Nonsyndromic Cleft Lip With Or Without Cleft Palate Research Articles

IRF6 and FGF1 polymorphisms in non-syndromic cleft lip with or without cleft palate in the Polish population.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common developmental defect that significantly affects the morphology and function of the stomatognathic system in children. The etiology of these birth defects is multifactorial, and single nucleotide polymorphisms (SNPs) in IRF6 and FGF1 have been associated with NSCL/P. This study aimed to evaluate whether SNPs in IRF6, namely rs2013162, rs642961, rs2235373, and rs34010 in FGF1, are associated with NSCL/P occurrence in the Polish population. The study included 627 participants: 209 children with NSCL/P and 418 healthy controls. DNA was isolated from saliva in the study group and from umbilical cord blood in controls. Genotyping of polymorphisms was performed using quantitative PCR. There was no statistically significant association of IRF6 gene variants with NSCL/P occurrence, although for rs2013162, AA genotype, odds ratio (OR) = 1.16 and for AC genotype, OR = 0.83; for rs642961, AA genotype, OR = 0.84 and for AG genotype, OR = 1.41; and for rs2235373, AA genotype, OR = 0.79 and for AG, OR = 0.85. In the instance of rs34010 polymorphism in FGF1, the presence of the AA genotype was statistically significant in reducing the risk of NSCL/P (OR = 0.31, p = 0.001). Genetic variation in FGF1 is an important risk marker of NSCL/P in the Polish population, which cannot be stated for the polymorphisms in the IRF6 gene.

Integrated Analysis of the Association Between Variants at PAX7 and NSCL/P in the Han Population.

Paired box 7 (PAX7) has been considered as a candidate gene for non-syndromic cleft lip with or without palate (NSCL/P). However, there is no research for the XXX, and previous studies concentrated on limited variants. This study aimed to conduct sufficiently dense and powerful scans of variants at PAX7 and explored the roles of variants at PAX7 in NSCL/P among the XXX. Targeted region sequencing was performed to thoroughly screen variations, followed by a two-phase association analysis. 159 NSCL/P cases and 542 controls were analyzed in phase 1. Then in phase 2, the validation study was performed using 1626 cases and 2255 controls. We also explored the roles of variants at PAX7 gene in NSCL/P subtypes. Additionally, indirect associations were found by calculating LD and haplotypes. The study was conducted in XXX. 159 NSCL/P cases and 542 controls were analyzed in phase 1. Then in phase 2, the validation study was performed using 1626 cases and 2255 controls. Blood samples were collected. To explore the association analysis between variants at PAX7 and NSCL/P in XXX. The results showed that rs2236810, rs114882979 and rs2236804 were significantly associated with NSCL/P, which were predicted to have regulatory functions. Besides, variants at PAX7 function differently in the NSCL/P subtypes. We also discovered a PAX7 missense variant, NM_001135254 p.A369 V (NM_002584.2:c.1106C > T). In summary, we confirmed 3 SNPs at PAX7 were significantly associated with NSCL/P in XXX and identified a missense variant, NM_001135254 p.A369 V (NM_002584.2:c.1106C > T).

