Rare Mutations in CCDC7 Contribute to Early-Onset Preeclampsia by Inhibiting Trophoblast Migration and Invasion

Abstract

Rare gene variants have been found to play a role in complex disorders. Preeclampsia, and especially early-onset preeclampsia, has a strong genetic link. However, the role of rare variants in the offspring of mothers with preeclampsia remains unclear. In this study, whole-exome sequencing (WES) was used to identify rare pathogenic variants in two families with early-onset preeclampsia. Two heterozygous rare variants in CCDC7, c.625C>T (p.R209C) and c.1015C>T (p.R339X), were detected in two families and were cosegregated in the offspring of preeclamptic pregnancies. We examined the spatiotemporal expression pattern of CCDC7 in human placental villi and the effects of CCDC7 on migration and invasion of trophoblast cells JEG-3. The quantitative real-time PCR and Western blot results showed that the expression of CCDC7 in placental villi was the lowest during the first trimester and increased as the pregnancy progressed. The CCDC7 p.R339X variant showed a decrease in mRNA and protein expressions. Loss-of-function assays showed that knockdown of CCDC7 suppressed the migration and invasion of JEG-3 cells. In conclusion, CCDC7 is a potential susceptibility gene for preeclampsia, which is key for the migration and invasion of trophoblast cells. Rare variants of preeclampsia in offspring may play a crucial role in the pathogenesis of preeclampsia and require further research.

Keywords: CCDC7; early-onset preeclampsia; migration and invasion; rare variants; trophoblast.

Figure 1

Family pedigree of people ((A), family A; (B), family B) enrolled in this study. Orange circles represent the preeclamptic mother, and black circles and boxes represent preeclamptic offspring. “+” and “−” indicate mutant allele and wild-type allele, respectively.

Figure 2

Rare variants of CCDC7 detected in two EOPE families and conservation analysis. (A) Variants c.625C>T (p.R209C) and c.1015C>T (p.R339*) were identified in family A and family B, respectively; (B) variants c.625C>T (p.R209C) and c.1015C>T (p.R339*) localized in exon 7 and exon 13, respectively; and (C) both of these two mutations are located in highly conservational sites across multiple species. Red represents the mutant site and “*” indicates a stop codon.

Figure 3

CCDC7 localization and expression in human placenta of different trimesters. (A) Immunofluorescence showed that CCDC7 localized in cytoplasm of cytotrophoblast, syncytiotrophoblast, and vascular epithelial cells in the human placenta and did not change during gestation; quantitative real-time polymerase chain reaction (B) and Western blotting (C) showed that the mRNA and protein levels of CCDC7 elevated with advancing gestational age. * p < 0.05, ** p < 0.01. Scale bars, 25 μm.

Figure 4

CCDC7 localization and expression in placenta from preeclampsia families and controls. (A) Immunofluorescence shows that the localization of CCDC7 in the placenta from the two preeclampsia families was the same as that in the controls. Quantitative real-time PCR (B) and Western blotting (C) showed that mRNA and protein levels of CCDC7 were decreased in the placenta from family B (p.R339*) and did not change in the placenta from family A (p.R209C) compared with those of age-matched controls (p.R339* vs. control2; p.R209C vs. control1). * p < 0.05; ns, not significant. Scale bars, 25 μm.

Figure 5

Effects of CCDC7 knockdown on the proliferation, migration, and invasion of JEG3 cells. Quantitative real-time PCR (A) and Western blotting (B) showed that shRNA1 and shRNA2 significantly decreased the expression level of CCDC7 mRNA and protein. Wound-healing assay (C,D) and transwell invasion assay (E,F) showed that the migration and invasion abilities of trophoblast cells decreased after knockdown of CCDC7. ** p < 0.01, *** p < 0.001. Scale bars, 50 μm in (C,E).