Loss ofRAD9Bimpairs early neural development and contributes to the risk for human spina bifida

Abstract

DNA damage response (DDR) genes orchestrating the network of DNA repair, cell cycle control, are essential for the rapid proliferation of neural progenitor cells. To date, the potential association between specific DDR genes and the risk of human neural tube defects (NTDs) has not been investigated. Using whole‐genome sequencing and targeted sequencing, we identified significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = .0241). Among the eight identified variants, the two frameshift mutants and p.Gln146Glu affected RAD9B nuclear localization. The two frameshift mutants also decreased the protein level of RAD9B. p.Ser354Gly, as well as the two frameshifts, affected the cell proliferation rate. Finally, p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and the two frameshift variants showed a decreased ability for activating JNK phosphorylation. RAD9B knockdowns in human embryonic stem cells profoundly affected early differentiation through impairing PAX6 and OCT4 expression. RAD9B deficiency impeded in vitro formation of neural organoids, a 3D cell culture model for human neural development. Furthermore, the RNA‐seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation. These results represent the first demonstration of a DDR gene as an NTD risk factor in humans.

Micronutrient imbalance and common phenotypes in neural tube defects

“…A number of mouse mutants for genes across different DDR pathways result in embryonic lethality or a variety of embryonic malformations including NTDs (Friedberg & Meira, 2006; Tran et al, 2012). For example, knockout of DDR genes Brca1 , Xrcc2 , p53 , and Rad9b result in embryos with NTD; however, the incidence for each of these examples is less than 100% (Armstrong et al, 1995; Cao et al, 2020; Deans, Griffin, Maconochie, & Thacker, 2000; Gowen, Johnson, Latour, Sulik, & Koller, 1996; Leloup et al, 2010; Sah et al, 1995). Knockout of Rad9b , a DDR repair gene involved in double strand break detection, in mouse ES cells results in an increase in genomic instability and decrease in cell viability when cells were challenged with a damaging agent (Leloup et al, 2010).…”

“…Although this work did not directly assess apoptosis, MTX‐induced NTDs show increased apoptosis (X. Wang et al, 2014), thus the accumulation of double‐strand breaks may be one factor in triggering apoptosis and eventual failure to close the neural tube in folate deficient mice. While in this paragraph we speculated on how micronutrient imbalance can lead to DNA damage‐induced apoptosis, evidence from Rad9b mutants and MTX‐induced NTDs suggest reduced expression of neural progenitor‐related genes may be a common phenotype when DDR is dysregulated (Cao et al, 2020; Pei et al, 2019). The reduced expression of neural progenitor genes is consistent with the enrichment of neural differentiation genes found in Cyp26b1 ‐deficient cells treated with ATRA (Li, Zhang, Chen, et al, 2018).…”

Unraveling the complex genetics of neural tube defects: From biological models to human genomics and back

“…As before, we also performed Sanger sequencing assays to validate any identified variants. In this cohort a heterozygous stop-gain variant of MED13L (Cao et al, 2020). To better appreciate the potential roles of RAD9B during embryonic development, embryoid bodies (Fong et al) were derived from hESCs.…”

“…To better appreciate the potential roles of RAD9B during embryonic development, embryoid bodies (Fong et al) were derived from hESCs. We were able to achieve a 60% reduction in RAD9B expression using small interfering RNAs (siRNAs) in an Oct4-GFP-hESC cell line (Cao et al, 2020). We methodologies to reflect known variants in human NTD candidate genes, and these hPSCs can serve as the initiating cells for creating neural tube organoids (Liu et al, 2019).…”

Corrigendum

“…In (Cao et al, 2020), Co‐corresponding author Richard H. Finnell has added a financial disclosure, which should correctly read as:…”