Identification of putative regulatory single-nucleotide variants in NTN1 gene associated with NSCL/P.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common polygenetic disease. Although genome-wide association studies (GWAS) identified NTN1 gene as a high-priority candidate of NSCL/P, the comprehensive genetic architecture of NTN1 weren't yet known. Thus, this study aimed to determine full-scale genetic variants of NTN1 for NSCL/P in Chinese Han people. Initially, targeted sequencing of NTN1 gene was performed on 159 NSCL/P patients to identify susceptible single nucleotide polymorphisms (SNPs) associated with NSCL/P. Then, association analysis and burden analysis were separately used to validate the common variants and rare variants identified among large size of samples (1608 NSCL/P cases and 2255 controls). Additionally, NSCL/P subtype association analysis was applied to elucidate the etiology discrepancy of non-syndromic cleft lip with palate (NSCLP) and non-syndromic cleft lip only (NSCLO). Lastly, bioinformatics analysis was performed to annotate and prioritize candidate variants. We found 15 NSCL/P-associated SNPs including rs4791774 (P = 1.10E-08, OR = 1.467, 95% CI: 1.286~1.673) and rs9788972 (P = 1.28E-07, OR = 1.398, 95% CI : 1.235~1.584) originally detected by previous GWASs in Chinese Han ancestry. Four NSCLO risk-associated SNPs and eight specific NSCLP associated SNPs were found. Three SNPs (rs4791331, rs4791774 and rs9900753) were predicted to locate at regulatory region of NTN1. Our study validated the association between NTN1 gene and pathogenesis of NSCL/P and reinforced the hypothesis that NSCLP have a different etiology from NSCLO. We also identified three putative regulatory SNPs in NTN1 gene.

Genetic Risk Assessment of Nonsyndromic Cleft Lip with or without Cleft Palate by Linking Genetic Networks and Deep Learning Models.

Recent deep learning algorithms have further improved risk classification capabilities. However, an appropriate feature selection method is required to overcome dimensionality issues in population-based genetic studies. In this Korean case-control study of nonsyndromic cleft lip with or without cleft palate (NSCL/P), we compared the predictive performance of models that were developed by using the genetic-algorithm-optimized neural networks ensemble (GANNE) technique with those models that were generated by eight conventional risk classification methods, including polygenic risk score (PRS), random forest (RF), support vector machine (SVM), extreme gradient boosting (XGBoost), and deep-learning-based artificial neural network (ANN). GANNE, which is capable of automatic input SNP selection, exhibited the highest predictive power, especially in the 10-SNP model (AUC of 88.2%), thus improving the AUC by 23% and 17% compared to PRS and ANN, respectively. Genes mapped with input SNPs that were selected by using a genetic algorithm (GA) were functionally validated for risks of developing NSCL/P in gene ontology and protein-protein interaction (PPI) network analyses. The IRF6 gene, which is most frequently selected via GA, was also a major hub gene in the PPI network. Genes such as RUNX2, MTHFR, PVRL1, TGFB3, and TBX22 significantly contributed to predicting NSCL/P risk. GANNE is an efficient disease risk classification method using a minimum optimal set of SNPs; however, further validation studies are needed to ensure the clinical utility of the model for predicting NSCL/P risk.

Rs28446116 in PTCH1 is associated with non-syndromic cleft lip with or without palate in the Ningxia population, China

ObjectivesTo investigate the association between PTCH1 single nucleotide polymorphism(SNP) and non-syndromic cleft lip with or without palate (NSCL/P) in the Ningxia Hui Autonomous region and predict the function of single nucleotide polymorphism through bioinformatics analysis. DesignA case-control analysis of 31 single nucleotide polymorphism locus alleles on PTCH1 gene (504 cases and 455 controls) was performed to explore the association between PTCH1 gene polymorphisms and non-syndromic cleft lip with or without palate in Ningxia region. Transcription factors, 3D single nucleotide polymorphism and other related information of single nucleotide polymorphism loci with statistical significance were screened by the case-control experiments, and then analyzed the corresponding transcription factors through the NCBI database. ResultsThe case-control study showed that 5 of the 31 single nucleotide polymorphism loci rs357564 (P = 0.0233), rs1805155 (P = 0.0371), rs28446116 (P = 0.0408), rs2282041 (P = 0.0439), rs56119276 (P = 0.0256) had statistically significant differences in allele frequencies between the case and control groups. Bioinformatics analysis revealed that EP300 and RUNX3, among the transcription factors associated with rs28446116, may be associated with the development of non-syndromic cleft lip with or without palate. ConclusionPTCH1 gene may be associated with the occurrence of non-syndromic cleft lip with or without palate in the Ningxia region, which may be related to the role of EP300 and RUNX3 in the development of cleft lip and palate.

