Spectrum of Perforin Gene Mutations in Familial Hemophagocytic Lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, ∼30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%–40% of the FHL cases and the FHL 1 locus on chromosome 9 for ∼10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes

Genetic studies of familial hemophagocytic lymphohistiocytosis

The main contribution from this thesis is a refined knowledge of locus heterogeneity in familial hernophagocytic lympbohistiocytosis (FHL) and a further characterization of corresponding clinical and immunological phenotypes. Familial hemophagocytic lymphohistiocytosis is a fatal autosomal recessive immune deficiency clinically characterized by fever, cytopenia, hepatosplenomegaly, and hemophagocytosis. A common feature of the pathophysiology is a defect in cytotoxic T lymphocyte (CTL) and natural killer (NK) cell cytotoxicity. Two different genes have been identified in FHL patients; the PRF1 gene and the MUNC13-4 gene. Perforin is a constituent of cytotoxic granules in CTLs and NK cells, released upon activation to cooperate with granzyme B (GZMB) in the induction of apoptosis in target cells. MUNC 13-4 functions in the same pathway by inducing degranulation of cytotoxic granules. In Paper I the aim was to investigate the mutational spectrum and to elucidate the relative contribution of PRF1 gene mutations in FHL. Results from direct DNA sequencing and linkage analysis for homozygosity in consanguineous families suggests that in no more than 35-40% of the FHL patients the disease can be explained by mutations in PRF1 gene in our material. Since these results intimate locus heterogeneity we investigated the presence of mutations in the GZMB and granulysin (GNLY) genes (Paper Il). None of the sequence variations disclosed associated with the disease, and thus we did not find any support for genetic defects in these genes explaining FHL. In order to characterize lymphocyte populations and PRF expression in FHL flow cytometry was used (Paper III). We observed diminished or reduced PRF expression correlating to mutations in the PRF1 gene. An interesting finding was a significant reduction in expression of circulating CD19+ B lymphocytes. A comprehensive study of genotype-phenotype correlations in large cohort of HLH patients revealed that PRF1 gene mutations were more frequent in the Middle East and Turkish populations (Paper IV). In addition, lymphadenopathy, jaundice and edema were found to be less frequent at presentation in patients without PRF1 gene mutations in the Nordic population compared to the non-Nordic population

Defective cytotoxic lymphocyte degranulation in syntaxin-11–deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8+ T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities