Copy Number Variants And Somatic Mutations Of Planar Cell Polarity Signaling Pathway Genes And Neural Tube Defects

BackgroundNeural tube defects(NTDs)are congenital malformations of the central nervous system resulting from a failure of the neural tube to close during the third-or fourth-weeks postfertilization.Although NTDs are considered as multifactorial disorders arising from complex interactions of genetic and environmental factors,genetic components are considered dominant factors contributing to the susceptibility of failed neural tube closure.Planar cell polarity(PCP)signaling pathway plays an essential role in the polarization and coordinated movement of cells during embryonic morphogenesis.Mutations of PCP genes could lead to NTD-related phenotypes in mice,and the digenic,trigenic,and oligogenic combinations of PCP variants may also cause mouse NTDs.At present,almost all of the reported human NTDrelated mutations are from studies that used DNA extracted from blood or saliva samples,and the NTD-related mutations identified in these studies were all considered as single-nucleotide germline mutations.At present,the importance of somatic mutations and copy number variants(CNVs)are well documented in neuro-developmental diseases and have been recognized recently.Considering that NTDs are a group of severe disabilities,and the parents of the NTDs cases are often normal,we aimed to identify the role of CNVs and somatic mutations of PCP pathway genes in the occurrence of NTDs.Objectives(1)To explore if rare de novo CNVs in PCP genes are related to NTDs occurrence.(2)To examine if somatic mutations in PCP pathway genes in central nervous tissues contribute to the occurrence of NTDs.MethodsThis study consisted of two parts:Part 1 focused on CNVs of PCP pathway genes,and part 2 focused on somatic mutations of PCP pathway genes.PART 1.CNVs of PCP pathway genes and NTDs(1)Copy number variation assayWe performed a quantitative analysis of copy numbers of all exon regions in the VANGL1,VANGL2,CELSR1,SCRIB,DVL2,DVL3,and PTK7 in DNA samples of umbilical cord tissues CNVplex assay,which is a high-throughput,multiplex CNV analysis method.Quantitative real-time PCR(qPCR)was carried out to confirm the results of the CNV analysis.(2)Bioinformatic analysisPearson’s chi-square test was used to examine the associations between the risk for NTDs and selected CNVs.A two-tailed P value of<0.05 was considered to be statistically significant.Statistical analysis was performed using the Statistical Package for the Social Sciences,version 18.0(SPSS Inc.,Chicago,IL,USA).PART 2.Somatic mutations of PCP pathway genes and NTDs(1)Human gene sequencing48 pairs of lesion sites and umbilical cord tissue DNA were collected from NTDs cases.Ion Torrent Personal Genome Machine(PGM)sequencing was performed to identify the somatic mutations.The targeted gene panel included 19 genes:CELSR1,DACT1,DVLs,FZD6,PRICKLE2,SCRIB,VANGLs,GRHL3,CDX1,CDX2,CTHRC1,FZD1,FZD2,MED12,SFRP1,SFRP2,SFRP5,SMURF1,SMURF2.The inclusion criteria of somatic mutation during this stage were:1)the mutation has a sequencing depth≥100x and alternate-allele read frequency in PGM>5%;2)p-value ≤5x 10-5;3)the frequency of the mutation in the general population was lower than 5%,and 4)the mutation was missense or loss of function.Sanger sequencing was used to validate mutations in the target genes.The source and distribution were further explored for the validated mutants when DNA samples from tissues of skin,heart,muscle,thymus,and lung of fetuses or blood of the parents were available.(2)Cellular functional studiesImmunostaining assays were carried out to examine the influence of target mutations on the subcellular localization of proteins.The western blotting assay was performed to examine the influence of variants on protein levels.Luciferase reporter assays were performed to examine the influence of variants on the expression of canonical and non-canonical WNT pathway signaling.Live-cell imaging for cell migration analysis was performed to measure the influence of variants on the cell migration process.All of the biological experiments were repeated at least twice.