Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.
Authors: Solomou Elena E. , Gibellini Federica , Chanock Stephen J. , Malide Daniela , Berg Maria , Visconte Valeria , Luppi Mario , Childs Richard , Green Spencer , Young Neal S. 
Published: April 2020

Abstract
Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls’ cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.