Association of single nucleotide polymorphisms in the IRF6 gene with nonsyndromic cleft lip with or without cleft palate in Kinh Vietnamese patients

The interferon regulatory factor 6 (IRF6) gene, which causes Van der Woude syndrome, is associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P). Single nucleotide polymorphisms (SNPs) in the IRF6 gene were identified as susceptibility indicators of this defect in several populations. To further evaluate its role in this birth defect, we conducted this study with the aim of identifying allele frequencies, genotype frequencies, and associations of 5 SNPs (rs2235373, rs2235371, rs2235375, rs2013162, and rs2236907) in the IRF6 gene with NSCL/P in Kinh Vietnamese patients. A total of 132 patients with NSCL/P and 132 healthy individuals were included in our study group. Direct sequencing was performed to genotype the tag SNPs. Genetic models were used to compare genotype and allele frequencies between the case and control groups. In the recessive model, the genotypes C/C of rs2236907, C/C of rs2013162, G/G of rs2235375, and A/A of rs2235373 were associated with an increased risk of NSCL/P, whereas there was no clear association between rs2235371 and the malformation in any genetic model. When subgroup analysis was performed, we observed a similar risk trend in the cleft lip and palate, cleft palate only and cleft lip only phenotypes. In haplotype analysis, haplotype models of 5 tag SNPs were associated with increased risks of this defect in all phenotypic models (ORGCGCC/CCAA = 23.64, 95% CI 12.28-45.49, p < 0.0001). These findings point to a considerable contribution of rs2236907, rs2013162, rs2235373, and rs2235375 to the NSCL/P defect in Kinh Vietnamese individuals.

Using whole exome sequencing to identify susceptibility genes associated with nonsyndromic cleft lip with or without cleft palate.

Cleft lip and palate is a common congenital birth defect in humans. Its incidence rate in China is as high as 1.82%, and is now a frequent deformity observed among the Chinese population; moreover, it varies across regions. Although the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been widely investigated, the results are inconsistent. The specific genes and mechanisms responsible for NSCL/P have not been fully understood. Whole exome sequencing (WES) is a new strategy for studying pathogenic genes. WES studies on NSCL/P have not been conducted in East China. Therefore, the aim of this study was to screen candidate genes of NSCL/P in East China using WES and analyze the temporal and spatial expressions of the candidate genes during embryonic palatal development. WES was performed in 30 children with NSCL/P from East China to screen candidate genes. A bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by immunohistochemistry and western blotting. After WES, 506,144 single-nucleotide variant sites were found. The results of database comparison, functional analysis, and mass spectrometry revealed that only the laminin alpha 5 (LAMA5) gene (site: rs145192286) was associated with NSCL/P. Immunohistochemistry results showed that LAMA5 expression in the medial edge epithelium changed with formation, lifting, and contact during palatogenesis. Almost no LAMA5 expression was detected in the palatal mesenchyme or after palatal fusion. Western blotting and immunohistochemistry results showed consistent trends. In conclusion, the WES results shows that the mutation at the site (rs145192286) of LAMA5 is associated with NSCL/P. The temporal and spatial expressions of LAMA5 during palatal development further demonstrate the involvement of this gene. Therefore, we speculate that LAMA5 is a new candidate pathogenic gene of NSCL/P. The identification of new pathogenic genes would help elucidate the pathogenesis of NSCL/P and provide a scientific basis for the prenatal diagnosis, prevention, and treatment of NSCL/P.

Mir152 hypomethylation as a mechanism for non-syndromic cleft lip and palate

ABSTRACT Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21–26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP.

