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. 2018 Apr;39(4):550-562.
doi: 10.1002/humu.23397. Epub 2018 Jan 19.

Genetic contribution of retinoid-related genes to neural tube defects

Huili Li  1   2 , Jing Zhang  1 , Shuyuan Chen  3 , Fang Wang  2 , Ting Zhang  2 , Lee Niswander  1
Affiliations

Genetic contribution of retinoid-related genes to neural tube defects

Huili Li et al. Hum Mutat. 2018 Apr.

Abstract

Rare variants are considered underlying causes of complex diseases. The complex and severe group of disorders called neural tube defects (NTDs) results from failure of the neural tube to close during early embryogenesis. Neural tube closure requires the coordination of numerous signaling pathways, including the precise regulation of retinoic acid (RA) concentration, which is controlled by enzymes involved in RA synthesis and degradation. Here, we used a case-control mutation screen study to reveal rare variants in retinoid-related genes in a Han Chinese NTD population by sequencing six genes in 355 NTD cases and 225 controls. More specific rare variants were found in exonic and upstream regions in NTD cases. The RA-responsive genes CYP26A1, CRABP1, and ALDH1A2 harbored NTD-specific rare variants in their upstream regions. Unexpectedly, the majority of missense variants in NTD cases were found in CYP26B1, which encodes a RA degradation enzyme, whereas no missense variants in this gene were found in controls. Functional analysis indicated that the CYP26B1 NTD variants were inefficient in the degradation of RA using assays of RA-induced transcription and RA-initiated neuronal differentiation. Our study supports the contribution of rare variants in RA-related genes to the etiology of human NTDs.

Keywords: CYP26B1; DNA sequencing; NTD; neural tube defects; retinoid-related genes.

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Conflict of interest statement

DISCLOSURE STATEMENT

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1. Retinoid related genes harbor rare variants in humans with neural tube defects
A) A schematic of the synthesis and degradation of retinoic acid from vitamin A with the enzymes involved in retinoid metabolism and binding indicated in blue font. The genes sequenced in the present study are denoted by underlining of the encoded proteins. B) Rare variants were found in all sequenced genes in the present cohort of NTD cases relative to controls. P value was obtained via Fisher’s exact test and false discovery rate (FDR) correction. C) Illustration for each symbol in D-F. D-F) Schematics show the genomic positions of rare variants found in upstream regions of CYP26A1, CRABP1, and ALDH1A2 genes relative to the retinoic acid response-element (RARE) and predicted transcription factor binding sites.
FIGURE 2
FIGURE 2. Rare variants found in the CYP26B1 gene in the NTD case:control cohort
A) CYP26B1 is located on chromosome 2 in the p13.2 genomic region. B) CYP26B1 contains 5–6 exons depending on alternative splicing. All cohort-specific rare variants found in the present study were visualized in the UCSC genome browser (GRCh37/hg19). Green font indicates rare variants identified in controls, red font indicates NTD-specific rare variants. C) Schematic of the CYP26B1 protein showing functional domains and the position of rare variants found in exonic regions indicated by red oval for NTD-specific variants or green oval for the single control individual. D) Sanger sequencing traces to confirm the potential damaging rare variants found in the CYP26B1 gene in NTD individuals. E) Conservation analysis by Clustal omega and location of NTD-specific variants. F) Predicted effect of the rare variants relative to protein function and amino acid charge.
FIGURE 3
FIGURE 3. CYP26B1 NTD-variants attenuate function as assessed by modulation of RA-mediated gene transcription
A–H) Real-time quantitative PCR assays were performed to test the mRNA levels of genes that are downstream of RA signaling and involved in neural development. NE-4C cells were transfected with CYP26B1 WT or mutant plasmids and treated for 24 hours with RA. Gapdh gene was used as a loading control. The level of expression is indicated as fold change relative to WT vector, which was set to 1. All experiments were from three biological replicates. ***: P < 0.0001; **: P < 0.01; *: P < 0.05; Student’s t-test.
FIGURE 4
FIGURE 4. CYP26B1 variants disrupt modulation of neural differentiation
Quantification of the immunofluorescent staining results of neuronal markers Tuj1 (A), Map2 (B) or Tbr2 (C) positive cells 7–8 days after RA treatment of NE-4C cells (Blank), or NE-4C cells transiently transfected with vector (Vector), wildtype CYP26B1 (WT), or different CYP26B1 variants. The percentage of immunopositive cells relative to all cells (Hoechst labeled) in a field of view were scored and then compared to cells transfected with WT CYP26B1. ***: P < 0.0001; **: P < 0.01; Student’s t-test. D) Typical images of the immunofluorescent staining used for calculations in A–C. For each genotype and neuronal marker, three to four replicates were performed.
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