FZD/VANGL Genes Polymorphisms Associated With Neural Tube Defects

Objective:To investigate the plausible role of PCP related genes VANGL and FZD in the pathogenesis of NTDs, we have selected SNPs from VANGL and FZD gene coding regions using bioinformatics tools. To detect associations among DNA variants and affection status of NTDs, we further screened those SNPs by DNA sequencing in112NTD patients and paired normal controls. In this study, we would like to provide experimental information on early screening, genetic consultant, prevention and treatment of NTDs base on DNA variants of the PCP pathways.Methods:(1) We searched coding region SNPs in VANGL1, VANGL2, FZD3, and FZD6genes, highly conserved regions were highlighted by the "Blocks" program, multiple alignments of sequences were performed by the CLUSTALW software, phylogenetic trees were further constructed. Finally, we have selected8coding region SNPs based on references and above bioinformatics searching.(2) We have collected whole blood samples from112NTDs patients and112normal controls (all samples were Han Chinese live in northern China). All subjects’ clinical characteristics were documented. Genomic DNA samples were extracted from whole blood samples for genotyping and DNA sequencing.(3) Direct DNA sequencing was employed to genotype8coding regions SNPs in4candidate genes. Allele and genotype frequencies were compared between cases and controls. Haplotype associations and genotype interactions among different SNPs were also performed. Prediction of2-D and3-D protein structure were conducted by SOPMA, SWISS-MODEL and CPHmodels platforms.Results:(1) Eight coding region SNPs were picked in4candidate genes, including346G>A (Alal16Thr) and1040A>C (Glu347Ala) in VANGL1,1209G>C (Gln403His) in VANGL2,435A>G (Leu145Leu) and702T>G (Pro234Pro) in FZD3,97A>G (Met33Val),1033A>C (Met345Leu), and1991C>A (Ala664Glu) in FZD6gene, respectively.(2) Two new polymorphisms were found by direct sequencing, including the codon116Ala/Pro (G>C) polymorphism in the VANGL1gene and the codon33(A>T) polymorphism in the FZD6gene. Interestingly, we have identified both G>A (Alal16Thr) and G>C (Alal16Pro) polymorphisms at codon116of VANGL1gene.(3) Significant associations were found between the VANGL1codon116polymorphism and NTDs (P=0.034and0.014for allele and genotype frequencies, respectively). The odd ratio of the GC genotype was9.918.(4) A three-SNP haplotype (A-G-C,97A>G,1033A>C,1991C>A) of FZD6gene was found associated with NTDs in the case-control study (P=0.0448). Since the odd ratio<1, it suggested that the haplotype could be a protection factor against NTDs.(5) Significant gene-gene interactions were found among the GC genotype of VANGL1codon116(Alal16Pro) and the GG genotype of codon145(Leu145Leu) polymorphism of FZD3gene, the AA genotype of FZD6gene codon33(Met33Val), the TG genotype of FZD6gene codon345(Met345Leu), and the CC genotype of FZD6gene codon664(Ala664Glu). P values were0.026,0.002,0.004, and0.01, respectively.(6) The3-D protein structure prediction showed that the VANGL1gene codon116Pro mutant had shortened a-helix than the wild type (Ala). The3-D structure change may have an impact on the biological function.Conclusion:(1) We have identified a new mutation of VANGL1(Alal16Pro) gene in Han Chinese. The GC genotype was significantly associated NTDs. Strong gene-gene interactions were found among the VANGL1codon116GC genotype, FZD3, and FZD6gene variants. It may suggest the plausible epistasis among PCP pathway genes. A3-SNP haplotype in the FZD6gene associated was negatively associated with NTDs, it could be a "protective" factor against the risk of neural tube defects.(2) Coding region mutations that identified in NTD patients could change3-D structures of PCP pathway genes. The structural change of VANGL1protein could have a dramatic impact on the biological functions of PCP pathway genes.(3) It is suggested that PCP pathway related genes VANGL1, FZD3, and FZD6variants associated the pathogenesis of NTDs. Although folic acid supplement has been proved to be an effective prevention of NTDs, however, individuals who carry PCP pathway gene mutations may not response to folic acid. Understanding genetic backgrounds of non-folic acid sensitive NTDs will pave the road of future personalized prevention and treatments.(4) In this study, we used bioinformatics tools to select, evaluate, validate and analyze SNPs from databases, and to predict protein structural changes for DNA variants. It provides a cost-effective strategy for SNP tagging and further deciphering of gene-disease associations.