FZD6 is a novel gene for human neural tube defects

Abstract

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6(-/-) mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3'-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs.

Figure 1

Rare nonsynonymous FZD6 variants. A: Schematic representations of FZD6 (RefSeq: NG_ 028909.1; NM_001164615.1) gene with the approximate locations of the identified rare nonsynonymous variants. FZD6 mutations absent in controls and predicted to have a functional effect are circled in red. The DNA mutation numbering is according to cDNA numbering with nucleotide +1 as the A of the ATG translation initiation codon in the reference sequence. Ex, exon. B: Topological model of the FZ6 protein. The approximate positions of the mutations identified are shown. Nonsynonymous FZD6 mutations absent in controls and predicted to have a functional effect are circled in red. CRD, cysteine-rich domain. C: Clustal W protein sequence alignment of human FZD6 with human FZD3 and orthologues from other species. Residues conserved between FZD and other family members are gray highlighted. Ensemble accession numbers of human FZD6 (hFZD6): ENSP000004290551; human FZD3 (hFZD3): ENSP000002400931, Pan troglodytes FZD6 (pFZD6): ENSPTRP000000437521, gorilla FZD6 (gFZD6): ENSGOP000000153811; mouse FZD6 (mFZD6): ENSMUSP000000229061, gallus FZD6 (gaFZD6): ENSGALP000000258431, Xenopus FZD6 (xFZD6): ENSXETP00000088811, Danio Rerio FZD6 (dFZD6): ENSDARP000000627021, and Drosophila FZD6 (drFZD6): FBpp00754851.

Figure 2

Electropherograms of nonsynonymous FZD6 mutations absent in controls and predicted to have a functional effect. The altered amino acids with their positions and corresponding nucleotide changes (capital letters) are shown with the sequencing trace data. Small letters indicate intronic sequence. The FZD6 p.Cys615X mutation is shown in the 3′→5′ direction and the complement nucleotide is shown. The other mutations are shown in sense (5′→3′) direction. WT, wild-type sequence; M, mutated sequence. ^*, stop codon.