Rs9891446 in NTN1 is associated with right-side cleft lip in Han Chinese population

ObjectivesThis study aimed to reveal the association between single-nucleotide polymorphism of NTN1 and subtypes of non-syndromic cleft lip with or without cleft palate (NSCL/P) in Han Chinese Population. DesignInitially, we selected three single-nucleotide polymorphisms (SNP) (rs4791331, rs4791774 and rs9891446) in NTN1 from previous genetic studies. Then we recruited two Han Chinese cohorts (2004 cases and 1823 controls) and divided cases into subgroups: non-syndromic right-side cleft lip, non-syndromic left-side cleft lip, non-syndromic bilateral cleft lip and non-syndromic cleft lip with palate to further evaluate the associations between the subtypes of NSCL/P and SNPs in NTN1. PLINK and Haploview program were utilized to analyze the data. ResultsIn the association analysis under additive model, we found that G allele at rs9891446 could specifically increase the risk of right-side cleft lip (P = 0.0073, OR = 1.44, 95%CI: 1.1-1.88), which was consistent with the results of association analysis under genotypic model. ConclusionThis study showed that rs9891446 of NTN1 was specifically associated with right-side cleft lip in Han Chinese, which indicates that different subtypes of non-syndromic cleft lip have distinct genetic background.

BMP4 Regulates EMT to be Involved in non-Syndromic Cleft lip With or Without Palate.

In the previous study, we identified bone morphogenetic protein 4 (BMP4) responsible for non-syndromic cleft lip with or without cleft palate (NSCL/P). We aimed to elucidate the effects and mechanisms of BMP4 on epithelial-mesenchymal transition (EMT) through Smad1 signaling pathway to be involved in NSCL/P. The human oral epidermoid carcinoma cells (KBs) were transfected with plasmids or small interfering RNA (siRNA) to build the models. The migration of the cells was evaluated by transwell assay. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the expressions of BMP4, E-cadherin, N-cadherin, EMT-related transcription factors snal1 and snal2, matrix metalloproteinase 2 (MMP2), MMP9, Smad1, and phosphorylated Smad1. In the overexpression group, the migration number of cells was increased significantly. The protein expression of E-cadherin was decreased significantly, while the protein expression level of the N-cadherin was increased significantly. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly higher. The expression level of Smad1 was not significantly changed, while the phosphorylation of Smad1 was significantly increased. In the BMP4-siRNA group, the migrating number cells was significantly decreased. The protein expression of E-cadherin was increased significantly, while the expression of N-cadherin was significantly decreased. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly lower than that of the control group. The expressions of Smad1 and phosphorylation of Smad1 were not significantly changed. BMP4 enhances cell migration and promotes cell EMT through Smad1 signaling pathway. Abnormal BMP4 mediates migration and EMT through other relevant signaling pathways resulting in NSCL/P. The study provides new insight into the mechanisms of NSCL/P associated with BMP4.n.

Association of soluble epoxide hydrolase 2 gene with the risk of non-syndromic cleft lip with or without cleft palate in western Han Chinese population.

This study aimed to explore the associations between soluble epoxide hydrolase 2 gene (EPHX2) variants and non-syndromic cleft lip with or without cleft palate (NSCL/P) in Chinese Han population. We recruited 159 NSCL/P cases from Chinese Han population and carried out targeted resequencing using the whole genome sequencing data of 542 healthy Chinese individuals from Novegene internal database as controls. We classified EPHX2 variants as common or rare according to their minor allele frequency and performed an association analysis for common variations and a burden analysis for rare variations. The lowest P-value in NSCL/P was observed at rs57699806 (P=0.000 13, OR=2.849 and 95% CI: 1.691-4.800), followed by rs4732723 (P=0.006 50, OR=0.662 and 95%CI: 0.491-0.892), rs7829267 (P=0.009 20, OR=1.496 and 95%CI: 1.117-2.005), rs721619 (P=0.011 00, OR=1.474 and 95%CI: 1.098-1.980), and rs7816586 (P=0.040 00, OR=1.310 and 95%CI: 1.015-1.691). The odds ratios suggested the C allele at rs4732723 as a protective factor for NSCL/P and the reference alleles at other single nucleotide polymorphisms (SNPs) as the risk factors for NSCL/P. Burden analysis showed no statistical significance (P>0.05). Through targeted resequencing, this study identified five SNPs named rs57699806, rs4732723, rs7829267, rs721619, and rs7816586 around the region of EPHX2 gene associated with NSCL/P in Chinese Han population. Four SNPs of rs57699806, rs4732723, rs7829267, and rs7816586 were first identified.

