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Objective: To provide novel insights into the aetiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) by integrating multi-omics data and exploring susceptibility genes associated with NSCL/P., Methods: A two-stage genome-wide association study (GWAS) of NSCL/P was performed, involving a total of 1,069 cases and 1,724 controls. Using promoter capture Hi-C (pCHi-C) datasets in human embryonic stem cells (hESC) and chromatin immunoprecipitation sequencing (ChIP-seq) in craniofacial tissues, we filtered out single nucleotide polymorphisms (SNPs) with active cis-regulation and their target genes. Additionally, we employed expression quantitative trait loci (eQTL) analysis to identify candidate genes., Results: Thirteen SNPs were identified as cis-regulation units associated with the risk of NSCL/P. Five of these were proven to be active in chromatin states in early human craniofacial development (rs7218002: odds ratio [OR] 1.50, P = 8.14E-08; rs835367: OR 0.78, P = 3.48E- 05; rs77022994: OR 0.55, P = 1.05E-04; rs961470: OR 0.73, P = 1.38E-04; rs17314727: OR 0.73, P = 1.85E-04). Additionally, pCHi-C and eQTL analysis prioritised three candidate genes (rs7218002: NTN1, rs835367: FGGY, LINC01135). NTN1 and FGGY were expressed in mouse orofacial development. Deficiencies in NTN1, FGGY and LINC01135 were associated with cleft palate and cleft lip, abnormal facial shape and bifid uvula, and abnormality of the face, respectively., Conclusion: Our study identified five SNPs (rs7218002, rs835367, rs77022994, rs961470 and rs17314727) and three susceptibility genes (NTN1, FGGY and LINC01135) associated with NSCL/P. These findings contribute to a better understanding of the genetic factors involved.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a complex disease with a strong genetic component. More than 40 loci have been identified to be associated with the risk of NSCL/P by genome-wide association studies (GWASs), but the majority of these variants are mapped to non-coding regions of the genome. Expression quantitative trait locus (eQTL) studies have increasingly been integrated with GWASs to identify target genes for these non-coding variants. In this study, we generated a unique, lip-specific eQTL dataset from 40 NSCL/P patients. A total of 5158 eQTL SNPs (eSNPs) -689 eQTL genes were identified after multiple corrections. Then, we integrated nominal eQTL SNPs with NSCL/P risk SNPs and identified 243 variants associated with the expression of 18 genes in lip tissues. Functional annotation analysis indicated that these risk eSNPs were significantly enriched in transcription regulation and chromatin open regions on the genome. These susceptible genes were enriched in cell fate determination, the pluripotency of stem cells, and Wnt signaling pathways. Finally, 8 of the 18 susceptible genes were differentially expressed in NSCL/P case-control studies. In summary, we have generated a unique lip-specific eQTL resource and identified multiple associations for NSCL/P risk loci, which should inform functional studies of NSCL/P biology., Competing Interests: The authors declare no conflict of interest.

Craniomaxillofacial developmental anomalies are one of the most prevalent congenital defects worldwide and could result from any disruption of normal development processes, which is generally influenced by interactions between genes and the environment. Currently, with the advances in genetic screening strategies, an increasing number of novel variants and their roles in orofacial diseases have been explored. Zebrafish is recognized as a powerful animal model, and its homologous genes and similar oral structure and development process provide an ideal platform for studying the contributions of genetic and environmental factors to human craniofacial malformations. Here, we reviewed zebrafish models for the study of craniomaxillofacial developmental anomalies, such as human nonsyndromic cleft lip with or without an affected palate and jaw and tooth developmental anomalies. Due to its potential for gene expression and regulation research, zebrafish may provide new perspectives for understanding craniomaxillofacial diseaseand its treatment., (© 2022 Wiley Periodicals LLC.)

Non-syndromic cleft lip with/without cleft palate (NSCL/P) is one of the most common birth defects in humans with an overall prevalence of one per 1000 live births. Due to genetic and environmental influences, the fusion of the lips or palate may be interrupted at any stage and cause a cleft. Over decades, dozens of susceptible genes and loci have been identified using multiple genetic approaches. Our group has collected samples of NSCL/P patients since 2008 and established the biobank. We discovered numerous susceptible loci related to the occurrence of NSCL/P in the Chinese population, such as 16p13.3, 1q32.2, 10q25.3 and 17p13.1. In addition, we performed functional studies on related loci and genes by using molecular biology, cell biology, animal models and other methods to provide a basis for the construction of the NSCL/P genetic map in the Chinese population and help to implement individualised prophylaxis and treatment. Future efforts will focus on identifying functional variants, investigating pathways and other interactions, and including phenotypic and ethnic diversity in research.

