Genetic, cellular and clinical studies of hemophagocytic lymphohistiocytosis

Abstract: Hemophagocytic lymphohistocytosis (HLH) is a life-threatening hyperinflammatory condition characterized by fever, cytopenia, hepatosplenomegaly, and sometimes hemophagocytosis. HLH is typically divided into two distinct groups, primary HLH and secondary HLH. Familial HLH (FHL), of autosomal recessive inheritance, is divided into type 2, 3, 4, and 5, caused by aberrations in PRF1, UNC13D, STX11, and STXBP2, respectively; all encoding proteins involved the perforin-mediated cytotoxic pathway. Consequently, patients with FHL display a defective NK cell cytotoxicity, one of the diagnostic criteria for HLH. The clinical presentation of the different forms of HLH can vary markedly, and the distinction between primary and secondary HLH is not always clear. In 1991, Henter et al. reported the first estimate of the annual incidence of FHL. Based on an increased awareness of HLH, with available diagnostic guidelines and increased clinical and biological understanding, we, in Paper I, hypothesized that the true incidence could be higher than previously estimated. However, somewhat surprisingly, the estimated annual incidence of primary HLH in Sweden was unchanged, 1.2 per million children less than 15 years of age, corresponding to 1.8 per 100 000 live born children. The annual incidence in patients aged less than 1 year was 11 per million children. The second part of Paper I aimed to provide a minimal incidence of primary HLH based on genetic findings and NK cell function consistent with primary HLH. Using these methods, twelve such patients referred to us were identified 2007-2011, giving a minimal incidence of 1.5 per million children aged less than 15 years in Sweden, corresponding to 2.2 patients per 100 000 live born children. These laboratory diagnostic tools may facilitate the diagnosis of primary HLH. In many patients with primary HLH, especially in patients of Scandinavian origin, the underlying molecular defect has not been identified. Thus, one of the major aims with this thesis was to provide a genetic diagnosis for these patients. Patients with Griscelli syndrome type 2, another autosomal recessive immunodeficiency associated with development of HLH, display a partial albinism in addition to the immunological defect. In Paper II, one out of 21 families diagnosed as having FHL, was identified with bi-allelic mutations in RAB27A, and thus instead affected by GS2. Three additional GS2 patients were also first diagnosed as having FHL and first later diagnosed with GS2, further stressing the importance of remembering GS2 among patients with HLH. The partial albinism in GS2 patients may easily be overlooked. In Paper III, Rab27a was shown to be required for NK cell cytotoxicity and degranulation induced by receptors both for natural cytotoxicity and antibody dependent cellular cytotoxicity, in contrast to what previously has been described. Furthermore, recruitment of Rab27a and Munc13-4 to perforin-containing granules was shown regulated by different receptor signals, with an inverse relationship between Rab27a and Munc13-4. In Paper IV, we describe the clinical presentation, the mutation spectrum, and NK cell function in patients with FHL type 5. Interestingly, a highly variable disease severity was observed among these patients, with an age at onset ranging from 2 months to 17 years. Furthermore, gastrointestinal symptoms, bleeding disorders, and hypogammaglobulinemia were present in about one third of the patients. Thus, we conclude that the clinical presentation of FHL type 5 can vary markedly, and that FHL5 should be considered also in patients with manifestations not typically associated with FHL. In Paper V, two non-coding aberrations in UNC13D were described to be causative of FHL type 3 in many patients of European origin, highlighting that aberrations outside the coding regions also can be a cause of disease. The first is a point mutation in intron 1 that selectively impairs UNC13D transcription in lymphocytes and the second is a 253-kb inversion straddling the UNC13D locus that affects the 3’end of the transcript. Both aberrations abolish the Munc13-4 expression. Taken together, this thesis provides genetic, cellular, and clinical findings of importance for the understanding of HLH. A genetic diagnosis, together with assessment of cytotoxic lymphocyte function, facilitates the diagnosis of patients with primary HLH and enables presymptomatic identification of affected individuals. Furthermore, a genetic diagnosis enables carrier testing and prenatal diagnosis in the affected families. Studies of cytotoxic lymphocyte function in these patients also provide fundamental insights in lymphocyte cytotoxic function and human immunology. A genetic diagnosis, together with an increased knowledge about the diverse clinical presentation, is highly valuable in the clinical management and for prompt initiation of adequate treatment in these life-threatening immunodeficiencies.