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: 10.45). 07/2005; 105(12):4700-6. DOI: 10.1182/blood-2004-12-4935
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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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    • "The pathogenic role of the g.272C>T variant has long been controversial [15], [16], although it is accepted that this SNV leads to reduced cytotoxic activity of the perforin protein due to incorrect folding that decreases its cleavage to the active form and increases its degradation [5], [7], [17], [18], [19]. Such a reduction in the level of activity could predispose an individual to late or atypical FHLH, and the development of anaplastic large cell lymphomas, B- and T-cell lymphomas and acute childhood lymphoblastic leukaemia carrying the BCR-ABL fusion gene [7], [20], [21], [22], [23]. "
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    ABSTRACT: NK/T-cell lymphoma (NKTCL) is the most frequent EBV-related NK/T-cell disease. Its clinical manifestations overlap with those of familial haemophagocytic lymphohistiocytosis (FHLH). Since PERFORIN (PRF1) mutations are present in FHLH, we analysed its role in a series of 12 nasal and 12 extranasal-NKTCLs. 12.5% of the tumours and 25% of the nasal-origin cases had the well-known g.272C>T(p.Ala91Val) pathogenic SNP, which confers a poor prognosis. Two of these cases had a double-CD4/CD8-positive immunophenotype, although no correlation was found with perforin protein expression. p53 was overexpressed in 20% of the tumoral samples, 80% of which were of extranasal origin, while none showed PRF1 SNVs. These results suggest that nasal and extranasal NKTCLs have different biological backgrounds, although this requires validation.
    PLoS ONE 03/2014; 9(3):e91521. DOI:10.1371/journal.pone.0091521 · 3.23 Impact Factor
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    • "Acute perforinopathy is caused by detrimental mutations in PRF1 or in proteins responsible for its delivery from the lumen of cytotoxic granules to the IS, UNC13D, STX11, or STXBP2 (4). The molecular basis of many disease-causing PRF1 mutations has been investigated directly using recombinant expression systems (23, 30–33), and is supported by the X-ray crystal structure of perforin (1). In contrast, the biochemical bases of pathological mutations in the other three proteins, Munc13-4, Syntaxin11, and Munc18-2, remain largely unexplored due to the lack of sufficiently informative experimental systems. "
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    ABSTRACT: Congenital perforin deficiency is considered a rare cause of human immunopathology and immune dysregulation, and classically presents as a fatal illness early in infancy. However, we propose that a group of related disorders in which killer lymphocytes deliver only partially active perforin or a reduced quantum of wild-type perforin to the immune synapse should be considered part of an extended syndrome with overlapping but more variable clinical features. Apart from the many rare mutations scattered over the coding sequences, up to 10% of Caucasians carry the severely hypomorphic PRF1 allele C272 > T (leading to A91V mutation) and the overall prevalence of the homozygous state for A91V is around 1 in 600 individuals. We therefore postulate that the partial loss of perforin function and its clinical consequences may be more common then currently suspected. An acute clinical presentation is infrequent in A91V heterozygous individuals, but we postulate that the partial loss of perforin function may potentially be manifested in childhood or early adulthood as "idiopathic" inflammatory disease, or through increased cancer susceptibility - either hematological malignancy or multiple, independent primary cancers. We suggest the new term "perforinopathy" to signify the common functional endpoints of all the known consequences of perforin deficiency and failure to deliver fully functional perforin.
    Frontiers in Immunology 12/2013; 4:441. DOI:10.3389/fimmu.2013.00441
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    • "The p.Asn252Ser PRF1 variant was first reported in a patient with FHL [6], and then identified in patient presenting with ALPS and NHL [18]. A functional analysis [38] and studies on a control population [19] have suggested that the p.Asn252Ser variant may be a benign polymorphism. p.Asn252Ser has since been proved to be a neutral PRF1 polymorphism. "
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    ABSTRACT: ABSTRACT: Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein.
    Hereditary Cancer in Clinical Practice 09/2011; 9(1):9. DOI:10.1186/1897-4287-9-9 · 1.47 Impact Factor
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