Objective: To explore susceptibility genes and pathways for non-syndromic cleft lip with or without cleft palate (NSCL/P)., Materials and Methods: Two genome-wide association studies (GWAS) datasets, including 858 NSCL/P cases and 1,248 controls, were integrated with expression quantitative trait loci (eQTL) dataset identified by Genotype-Tissue Expression (GTEx) project in whole-blood samples. The expression of the candidate genes in mouse orofacial development was inquired from FaceBase. Protein-protein interaction (PPI) network was visualized to identify protein functions. Go and KEGG pathway analyses were performed to explore the underlying risk pathways., Results: A total of 233 eQTL single-nucleotide polymorphisms (SNPs) in 432 candidate genes were identified to be associated with the risk of NSCL/P. One hundred and eighty-three susceptible genes were expressed in mouse orofacial development according to FaceBase. PPI network analysis highlighted that these genes involved in ubiquitin-mediated proteolysis (KCTD7, ASB1, UBOX5, ANAPC4) and DNA synthesis (XRCC3, RFC3, KAT5, RHNO1) were associated with the risk of NSCL/P. GO and KEGG pathway analyses revealed that the fatty acid metabolism pathway (ACADL, HSD17B12, ACSL5, PPT1, MCAT) played an important role in the development of NSCL/P., Conclusions: Our results identified novel susceptibility genes and pathways associated with the development of NSCL/P., (© 2020 Wiley Periodicals LLC.)

Although several genome-wide association studies (GWAS) of non-syndromic cleft lip with or without cleft palate (NSCL/P) have been reported, more novel association signals are remained to be exploited. Here, we performed an in-depth analysis of our previously published Chinese GWAS cohort study with replication in an extra dbGaP case-parent trios and another in-house Nanjing cohort, and finally identified five novel significant association signals (rs11119445: 3' of SERTAD4, P = 6.44 × 10 -14 ; rs227227 and rs12561877: intron of SYT14, P = 5.02 × 10 -13 and 2.80 × 10 -11 , respectively; rs643118: intron of TRAF3IP3, P = 4.45 × 10 -6 ; rs2095293: intron of NR6A1, P = 2.98 × 10 -5 ). The mean (standard deviation) of the weighted genetic risk score (wGRS) from these SNPs was 1.83 (0.65) for NSCL/P cases and 1.58 (0.68) for controls, respectively (P = 2.67 × 10 -16 ). Rs643118 was identified as a shared susceptible factor of NSCL/P among Asians and Europeans, while rs227227 may contribute to the risk of NSCL/P as well as NSCPO. In addition, sertad4 knockdown zebrafish models resulted in down-regulation of sox2 and caused oedema around the heart and mandibular deficiency, compared with control embryos. Taken together, this study has improved our understanding of the genetic susceptibility to NSCL/P and provided further clues to its aetiology in the Chinese population., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aim: To investigate the associations between single nucleotide polymorphisms (SNPs) in miRNA regulome and nonsyndromic orofacial clefts. Materials & methods: The associations were evaluated by logistic regression model in stage I (504 cases and 455 controls) and stage II (1500 cases and 1386 controls). Functional experiments including luciferase activity assay, cell apoptosis and proliferation, serum miRNA expression, and mouse embryo RNA sequencing were performed. Results: Rs3830766 in the enhancer of hsa-miR-4260 was associated with cleft lip only (CLO) and enhancer activity. Hsa-miR-4260 expression decreased in the serum of CLO. Overexpression of miR-4260 inhibited cell proliferation and promoted cell apoptosis. UBB was the target gene of hsa-miR-4260. Conclusion: Rs3830766 in the hsa-miR-4260 enhancer that can interact with UBB was relevant to CLO.

