly CTLA4 fusion proteins (abatacept and belatacept). These fusion proteins bind to CD80 and CD86 and inhibit CD28-mediated immune activation.

In conclusion, we have detected a mutation in the CTLA4 gene with corresponding T cell aberrations in this family through the new manifestation PRCA. These findings may be of clinical importance for the family we have characterized as well as for other patients with similar manifestations.