(3)Mouse CRISPR/Cas9 editingSix-week-old female C57BL/6 mice were used to provide the embryos on which to perform CRISPR/Cas9 assay.The six-week-old female mice mated with males,and then the zygotes were obtained the next day and cultured.Zygotes were injected with a mixture of Cas9 protein,Med 12 sgRNA,and SSODN to the targeting site.The injected zygotes were then transferred to pseudopregnant mice(32 zygotes per mouse for a total of 6 mice)to be carried to parturition.Mice were dissected at embryonic day 12.5 to screen the embryos for thepresence of any malformations.NTDs and embryo lethality was recorded.The Sanger sequencing method was performed to confirm the genetic variants in the mice,while wholeexome sequencing was used to make sure that the NTD phenotypes of knock-in mice were caused by Med12 variants rather than other NTD-causing genes.ResultsPART 1.CNVs of PCP pathway genes and NTDs(1)CNV analysis:A total of 16 CNVs were identified among 11 NTD probands.These CNVs were found at 5 loci involving DVL2(exons 1-15),VANGL1(exons 1-7,exon 8),and VANGL2(exons 5-8,exon 7-8).One CNV(DVL2 exons 1-15)was a duplication,and the remaining 15 were deletions.Eleven of the 16 CNVs were confirmed by qPCR.Relative copy number gains were found on exons 1-15 of DVL2 in one AN case;relative copy number losses were found on exons 1-7 of VANGL1 in two SB cases,and on exon 8 of VANGL1 in 8 NTD cases with different phenotypes.(2)Association analysis of rare de novo CNVs in VANGL1 and DVL2 with the risk of NTDs:Maternal and paternal inheritance was examined.Two CNVs were found to be de novo.The two de novo CNVs included a loss of exons 1-7 of VANGL1 in one SB case and a gain of exons 1-15 of D VL2 in one AN case.CNVs of VANGL1 and DVL2 public databases were used as controls.Both CNVs showed frequencies of 0.01%in the control population,indicating rare mutations in unaffected individuals.Compared with controls,the frequency of VANGL1 exon 1-7 deletion(1.14%)and DVL2 exon 1-15 duplication(0.57%)showed significant(P<0.05)enrichments in our NTDs cohort.PART 2.Somatic mutations of PCP pathway genes and NTDs(1)Human gene sequencingA total of 27 protein-altering somatic single nucleotide variants located in 15 PCP genes were identified;17 of them were predicted to be harmful to protein functions,and 10 had alternate-allele read frequencies greater than 40%.16 were novel,and 11 mutations were rare in control populations with minor allele frequency(MAF)<1%.Sanger sequencing showed 10 mutations were validated.The mutations CELSR1 c.6375G>C,DACT c.1192C>T,DVL2 c.l195A>G,FZD6 c.1991A>C and c.262C>G,VANGL1 c.1121G>A and MED12 c.5344C>T only occurred in neural tube lesion tissues,but not in other control tissues.Based on the bioinformatics data,we selected FZD6 c.262C>G(p.Gln88Glu),VANGL1 c.1121G>A(p.Arg374His),MED 12 c.5344C>T(p.Arg1782Cys)on which to perform further functional studies.(2)Cellular functional studiesFZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus and disrupted the colocalization of CELSR1 and FZD6.This mutation also affected the expression of non-canonical WNT signaling.VANGL1 p.Arg374His impaired co-localization of CELSR1 and VANGL1,increased the protein levels of VANGL1,and influenced cell migration.MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical WNT signaling pathway.(3)Mouse CRISPR/Cas9 editingCRISPR/Cas9 mutagenesis editing was employed to generate Med12 p.Arg1784Cys(homologous position of p.Arg1782Cys in human)knock-in mice.12 viable embryos and 1 dead embryo were observed.10 embryos were male,and 2 were female.Of the 10 male embryos,2 were Med12 p.Arg1784Cys knock-in hemizygotes with NTD phenotypes.The others were wildtype and no obvious structural malformations.The two female embryos were wildtype.The two hemizygous Med12 p.Arg1784Cys/Y mouse embryos exhibited 100%penetrant NTD phenotypes,including exencephaly and spina bifida,and curly tails.The result indicates that Med12 p.Arg1784Cys found in a human NTD patient can also cause NTDs in mice.Conclusions(1)Rare de novo CNVs in the PCP genes VANGL1 and DVL2 are associated with human NTDs.(2)Somatic mutations of PCP pathway genes(e.g.,VANGL1,FZD6,and MED12)in neural tissues are one of the genetic mechanisms underlying NTD occurrence.