Previous genome-wide association study of nonsyndromic cleft lip with or without cleft palate (NSCL/P) identified a susceptible variant (rs4791774). We hypothesized that the functional single nucleotide polymorphism (SNP) may be in linkage disequilibrium with this lead SNP. The potential functional SNP (rs4791331) was identified by bioinformatic analysis. A case-control study with 891 orofacial cleft cases and 830 controls was designed to investigate its association with orofacial cleft. The allele-specific DNA-protein binding preference was predicted by JASPAR database. Cell proliferation, cycle and apoptosis, luciferase activity and netrin-1 (NTN1) expression were examined after transfection with the rs4791331 C/T vector in HEK-293 and HEPM cell lines. Forty-six lip tissues of NSCL/P patients were collected to detect NTN1 expression. ntn1a knockout zebrafish models were generated by CRISPR/Cas9 and observed with micro-CT. In the case-control study, the rs4791331-T allele was associated with an increased risk of nonsyndromic orofacial cleft (OR = 1.41, 95% CI = 1.19-1.68), as well as the subgroups cleft lip only (OR = 1.46, 95% CI = 1.14-1.87) and cleft lip and palate (OR = 1.58, 95% CI = 1.27-1.96). The T allele of rs4791331 exhibited anti-apoptotic effects and promoted cell cycle progression at the G1/S transition. Decreased enhancer activity and reduced NTN1 expression following transfection of the T allele were observed. Carriers of the CT/TT genotypes showed significantly lower expression of NTN1 than CC carriers. The ntn1a -/- zebrafish showed relatively wider intermaxillary fissures. These results indicate that rs4791331 (C > T) disrupted motif binding and led to abnormal expression of NTN1, which may be involved in the development of NSCL/P.

Nonsyndromic cleft lip with/without cleft palate (NSCL/P) is one of the most common human congenital defects. Rs2262251 (G>C) in long noncoding RNA (lncRNA) RP11-462G12.2 is in high linkage disequilibrium with rs8049367, which was identified in our previous genome-wide association study on NSCL/P, and is a potential causative single-nucleotide polymorphism (SNP) for NSCL/P. To test these hypotheses, rs2262251 was evaluated in another cohort of 1,314 cases and 1,259 controls. Rs2262251 was associated with NSCL/P risk (p = .003). However, no association was detected for cleft palate only. SNP rs2262251 affected the structure and expression of lncRNA RP11-462G12.2 in HEK293 and HEPM cells and in lip tissues from patients with NSCL/P. Overexpression of the rs2262251 G allele contributed to reducing the number of cells in the G0/G1 phase, inhibiting cell apoptosis, and promoting cell proliferation in vitro. The rs2262251 C allele regulated the expression of miR-744-5p and its target gene IQSEC2, both of which were expressed in human lip tissues, and showed reverse correlation during mouse lip development. Taken together, these findings suggest that rs2262251 is associated with the risk of NSCL/P and participates in a lncRNA-miRNA-mRNA regulatory axis in which miR-744-5p and IQSEC2 combine to control NSCL/P development., (© 2019 Wiley Periodicals, Inc.)

