Heterozygosity for the common perforin mutation, p.A91V, impairs the cytotoxicity of primary natural killer cells from healthy individuals

Abstract

The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, inheritance of the common PRF1 polymorphism, p.A91V, (c.272C>T) found in 8-9% of the Caucasian population, with another mutated allele resulting in reduced PRF function or trafficking, has been shown to result in hyperinflammatory diseases and/or haematological cancers. In this study, we sought to investigate the function of p.A91V on a wild-type (WT) perforin background. We first developed an assay that distinguishes the relative levels of transcription of individual PRF1 alleles, including p.A91V. The p.A91V allele was seen to be expressed at similar levels as the WT allele in primary human natural killer (NK) cells, ruling out that allelic expression imbalance influenced their function. We then demonstrated that the p.A91V mutation results in protein misfolding and an appreciable reduction in NK-cell cytotoxicity in healthy carriers of p.A91V. We propose that this level of cytotoxic dysfunction may readily account for the predisposition to immune-mediated disease in individuals homozygous for p.A91V. Also, the fact that monoallelic mutations of PRF1 decrease NK-cell cytotoxicity should be considered in individuals presenting with the manifestations of immune deficiency states that impinge on NK-cell cytotoxicity.

Missense mutations in the perforin (PRF1) gene as a cause of hereditary cancer predisposition

“…16,17 Although misfolded A91V PRF is detectable by standard methodologies in healthy A91V heterozygotes, 17 earlier reports suggested that A91V homozygote patients (or those who co-inherited A91V together with a null mutation) have severely reduced or absent PRF levels. 16,18 In analyzing patient II-3, who had bi-allelic A91V mutations but has remained healthy, we demonstrated, for the first time, that A91V homozygosity is indeed compatible with reduced, but still detectable PRF levels (Fig.…”

“…23 Although hereditary cancer syndromes are typically autosomal dominant with incomplete penetrance, the pattern of inheritance is less clear in this family, with both mono-allelic and bi-allelic carriers of PRF1 mutations developing disease. One explanation for this variable pattern in inheritance is that PRF mutants such as A91V have been previously shown to exhibit dominant negative activity, 17,24 and thus may cause pathology when only present on one allele. Furthermore, although PRF1 mutations may be acting alone, it is possible that another undetected mutation may be present in this family.…”

“…Such in vitro studies on PRF mutants have previously been shown to be strong predictors of their behavior in vivo in carriers of these mutations. 11,17 Whereas, the complete loss of PRF function typically presents in early childhood as FHL2, the subtotal loss of PRF activity causes systemic inflammatory disease that is delayed beyond infancy (as in the index case II-1) or may present as a different immunopathology. 6 In these instances, consistent with Burnet's hypothesis of tumor immune surveillance, 19 reduced cytotoxic lymphocyte activity caused by partial loss of PRF may be expected to predispose to neoplasia, and this is indeed supported by previous studies.…”

Modulatory effects of perforin gene dosage on pathogen-associated blood-brain barrier (BBB) disruption

“…Typically, perforin deficiency is correlated with the onset of other diseases coined perforinopathies [28–30, 44]. However, there is a subset of individuals that have decreased perforin expression and activity but live otherwise healthy lives [24–26]. This is most likely due to the perforin allele having extensive SNVs [18–24, 27, 45].…”

“…The most notable of these SNVs is 272C>T resulting in an alanine to valine mutation in the amino acid at position 91 (A91V). Currently, it is reported that 5–20 % of the Caucasian population has this mutation which results in a 50 % decrease in perforin activity when homozygous [26]. The consequence of humans harboring perforin SNVs, resulting in reduced activity, remains to be defined.…”