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. 2021 Mar 4:9:641831.
doi: 10.3389/fcell.2021.641831. eCollection 2021.

Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects

Tian Tian  1   2 , Xuanye Cao  3 , Yongyan Chen  1   2 , Lei Jin  1   2 , Zhiwen Li  1   2 , Xiao Han  3 , Ying Lin  3 , Bogdan J Wlodarczyk  3 , Richard H Finnell  3   4 , Zhengwei Yuan  5 , Linlin Wang  1   2 , Aiguo Ren  1   2 , Yunping Lei  3
Affiliations

Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects

Tian Tian et al. Front Cell Dev Biol. .

Abstract

Background: Neural tube defects (NTDs) are among the most common and severe congenital defects in humans. Their genetic etiology is complex and remains poorly understood. The Mediator complex (MED) plays a vital role in neural tube development in animal models. However, no studies have yet examined the role of its human homolog in the etiology of NTDs.

Methods: In this study, 48 pairs of neural lesion site and umbilical cord tissues from NTD and 21 case-parent trios were involved in screening for NTD-related somatic and germline de novo variants. A series of functional cell assays were performed. We generated a Med12 p.Arg1784Cys knock-in mouse using CRISPR/Cas9 technology to validate the human findings.

Results: One somatic variant, MED12 p.Arg1782Cys, was identified in the lesion site tissue from an NTD fetus. This variant was absent in any other normal tissue from different germ layers of the same case. In 21 case-parent trios, one de novo stop-gain variant, MED13L p.Arg1760∗, was identified. Cellular functional studies showed that MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical-WNT signaling pathway. The Med12 p.Arg1784Cys knock-in mouse exhibited exencephaly and spina bifida.

Conclusion: These findings provide strong evidence that functional variants of MED genes are associated with the etiology of some NTDs. We demonstrated a potentially important role for somatic variants in the occurrence of NTDs. Our study is the first study in which an NTD-related variant identified in humans was validated in mice using CRISPR/Cas9 technology.

Keywords: CRISPR/Cas9; MEDs; de novo variant; neural tube defects; somatic variants.

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Conflict of interest statement

RF and BW formerly consulted with the now dissolved TeratOmic Consulting LLC. RF also receives travel funds to attend editorial board meetings of the Journal of Reproductive and Developmental Medicine published out of the Red Hospital of Fudan University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The identified variants of MED12 in human samples. (A) Sanger sequencing map of the MED12 c.5344C > T in different organs. (B) Conserved domains and the total scheme of the target genes and positions of the identified mutations. (C) A partial alignment of human target MED12 protein with other orthologous sequences.
FIGURE 2
FIGURE 2
The identified variants of MED13L in human samples. (A) Whole-exome-sequencing map and Sanger sequencing map of MED13L c.5278C > T. (B) Conserved domains and the total scheme of the target genes and positions of the identified mutations. (C) A partial alignment of human target MED13L protein with other orthologous sequences. (D) The Sanger sequencing map of PPP5C and NCKAP1L genes.
FIGURE 3
FIGURE 3
The influence of MED12 p.Arg1782Cys on MED12 subcellular location and protein levels. (A) The subcellular localization of wildtype of MED12 and mutated MED12. MDCK-II cells were transfected with GFP-MED12 (WT and mutants) for 48-h incubation and then were imaged under a deconvolution microscope. WTU: wildtype. The staining assay was performed twice. (B) The variant affected the protein level of MED12. HEK293T cells were transfected with GFP-MED12 (WT and mutants) for 48 h, and then western blotting assays were performed to determine the protein levels. The predicted sizes of WT and mutant constructs are 260 KD. The western blotting assay was repeated three times, and t-tests on the relative protein levels to GAPDH were conducted to assess potential differences between WT and the mutant group. *p < 0.05. Ns: not significant. Three replicates of the transfections and western blotting assays were performed.
FIGURE 4
FIGURE 4
The influence of MED12 p.Arg1782Cys on the expression of WNT signaling pathway. (A) The variant of MED12 affected the non-canonical WNT signaling pathway. Luciferase assay was performed among HEK293T cells. Top-flash relative signal to Renilla represents the canonical WNT signaling. *p < 0.05. Ns: not significant. (B) The effect of the variant of MED12 on the canonical WNT signaling pathway. Luciferase assay was performed among HEK293T cells. AP1 represents non-canonical WNT signaling.
FIGURE 5
FIGURE 5
The knock-in of MED12 c.5344C > T (p.Arg1784Cys) on mouse embryogenesis. (A) The knock-in of MED12 c.5344C > T (p.Arg1784Cys) produced mouse embryos displaying exencephaly and spina bifida phenotypes. The lesion site of exencephaly was indicated by the red arrow, while the spina bifida lesion site was indicated by the white arrow. The phenotypes of mice were observed and captured under a 6X microscope. (B) DNA Sanger sequencing on the CRISPR/Cas9 Mice.
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