Background: Non-syndromic supernumerary teeth (NSST) or hyperdontia may share common genetic determinants with non-syndromic cleft lip with or without palate (NSCL/P). The aim of this study was to test the associations between five genome-wide-associated NSCL/P-susceptible single nucleotide polymorphisms (SNPs) (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) and the occurrence of NSST., Materials and Methods: A total of 163 cases and 326 controls were recruited and their genomic DNA was extracted from blood samples. Five NSCL/P-susceptible SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan method. Odds ratio (OR) and 95% confidence interval (CI) were used to estimate the associations between the SNPs and the risk of NSST by PLINK software., Results: Rs4791774 (A > G) and rs13041247 (T > C) were associated with risk of NSST (rs4791774: P add  = 0.011, OR, 95% CI = 0.62, 0.43-0.90; rs13041247: P homo  = 0.031, OR, 95% CI = 1.79, 1.05-3.05) and one supernumerary tooth (rs4791774: P dom  = 0.009, OR, 95% CI = 0.56, 0.36-0.87; rs13041247: P homo  = 0.034, OR, 95% CI = 1.82, 1.05-3.15). Rs4791774 (A > G) was also showed association with risk of upper arch supernumerary teeth only (P add  = 0.010, OR, 95% CI = 0.60, 0.41-0.89)., Conclusion: Non-syndromic cleft lip with or without palate-susceptible loci rs4791774 (A > G) and rs13041247 (T > C) were associated with the risk of supernumerary teeth., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Objective: Non-syndromic tooth agenesis (NSTA) may share common genetic factors with non-syndromic cleft lip with or without cleft palate (NSCL/P). Single-nucleotide polymorphisms (SNPs) were associated with individual's susceptibility to these anomalies. We selected five NSCL/P-associated SNPs from our previous genome-wide association study (GWAS) to test for the associations with NSTA., Materials and Methods: A total of 677 NSTA cases and 1,144 healthy controls were recruited in this case-control study. Five genome-wide NSCL/P-associated SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan platform and evaluated for the associations with NSTA using plink software., Results: No significant associations between these SNPs and risk of NSTA were observed in the overall analysis and subgroup analysis with the number of missing teeth. However, in the subgroup analysis by tooth position, rs8049367 was nominally associated with mandibular premolar agenesis (Dominant model: OR dom  = 0.66, 95% CI dom  = 0.47-0.93, p dom  = 0.016; Heterozygote model: OR het  = 0.60, 95% CI het  = 0.41-0.88, P het  = 0.008). Rs4791774 showed a nominal association with congenitally missing maxillary canine (Dominant model: OR dom  = 0.53, 95% CI dom  = 0.28-0.98, p dom  = 0.041; Heterozygote model: OR het  = 0.50, 95% CI het  = 0.26-0.97, P het  = 0.041) and premolar (Additive model: OR = 0.59, 95% CI = 0.36-0.96, p = 0.035)., Conclusion: This study showed that NSCL/P susceptible loci rs8049367 and rs4791774 were probably associated with the risk of NSTA., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: The genetic etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) has not been fully clarified to date. Epidermal growth factor receptor (EGFR) was reportedly involved in its biological establishment and regulation of cell migration during the embryonic stage., Methods: We selected a super pathway of endocytic trafficking of EGFR and investigated the associations of single-nucleotide polymorphisms (SNPs) in the super pathway with the risk of NSCL/P by analyzing our published genome-wide association study (GWAS) data from 504 NSCL/P individuals and 455 controls. After the false discovery rate (FDR) control, we conducted linkage disequilibrium (LD) analyses and conditional regression analyses to obtain independent lead SNPs. We performed LD analyses between the lead SNPs and the reported SNPs to find novel ones from our study. We annotated the lead SNPs and investigated their mapped genes in silico., Results: A total of 82 SNPs showed a statistical association with the risk of NSCL/P after FDR control. They contained three reported SNPs which were g.117068049G>A (rs7078160), g.117086783C>G (rs10886040), and g.117101266G>T (rs17095681). Four independent lead SNPs were obtained, including g.116979803 T>C (rs1905539) and g.117037960A>G (rs7902502) at 10q25.3, g.35720163G>C (rs75656820) at 17q12, and g.156864512G>A (rs1800877) at 1q23.1. Three of them were in low LD (r 2  < 0.5) with the reported SNPs except g.117037960A>G (rs7902502), so these three were newly identified. Lead SNPs were mapped to three genes: SHTN1, AP2B1, and NTRK1. The three genes were relatively more highly expressed in the human craniofacial region and in the proximal maxillary location during the craniofacial development stage of the embryonic mouse., Conclusion: Our results suggested that SHTN1, AP2B1, and NTRK1 might be associated with the development of NSCL/P., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

microRNAs (miRNAs) are widely involved in craniofacial development, and genetic variants of miRNAs may be associated with the risk of nonsyndromic orofacial cleft (NSOC). Here, we systematically selected five single nucleotide polymorphisms (SNPs) of miRNAs and investigated the associations between these variants and NSOC susceptibility in a two-stage case-control study including 1,406 NSOC patients and 1,578 controls from the Chinese population. We found that compared with the C allele, the rs2910164 G allele of pre-miR-146a was associated with an increased risk of NSOC (additive model: odds ratio [OR] = 1.17, 95% confidence interval [CI]: 1.06-1.30, P = 0.002), including both cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO). Bioinformatic prediction and functional assays revealed that the C allele of rs2910164 was significantly associated with inhibited HEK-293 and HEPM cell proliferation and decreased abundance of TRAF6. Both miR-146a and TRAF6 were expressed in the lip tissue samples of NSOC patients, and a moderate inverse correlation was observed between them. Taken together, these results demonstrated that miR-146a/rs2910164 is associated with susceptibility to NSOC, providing novel insights into the genetic etiology and underlying biology of NSOC., (© 2018 Wiley Periodicals, Inc.)