Arhgap29 is Required for Proper Palatogenesis and its Loss in Ectodermal‐Derived Cells Results in a Kinked Tail Phenotype

Craniofacial deformities, specifically non‐syndromic cleft lip with or without palate (NSCL/P), are among the most common class of birth defects. Complex interactions between genetic and environmental factors contribute to the development of NSCL/P. Many genes have been associated with this disease, including ARHGAP29. ARHGAP29, Rho GTPase Activating Protein (GAP) 29, functions in the cyclical regulation of the small GTPase RhoA. Previous studies have identified genetic variants of ARHGAP29 in NSCL/P and have found that ARHGAP29 levels were reduced in mice deficient for Irf6. Data from these studies suggest a role for ARHGAP29 in craniofacial morphogenesis but it remains unknown if ARHGAP29 is required for, or regulates craniofacial development. ARHGAP29 is detected in many tissues, including the oral epithelium. Using the Keratin14‐driven Cre‐recombinase allele, Krt14Cre;Arhgap29fl/fl mice displayed a delay in palatal shelf elevation and did not exhibit a cleft palate. We hypothesized that the absence of a cleft palate was due to the late activation of the Krt14‐Cre driver. EctCre drives Cre recombinase specifically in the ectoderm a couple of days before the Krt14‐Cre. Our results show that EctCre;Arhgap29fl/fl mice survive to birth and display a non‐mendelian pattern of inheritance. Interestingly, EctCre;Arhgap29fl/fl mice display a fully penetrant kinked tail phenotype, which has been described in other murine models of NSCL/P, and has been attributed to neural tube and skeletal defects. Additionally, at embryonic (E) day 14.5, while all the wild‐type embryos showed adherent palatal shelves, 75% of EctCre;Arhgap29fl/fl mice displayed palatal shelves not elevated or adherent. Our preliminary data also identified one EctCre;Arhgap29fl/fl embryo out of 3 total analyzed with a cleft palate at E18.5. These data show that ARHGAP29 is required for on‐time palatal shelves movement, supporting a role for ARHGAP29 in craniofacial development.

Vitamin B12 Transport Genes and Nonsyndromic Cleft Lip With or Without Cleft Palate in Chile.

The aims of this study were to assess the association between polymorphisms within genes involved in vitamin B12 transport and nonsyndromic cleft lip with or without cleft palate (NSCL/P) and global DNA methylation in Chile. From 247 cases and 453 controls, we obtained variant genotypes for CBLIF, CUBN, AMN, ABCC1, CD320, and TCN2 from a single nucleotide polymorphisms array. Global DNA methylation in 95 controls was obtained through LINE-1 methylation. After multiple comparison corrections, only rs780807 in CUBN remains associated with NSCL/P at dominant model (OR 0.564, p-value = 0.0006, q-value = 0.0450). Carriers of protective allele showed lower levels of DNA methylation than non-carriers (p = 0.0259). Further studies are necessary in order to explain relations with the phenotype and DNA methylation due to the absence of functional evidence for rs780807 in CUBN.

Periconceptional use of vitamin A and the risk of giving birth to a child with nonsyndromic orofacial clefts-A meta-analysis.

