Novel mutations in the PLCZ1 gene associated with human low or failed fertilization

Abstract

Background: Fertilization failure (FF) is a complex reproductive disorder characterized by the failure of pronuclei formation during fertilization. In addition to some cases caused by iatrogenic problems and known genetic factors, there are still many unexplained aspects of FF. Here, we aimed to assess the clinical and genetic characteristics of two families experiencing primary infertility with FF.

Methods: We have characterized two families from China. All of the infertile couples presented with similar clinical phenotypes, that is, partial or total fertilization failure in repeated cycles. We performed Sanger sequencing of their WEE2, TLE6, and PLCZ1 genes, and further bioinformatics and functional analyses were performed to identify the pathogenic elements of the variants.

Results: We identified novel compound heterozygous mutations c.1259C>T (p.P420L) and c.1733T>C (p.M578T) in the PLCZ1 gene in a male patient of family 1 with total fertilization failure, and another novel homozygous mutation c.1727T>C (p.L576P) in the same gene in a male patient of family 2 with partial fertilization failure. These three novel mutations were absent in the control cohort and in the databases. The amino acids were conserved at their positions among six different species. All mutant amino acids were located in key domains and were predicted to impair hydrolytic activity and lead to PLCZ1 dysfunction. Further functional detection revealed that the three mutations could significantly impair the catalytic activity of PLCZ1.

Conclusions: We identified three novel mutations in PLCZ1 associated with partial and total fertilization failure and have provided new evidence about the genetic basis of FF.

Keywords: PLCZ1; fertilization failure; infertility; low fertilization; mutation.

Figure 1

Partial sequencing results of novel mutations in FF patients for the PLCZ1 gene. (a) Pedigree of the first family. Filled square indicates the FF patient (II:3). Arrow indicates the proband. Open squares or circles indicate normal family members. (b) Pedigree of the second family. Filled square indicates the FF patient (II:1). Arrow indicates the proband. Open squares or circles indicate normal family members. (c) The left arrow points to the wild‐type c.1259C in a control sample and the wild‐type codon is underlined; the right arrow points to the heterozygous c.1259C>T (p.P420L) mutation in the patient of family 1 (proband), and the mutated codon is underlined. (d) The left arrow points to the wild‐type c.1727T in a control sample, and the right arrow points to the homozygous c.1727T>C (p.L576P) mutation in the patient of family 2 (proband). (e) The left arrow points to the wild‐type c.1733T in a control sample, and the right arrow points to the heterozygous c.1733T>C (p.M578T) mutation in the patient of family 1 (proband), and the mutated codon is underlined

Figure 2

Bioinformatic analysis of novel mutations in PLCZ1. (a) Comparison of the human PLCZ1 amino acid sequence with five different species (Macaca fascicularis, Bos taurus, Rattus norvegicus, Mus musculus, and Gallus gallus). Pro420, Leu576, and Met578 are conserved in PLCZ1. (b) Schematic illustration of the domains in PLCZ1. The wild‐type PLCZ1 protein has 608 amino acids and contains EF hand domain, two catalytic domains (X‐box and Y‐box) and C2 domain. Three novel mutations identified in our study are highlighted in red, and other known mutations found in FF patients are highlighted in black. (c) Overall structure of hPLCZ1. EF hand domain, C2 domain, and catalytic domain are shown as yellow, white, and blue cartoons, respectively. Ca atoms of P420, L576, and M578 are shown as pink spheres. The residue ID is labeled nearby the corresponding residues. (d) Zoom‐in view of hPLCZ1 structure. P420, L576, M578, and hydrophobic core residues are shown as purple and green sticks, respectively. The residue ID is labeled nearby the corresponding residues

Figure 3

Functional analysis of the identified mutations in PLCZ1. (a) Schematic diagram of plasmids for expressing the wild‐type and mutant PLCZ1 fused with EGFP via a flexible linker. (b) Fluorescence microscopy images of EGFP expression in HEK293T cells 40 hr post transfection. Blank stands for negative control where cells are non‐transfected. Scale bar =100 μm. (c) qRT‐PCR analysis of the transcriptional levels of PLCZ1 relative to GAPDH in transfected HEK293T cells. Blank stands for negative control where cells are non‐transfected. (d) qRT‐PCR analysis of the transcriptional levels of PLCZ1 relative to NeoR in transfected HEK293T cells. Blank stands for negative control where cells are non‐transfected. (e) Representative flow cytometry histogram of EGFP signal of cells 40 hr post transfection. Blank stands for negative control where cells are non‐transfected. (f) Quantification of EGFP fluorescence intensity in transfected HEK293T cells as determined by cytometry analysis. (g) Western blot analysis of EGFP expression levels in transfected HEK293T cells. Blank stands for negative control where cells are non‐transfected. (h) Quantification of PLCZ1‐EGFP fusion protein expression levels based on of the intensity of bands of Western blot. Blank stands for negative control where cells are non‐transfected. (i) Determination of the catalytic activity of over‐expressed the wild‐type and mutant PLCZ1. Asterisks (*) indicate significant differences (p < 0.01).