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect due to abnormal orofacial development. Previous studies report abnormal sonic hedgehog (SHH) signalling activity during NSCL/P pathogenesis and propose several genes in the SHH pathway as candidate risk genes. As such, we focussed on GLI3, a downstream modulator of the SHH pathway. In the present study,we genotyped 34 tag SNPs covering GLI3 and performed association analysis with NSCL/P in 504 cases and 455 healthy controls. Our preliminary results identified risk variants of GLI3 that are associated with NSCL/P susceptibility in a Chinese population. In particular, rs3801161 and its haplotypes rs3801161-rs7785287 displayed significant association with NSCL/P and survived Bonferroni correction for multiple comparisons. The robustness of the association between GLI3 and NSCL/P is worth further examination in the future across different populations.

Orofacial clefts are among the most common birth defects in humans worldwide. A large-scale, genome-wide association study (GWAS) in the Chinese population recently identified several genetic risk variants for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We selected 16 significant SNPs from the GWAS I stage (P < 1.00E-5) that had not been replicated to validate their association with NSCL/P in 1931 NSCL/P cases and 2258 controls. Ultimately, we identified a NSCL/P susceptibility loci (rs17095681 at 10q25.3, intron of SHTN1 and 27.2 kb downstream of VAX1, P meta  = 3.80E-9, OR = 0.64) in Chinese Han and Hui populations. This locus was not high LD with the reported loci in 10q25.3. It was a newly identified independent locus in 10q25.3 associated with NSCL/P. These results imply that SHTIN1 may involve in the pathogenesis of NSCL/P advance our understanding of the genetic susceptibility to NSCL/P.

Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects worldwide and is characterized by abnormalities of the orofacial structure. Syndromic CL/P is mainly caused by Mendelian disorders such as Van der Woude Syndrome (VWS). However, >70% of CL/P cases are nonsyndromic, characterized by isolated orofacial cleft without any known syndrome. The etiology of nonsyndromic CL/P (NSCL/P) remains elusive, but it has been suggested that causative genes of syndromic CL/P might also contribute to NSCL/P. As such, the VWS causative gene IRF6 has been extensively studied in NSCL/P. Recently, GRHL3 was identified as another VWS causative gene. Thus, it may be a novel candidate gene for NSCL/P. In the present study, we genotyped 10 tag SNPs covering GRHL3 and performed association analysis with NSCL/P in 504 cases and 455 healthy controls. Our preliminary results identified rs10903078, rs4638975, and a haplotype rs10903078-rs6659209 of GRHL3 that exceeded the significance threshold (p<0.05), though none survived Bonferroni correction for multiple comparisons. As the first study between GRHL3 and NSCL/P, the contribution of this gene to NSCL/P etiology should be interpreted with caution based on existing evidence. Further, the robustness of association between GRHL3 and NSCL/P should be further validated in expanded cohorts., (Copyright © 2016. Published by Elsevier B.V.)

We hypothesized that microRNA binding site single nucleotide polymorphisms (SNPs) in fibroblast growth factors (FGFs) and their receptor genes (FGFRs) may affect microRNA and mRNA interactions and are thereby associated with susceptibility of non-syndromic orofacial cleft (NSOC). Ten SNPs among the FGF and FGFR genes were selected and their associations with NSOC susceptibility were investigated in a case-control study of 602 patients with NSOC and 605 healthy controls. FGF2/rs1048201, FGF5/rs3733336 and FGF9/rs546782 showed suggestive association with NSOC susceptibility. In the combination analysis, the observed odds ratios (ORs) decreased with the number of protective alleles (rs1048201-T, rs3733336-G and rs546782-T) but were not statistically significant beyond the first comparison. Hsa-miRNA-496, hsa-miRNA-145 and hsa-miRNA-187 were predicted to be miRNAs with binding sites within/near these SNPs and were expressed in lip tissues. Decreased FGF2, FGF5 and FGF9 expression was observed in three cell lines transfected with the corresponding miRNAs. Moreover, the three SNPs could contribute to differential binding efficacy between hsa-miRNA-496 and FGF2, hsa-miRNA-145 and FGF5, hsa-miRNA-187 and FGF9 in luciferase assay. The results suggest that FGF2/rs1048201, FGF5/rs3733336 and FGF9/rs546782 are associated with the risk of NSOC and that these miRNA-FGF interactions may affect NSOC development.

