The novel p.Gly306Asp perforin mutation causes Familial Hemophagocytic Lymphohistiocytosis type 2 (FHL-2) probably due to a critical role of Gly306 in the pore-forming perforin domain.

Abstract

Background: Three substitutions at either Gly305 or Gly306 within the membrane attack/complex perforin domain (MACPF) of perforin have been previously identified in a number of patients with hemophagocytic lymphohistiocytosis (HLH). However, their pathogenic impact remains unclear since all the cases reported so far carried heterozygous genotypes and showed very heterogeneous clinical presentations. Here, we report a new substitution (p.Gly306Asp) and use in silico tools to elucidate the pathogenic mechanisms and severity associated with human Gly306 and Gly305 mutations. Methods: The immunological workup included perforin expression and perforin gene (PRF1) mutation analysis. Computer algorithms based on conservation, secondary, and tertiary protein structure analyses were applied to assess the role of the mutations in disease pathogenesis. Results: In our patient, we found a previously undescribed homozygous c. 917G>A (p.Gly306Asp) mutation in the PRF1 gene that was associated with null perforin expression in her natural killer lymphocytes. Sequence alignments revealed that Gly306 and Gly305 are highly conserved positions among vertebrate perforins, as well as in other related pore-forming proteins such as bacterial cytolysins. Further in silico analyses consistently predicted mutations in these 2 positions to be pathogenic due to diminished stability of the perforin molecule. Conclusion: Age of HLH onset, severity of the disease and undetectable perforin in our p.Gly306Asp homozygous patient along with the in silico results unmask this novel mutation as highly detrimental. Our results highlight the need of combining all clinical features, in vitro phenotypes and computer based approaches to classify human perforin mutations accurately. Statement of novelty: The study of these PRF1 mutations points to an important role of the 2 glycine amino acids (Gly305 and Gly306) in the molecular stability of perforin, which may also be likely in other pore-forming proteins. Our in silico results conclude that the pathogenicity of mutations in highly conserved Gly305 and Gly306 is likely to be associated with a serious destabilization of the native perforin conformation.