A novel homozygous mutation of phospholipase C zeta leading to defective human oocyte activation and fertilization failure

Abstract

Study question: Is a novel homozygous phospholipase C zeta (PLCζ), c.1658 G>C; p. R553P mutation in the C2 domain associated with the outcomes of recurrent fertilization failure after ICSI?

Summary answer: PLCζ, c.1658 G>C led to defective human oocyte activation and fertilization failure, while this mutation in the C2 domain of PLCζ did not compromise concentration, motility and chromosome ploidy of sperm.

What is known already: Sperm-specific PLCζ is now widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations, which are essential for egg activation during mammalian fertilization. Thus far, few genetic studies have shown that different point mutations in the PLCζ gene are associated with male infertility.

Study design, size, duration: This was a basic medical research to assess pathogenicity for novel mutation in the C2 domain of PLCζ during human fertilization.

Participants/materials, setting, methods: Single-cell omics were applied to analyze the DNA methylation state of the fertilization failure oocytes and the ploidy of the patient's sperm. Whole genome sequencing data for the patient were analyzed for mutations in PLCζ. Sanger sequencing confirmed the presence of a rare variant, and then the mutant and wild-type PLCζ mRNA were injected to observe oocyte activation.

Main results and the role of chance: The fertilization failure oocytes (n = 4) were triploid and lacking proper DNA demethylation. The whole genome sequencing analysis revealed a novel missense homozygous mutation in PLCζ, c.1658 G>C; p. R553P, which leads to the conversion of arginine 553 to proline. This point mutation does not affect the production of the corresponding protein in sperm. However, microinjection of the mRNA transcribed from the PLCζ R553P mutation gene failed to trigger oocyte activation and the subsequent embryo development.

Limitations, reasons for caution: Only one patient with PLCζ mutations was available because of its rare incidence.

Wider implications of the findings: Notably, we discovered a novel homozygous mutation in PLCζ, which results in an abnormal conformation at the C2 domain of the PLCζ protein. Our findings indicate an essential role of PLCζ in human fertilization and the requirement of a normal structure of C2 domain in PLCζ-mediated physiological function.

Study funding/competing interest(s): This project is funded by the National Natural Science Foundation of China (31571544, 31871482, 31871447) and National Key Research and Development Program (2018YFC1004000, 2017YFA0103801). All authors declared no competing interests.

Trial registration number: Not applicable.

Keywords: C2 domain; ICSI; fertilization failure; phospholipase C zeta; point mutation.