BackgroundWe conducted a meta‐analysis of observational epidemiological studies to evaluate the association between periconceptional use of vitamin A and the risk of giving birth to a child with nonsyndromic orofacial clefts (NSOFCs).MethodsWe carried out a systematic literature search of Embase, PubMed, Web of Science, Google Scholar, and OpenGrey from inception to June 30, 2021. Two reviewers independently evaluated the studies that met the inclusion criteria and filled out an abstraction form for each study. Study quality was assessed using the Newcastle‐Ottawa Assessment Scale (NOS). Adjusted estimates were pooled with an inverse variance weighting using a random‐effects model. Heterogeneity and publication bias were assessed using the Cochran's Q test and funnel plot, respectively.ResultsA total of six case–control studies with moderate risk of bias were included. The pooled OR showed a 20% reduction in the risk of NSOFCs for periconceptional use of vitamin A which was not statistically significant (OR = .80; 95% CI .54–1.17, p = .25). For nonsyndromic cleft lip with or without cleft palate (NSCL/P), the studies were homogenous, and the pooled estimate showed a 13% risk reduction, which was significant (OR = .87; 95% CI .77–.99, p = .03). For nonsyndromic cleft palate only (NSCPO), the pooled estimate showed a 33% lower likelihood, which was not statistically significant (OR = .67; 95% CI .42–1.08, p = .10).ConclusionOur results suggest a possible protective effect for the periconceptional use of vitamin A on the risk of NSCL/P. This finding should be investigated further in prospective studies across multiple populations.

Long non-coding RNAs MALAT1 and NEAT1 in non-syndromic orofacial clefts.

Long non-coding RNAs (lncRNAs) are thought to play important roles in non-syndromic orofacial clefts (NSOFC). Clinical diagnosis was categorized as either non-syndromic cleft lip with or without cleft palate (NSCL/P), or non-syndromic cleft palate only (NSCPO). Tissues excised from the trimmed wound edge were reserved as experimental samples; adjacent normal control was used as a positive control, and tissue from healthy individuals was used as a blank control. Target lncRNAs in the collected tissues were identified using microarrays and quantitative reverse transcription PCR (RT-qPCR). Immunohistochemical (IHC) staining and RT-qPCR were used to verify the target mRNAs. Pathway, gene ontology (GO) enrichment, and TargetScan predictions were employed to construct competing endogenous RNA networks (ceRNA networks) and explore their potential functions. RNA-Seq revealed 24 upregulated and 43 downregulated lncRNAs; MALAT1 and NEAT1 were screened and validated using RT-qPCR. Common NSOFC risk factors were positively correlated with MALAT1 and NEAT1 expression. Bioinformatics predicted four ceRNA networks; GO enrichment focused on their potential functions. RT-qPCR and IHC data were consistent with respect to expression levels of proteins and the mRNAs that encode them. As MALAT1 and NEAT1 are associated with the severity of NSOFC, they represent potential therapeutic targets and prognostic biomarkers.

Pathway analysis identified a significant association between cell-cell adherens junctions-related genes and non-syndromic cleft lip/palate in 895 Asian case-parent trios

ObjectivesTo study the association between the seven cell-cell adherens junctions (AJs) -related genes (CDH1, CTNND1, CTNNA1, ESRP1, ESRP2, PLEKHA5, and PLEKHA7) and non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Asian population. DesignThe study included 895 NSCL/P case-parent trios of Asian ethnicity drawn from an international consortium established for a genome-wide association study. We performed association analysis by applying the genotypic transmission disequilibrium test for each of the 144 single nucleotide polymorphisms (SNPs), the versatile gene-based association study for the combined effects of all SNPs, all in or around the seven target genes, and the versatile pathway-based approach for the combined effects of these seven target genes, consecutively. ResultsIn our analysis, we found neither a significant SNP-based nor gene-based association with NSCL/P for any of the 144 relevant SNPs or the seven target genes. However, novel evidence of a significant association (P = 6.00 × 10−6) with NSCL/P was observed in the combined effects of these seven target genes in the versatile pathway-based analysis. ConclusionsOur results were consistent with the findings of previously published human sequencing studies and reinforced the role of these cell-cell AJs-related genes in the pathogenesis of NSCL/P that may be replicated using data from other genome-wide association studies. This pathway approach better reflects our current understanding of the biological mechanisms underlying the aetiology of NSCL/P.

