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.

A systematic review of the validated monogenic causes of human male infertility: 2020 update and a discussion of emerging gene-disease relationships

“…The shift away from Sanger sequencing to NGS methods is continuing to take place, and 71% of all sequencing studies used NGS in 2020 (Figure 2C), which constitutes an increase of 90% since 2017. While WGS has started to emerge in the field (recent examples: Bedoni et al 2016;Wang et al 2017;Yuan et al 2020;Arafat et al 2020), WES is currently still the predominant method over panel sequencing and whole genome sequencing (75% vs. 19% and 6%, respectively).…”

The Role of Genetics and Oxidative Stress in the Etiology of Male Infertility—A Unifying Hypothesis?

“…Of course, motility is not the only attribute of sperm function susceptible to interference by genetic and epigenetic mutations. For example, we know that genetic defects in sperm PLCζ, which activates the generation of calcium transients in the fertilized oocyte, impair human oocyte activation and fertilization ( 59 ). Fertilization failure has also been associated with mutations in CATPSERE (CatSper-epsilon), a component of the sperm calcium channel ( 60 ) as well as polymorphisms in the mitochondrial genes MT-ATP6 and MT-CYB ( 61 ).…”

Diagnosis and Treatment of Male Infertility-Related Fertilization Failure

“…PLCζ specifically lacks the Src homology (SH) domain and Pleckstrin homology (PH) domain, which makes it the smallest PLC isozyme known with a protein length of 608 amino acids and molecular mass of 70 kDa [128]. Up to now, 21 different mutations in the PLCZ1 gene have been reported to cause low or total fertilization failure after ICSI [40,41,45,71,86,[129][130][131][132][133][134] (Table A1). These mutations have been found throughout all regulatory regions of the PLCζ protein (Figure 4), affecting the protein function in different manners.…”

“…Three proteins predicted to be damaging have been found in the C2 domain (p.I489F, p.S500L, p.R553P). While p.I489F and p.R553P result in reduced oocyte activation rates after an injection of mutant human PLCζ cRNA into mouse and IVM human oocytes, respectively [86,131], p.S500L does not influence fertilization rates when injected into IVM human oocytes [40]. However, the latter mutation has been detected in 9/37 (24.3%) patients with fertilization failure analyzed by the same study [40], compared to its presence in 3% of the general population (minor allele frequency reported by the ExAc database) which suggests its direct association to the phenotype.…”