Pathogenic homozygous PLCZ1 mutation reduces sperm protein levels with a simulated reduction of PIP2 binding: a case report

Abstract: Mammalian oocyte activation is driven by intracellular calcium (Ca2+) oscillations induced by sperm-specific phospholipase C zeta (PLCζ). Sperm PLCζ deficiency is linked to male infertility caused by oocyte activation deficiency (OAD). Using whole exome sequencing, we report finding a previously reported loss of activity pathogenic mutation of PLCZ1 from two unrelated males in a separate ethnicity, homozygous in both cases. We examined sperm PLCζ using immunoblotting and immunofluorescence to compare levels with fertile control sperm. We also utilised bioinformatic tools to characterise the effect of this variant. The variant caused a guanine (G) to adenine (A) change at position 1154 (c.1154G>A), resulting in an arginine (R) to glutamine (Q) change at amino acid 385 (p.Arg385Gln;R385Q), disrupting the local protein fold in the Y domain of the active site in a highly deleterious and pathogenic manner. A previous study identified this mutation as heterozygous, while we identified this variant as homozygous in an ethnically distinct population from this previous study. Sperm PLCζ was significantly reduced compared to controls. The R385Q change indicated a reduction of PIP2 interaction with PLCζ following docking analyses. Pathogenic PLCζ mutation may be common in men with repeated fertilisation failure. Raw ejaculate analysis, rather than density gradient washed sperm, could provide a more accurate assessment of PLCζ levels, especially in men with low sperm count or ejaculate volume. Such pathogenic cases may underlie higher proportions of sperm exhibiting abnormal PLCζ localisation and could stand to aid a larger number of patients seeking fertility treatment. Lay summary: In this study, we examined two unrelated men who had been unable to have children despite multiple IVF (in vitro fertilisation) attempts. We discovered that both men carried the same mutation in a gene called PLCZ1. This gene normally produces a protein in sperm that tells the egg to begin developing into an embryo, but because of the mutation in these men, the sperm could not deliver that message to the egg. Interestingly, this same mutation had previously only been reported in Chinese patients, and this was the first time it was found in men from a different ethnic background, suggesting that this mutation may be a globally occurring mutation. This finding could help doctors recognise similar fertility problems in other patients and could lead to more effective treatments in the future.