Genome-wide Interaction Study Implicates VGLL2 and Alcohol Exposure and PRL and Smoking in Orofacial Cleft Risk.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect, affecting approximately 1 in 700 births. NSCL/P has complex etiology including several known genes and environmental factors; however, known genetic risk variants only account for a small fraction of the heritability of NSCL/P. It is commonly suggested that gene-by-environment (G×E) interactions may help explain some of the “missing” heritability of NSCL/P. We conducted a genome-wide G×E interaction study in cases and controls of European ancestry with three common maternal exposures during pregnancy: alcohol, smoking, and vitamin use using a two-stage design. After selecting 127 loci with suggestive 2df tests for gene and G x E effects, 40 loci showed significant G x E effects after correcting for multiple tests. Notable interactions included SNPs of 6q22 near VGLL2 with alcohol and 6p22.3 near PRL with smoking. These interactions could provide new insights into the etiology of CL/P and new opportunities to modify risk through behavioral changes.

Targeted Re-Sequencing of the 2p21 Locus Identifies Non-Syndromic Cleft Lip Only Novel Susceptibility Gene ZFP36L2.

rs7590268 present on the 2p21 locus was identified to be associated with non-syndromic cleft lip with or without cleft palate (NSCL/P) in several populations, including the Chinese Han population, indicating that 2p21 was a susceptibility locus for NSCL/P. However, previous studies have only identified common single-nucleotide polymorphism (SNP) within the THADA gene, neglecting the rare variants and other genes in 2p21; thus, this study was designed to investigate additional variants and novel susceptibility genes in 2p21. A total of 159 NSCL/P patients and 542 controls were recruited in the discovery phase, whereas 1830 NSCL/P patients and 2,436 controls were recruited in the replication phase. After targeted region sequencing, we performed association and burden analyses for the common and rare variants, respectively. Furthermore, RNA-seq, proliferation assay and cell cycle analysis were performed to clarify the possible function of the candidate gene ZFP36L2. Association analysis showed that four SNPs were specifically associated with non-syndromic cleft lip only (NSCLO) and two SNPs were associated with both NSCLO and NSCL/P. Burden analysis indicated that ZFP36L2 was associated with NSCLO (p = .0489, OR = 2.41, 95% CI: 0.98–5.90). Moreover, SNPs in the ZFP36L2 targeted gene JUP were also associated with NSCLO. ZFP36L2 also inhibited cell proliferation and induced G2 phase arrest in the GMSM-K cell line. Therefore, we proposed that ZFP36L2 is a novel susceptibility gene of NSCLO in the 2p21 locus, which could lead to NSCLO by modulating cell proliferation and cycle.

MTHFR and MDR1 Gene Polymorphisms in Yakut Patients with Non-Syndromic Orofacial Clefts