Located at 15q22 a susceptibility region for nonsyndromic orofacial clefts (NSOC), TPM1 encodes a group of highly conserved ubiquitous actin-binding proteins involved in the muscle contraction and cytoskeleton organization. Considering the multiple functions of TPM1 gene, we investigated the potential relationship between TPM1 polymorphisms and risk of NSOC in a Chinese Han population. Four tag single nucleotide polymorphisms (tSNPs) of TPM1 (rs11071720, rs3803499, rs12148828, and rs1972041) were selected to conduct a case-control study with 673 NSOC patients and 705 unrelated healthy controls from a Chinese Han population. The SNPs were genotyped by the IPLEX Sequenom MassARRAY platform. SNP rs1972041GA showed a decreased risk of NSOC in heterozygotes (P = 0.038, OR = 0.77, 95%CI = [0.61, 0.99]). Further stratified analysis revealed an enhanced protective effect of the minor allele G at rs197204 on lip with cleft palate (CLP) and cleft lip with or without cleft palate (CL/P) groups under a codominant or dominant model. No association was observed between the remaining three markers (rs11071720, rs3803499, and rs12148828) and NSOC as well as its subgroups. TPM1 polymorphisms might contribute to the etiology of NSOC, and more emphasis should be placed on TPM1 during craniofacial development., (© 2016 Wiley Periodicals, Inc.)

Nonsyndromic cleft lip with or without a cleft palate (NSCL/P) is among the most common human congenital birth defects and imposes a substantial physical and financial burden on affected individuals. Here, we conduct a case-control-based GWAS followed by two rounds of replication; we include six independent cohorts from China to elucidate the genetic architecture of NSCL/P in Chinese populations. Using this combined analysis, we identify a new locus at 16p13.3 associated with NSCL/P: rs8049367 between CREBBP and ADCY9 (odds ratio=0.74, P=8.98 × 10(-12)). We confirm that the reported loci at 1q32.2, 10q25.3, 17p13.1 and 20q12 are also involved in NSCL/P development in Chinese populations. Our results provide additional evidence that the rs2235371-related haplotype at 1q32.2 could play a more important role than the previously identified causal variant rs642961 in Chinese populations. These findings provide information on the genetic basis and mechanisms of NSCL/P.

Background: The axis inhibition protein 2 (AXIN2) is an important regulator of β-catenin degradation in the Wnt pathway, which plays a key role in craniofacial morphogenesis. The goal of this study was to investigate the potential relationship between AXIN2 polymorphisms and the risks of non-syndromic orofacial clefts (NSOC) in a Chinese Han population., Methods: Four polymorphisms of AXIN2 (rs2240307, rs11867417, rs2240308, and rs7591) were selected to perform a case-control study with 599 NSOC cases and 602 healthy individuals from a Chinese Han population. The single nucleotide polymorphisms (SNPs) were genotyped on basis of double ligation and multiplex fluorescence PCR., Results: Weak associations were found between these four SNPs and the risk of NSOC. Further stratified analysis showed that the overall genotype frequencies of rs2240307 were different between the cleft palate only (CPO) group and the control group (P = 0.048), and GG genotype markedly contributed to the susceptibility to CPO (OR = 3.22, 95% CI = 1.13-9.18). The similar effect was also observed on GA/AA genotype compared with GG homozygote (OR = 0.30, 95% CI = 0.11-0.84). The results of LD analysis between each pair of SNPs revealed that two SNPs (rs11867417 and rs7591) were in a LD block (r(2) > 0.8). But no statistically significant was found between cases and controls from haplotype analysis in these two loci., Conclusions: The borderline results gave us a hint that rs2240307 contributed to the susceptibility to CPO in a Chinese Han population, which was conductive to improving our awareness of the causes of NSOC., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)