Voskoboinik, I., Thia, M. C. & Trapani, J. A. A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis. Blood 105, 4700-4706

Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, VIC 8006, Australia. <>
Blood (Impact Factor: 10.43). 07/2005; 105(12):4700-6. DOI: 10.1182/blood-2004-12-4935
Source: PubMed

ABSTRACT Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms.

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    • "A second, more common outcome (n = 14) was presynaptic dysfunction, which we have previously defined as reduced expression and/or PRF degradation or truncation in the RBL cells (Voskoboinik et al., 2004). As was observed previously with FHL-associated mutations (Ishii et al., 2005; Voskoboinik et al., 2005b), this group of substitutions invariably led to a marked loss of function (Table S1). Most commonly, presynaptic dysfunction occurred at residues that showed extremely high conservation between species as divergent as humans and fish (for instance, residues 189, 220, 221, 231, or 239), presumably because they are critical for protein folding during its biosynthesis. "
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    ABSTRACT: Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.
    Immunity 06/2009; 30(5):684-95. DOI:10.1016/j.immuni.2009.03.016 · 19.75 Impact Factor
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    • "was homozygous for the novel missense mutation c . 112G4A ( Val38Met ) and homozygous for the known mutation c. 272C4T ( Ala91Val ) , which was initially described as a nonpathogenic variant [ Feldmann et al . , 2002 ] but was later proposed to contribute to the disease [ Busiello et al . , 2004 ; Clementi et al . , 2002 ; Trambas et al . , 2005 ; Voskoboinik et al . , 2005 ] . Interestingly , the patients with mutations in PRF1 included two with a very - late - onset form of the disease ( Patients 17 and 18 ) . They were compound heterozygous for missense mutations and an in - frame deletion , respectively . In total we detected PRF1 mutations in three of 23 patients from Germany , 14 of 32 of Turkish ori"
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    ABSTRACT: Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal-recessive disease that affects young children. It presents as a severe hyperinflammatory syndrome with activated macrophages and T lymphocytes. Mutations in the perforin 1 gene (PRF1) were found in FHL-2 in 15-50% of all cases. Defective granule exocytosis caused by mutations in the hMunc13-4 gene (UNC13D) has been described in FHL-3. FHL-4 patients have mutations in STX11, a t-SNARE involved in intracellular trafficking. We analyzed a large group of 63 unrelated patients with FHL of different geographic origins (Turkey:32; Germany:23; others:8) for mutations in STX11, PRF1, and UNC13D. We identified mutations in 38 samples (20 in PRF1, 12 in UNC13D, and six in STX11). Of 32 patients from Turkey, 14 had mutations in PRF1, six had mutations in UNC13D, and six had mutations in STX11. The mutation Trp374X in PRF1 was found in 12 patients from Turkey and was associated with a very early onset of the disease below the age of 3 months in all cases. In contrast, three of 23 and four of 23 patients from Germany, and three of eight and two of eight from other origins showed mutations in PRF1 and UNC13D, respectively, but none in STX11. Thus, FHL-2, FHL-3, and FHL-4 account for 80% of the HLH cases of Turkish origin, and for 30% of German patients. Furthermore, we identified mutations in RAB27A in three patients with FHL-related Griscelli syndrome type 2. In functional studies using a mammalian two-hybrid system we found that missense mutations Ala87Pro in Rab27a and Leu403Pro in hMunc13-4 each prevented the formation of a stable hMunc13-4/Rab27a complex in vitro. Our findings demonstrate extensive genetic and allelic heterogeneity in FHL and delineate an approach for functionally characterizing missense mutations in RAB27A and UNC13D.
    Human Mutation 01/2006; 27(1):62-8. DOI:10.1002/humu.20274 · 5.05 Impact Factor
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    ABSTRACT: We screened 100 children with acute lymphoblastic leukemia (ALL) to assess the incidence of single amino acid change A91V in perforin. Heterozygous A91V was found in 12/100 patients and 5/127 controls (OR, 3.4; 95%CI: 1.15-9.95; p=0.014). A91V is a novel and frequent predisposing factor for childhood ALL.
    Haematologica 06/2005; 90(5):697-8. · 5.87 Impact Factor
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