A FUNCTIONAL ANALYSIS OF THE PUTATIVE POLYMORPHISMS A91V AND N252S, AND 22 MISSENSE PERFORIN MUTATIONS ASSOCIATED WITH FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS. Short Title: Analysis of perforin mutations associated with HLH

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 to wild-type, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLHassociated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained ~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 a 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 two most common perforin polymorphisms. For personal use only. on September 14, 2017. by guest www.bloodjournal.org From