The aim of our study was to investigate the relationship between the MDR1 and MTHFR gene polymorphisms and non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Yakut population in the Republic of Sakha (Yakutia). Methods and Results: The sample of examined persons consisted of 60 children with NSCL/P. The NSCL/P group was divided into the CLP (cleft lip with cleft palate) subgroup (n=31), CLO (cleft lip only) subgroup (n=14), and CPO (cleft palate only) subgroup (n=15). The comparison group (control) included 174 healthy volunteers who did not have relatives with OFCs. The study of the MDR1 rs1045642 SNP and the MTHFR rs1801133 SNP was performed by PCR and RFLP analysis. Analysis of the frequency distribution of alleles and genotypes depending on the severity of clefts showed that the carriage of the TT homozygous genotype of the MDR1 rs1045642 SNP was associated with significant risk for the development of NSCL/P (OR=2.52, 95% CI: 1.19-5.32, P=0.02). Analysis of the recessive model (TT vs CC + TC) also found a significant risk of NSCL/P with the TT genotype carriage (OR=2.20, 95% CI: 1.06-4.57, P=0.04). Analysis of the over-dominant model (TC vs TT + CC) showed that the heterozygous TC genotype had a protective effect (OR=0.41; 95% CI: 0.22-0.77, P=0.01) on the development of NSCL/P. Subgroup analysis according to NSCL/P subtypes (CLO, CPO and CLP) showed that the MDR1 rs1045642 SNP was significantly associated with a high risk of CPO in three genetic models: heterozygous [(TT vs TC): OR=5.03; 95% CI: 1.55-16.32; P=0.01], recessive [(TT vs CC + TC): OR=3.96; 95% CI: 1.32-11.95; P=0.02], and over-dominant [(TC vs TT + CC): OR=0.23; 95% CI: 0.08-0.66; P=0.01]. Conclusion: A study of two SNPs in the MDR1and MTHFR genes revealed a statistically significant increased risk for NSCL/P in carriers of the TT genotype of the MDR1 rs1045642 SNP.

Association of the IRF6 rs2235371 and rs861019 Polymorphisms with Non-Syndromic Cleft Lip with or without Cleft Palate in the Yakut Population

Background: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects. NSCL/P can be broadly divided into cleft lip only (CLO), cleft palate only (CPO), and cleft lip with cleft palate (CLP) based on clinical presentation. The aim of this study was to investigate the relationship between the IRF6 gene polymorphisms and non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Yakut population. Methods and Results: In 23 OFC patients and 58 unrelated control subjects from the Yakut population, we tested two SNPs (rs2235371 and rs861019) with a minor allele frequency of more than 5% in the candidate gene IRF6. We found that the SNP marker rs861019 showed significant differences in allele frequencies (OR=2.07, 95%CI: 1.01–4.23, P=0.04) between the NSCL/P patients and the comparison group. Analysis of allele frequencies for rs861019 SNP in subgroups showed that there was a difference in the frequency between CLP and control (OR=5, 95% CI: 1.61-15.53, P=0.11); however, this result was not significant. Genotype analysis showed significant differences in patients from the CLP subgroup in comparison with controls for homozygous (AA compared with GG) (OR=9.00, 95% CI: 1.03–78.58, P=0.03), heterozygous (GA compared with GG) (OR=5.50, 95% CI: 1.05-28.75, P=0.04,), recessive (GG compared with GA + AA) (OR=6.67, 95% CI: 1.61-27.58; RR=4.78, 95% CI: 1.42–16.10, P=0.008,) and co-dominant (GG compared with GA, compared with AA) (P=0.02) inheritance models. Diplotype analysis showed that the NSCL/P group was more likely to have the [CC]-[GG] diplotype than the comparison group. This diplotype carries the risk GG genotype (rs861019) (30.4%) and does not carry the risk T allele(rs2235371). In the CLP subgroup, two diplotypes ([CT]-[GG] and [CC]-[GG]) were found more often than in the comparison group. Both diplotypes carry the risk GG genotype(rs861019; 33.3%). In the CPO subgroup, the [CT]-[GG] diplotype was more common. In the CLO subgroup, only two diplotypes ([CC]-[GA] and [CC]-[GG]) were found, both of which were more common than in the comparison group (75% and 25%).. It is likely that these results for the CLO and CPO subgroups were influenced by the small size of both samples. Unlike the NSCL/P and CLP groups, in these samples, diplotypes with the homozygous genotype GG (rs861019) without the homozygous genotype TT (rs2235371) were more common. Diplotypes with a homozygous genotype of the TT risk allele were not found in the studied groups except for the comparison group, where the [TT]-[AA] diplotype was represented by a low frequency (0.17%). Conclusion: The present study provides strong statistical support (for the first time to our knowledge) that genetic variants of the IRF6 rs861019 SNP are associated with NSCL/P in Yakuts.