Nonsyndromic Cleft Lip With Or Without Cleft Palate Research Articles

Alterations of senescence-associated markers in patients with non-syndromic cleft lip and palate

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common craniofacial anomalies. Abnormal Alu methylation in DNA of the pregnant mother may influence the abnormal development of the child. This study aimed to examine Alu methylation and cellular senescence in NSCL/P patients and their mothers as well as the correlation with the severity of NSCL/P. A total of 39 patients with NSCL/P and 33 mothers were enrolled. Of these patients, 6 were cleft lip only (CLO), 9 were cleft palate only (CPO), and 24 were cleft lip and palate (CLP). Alu methylation and senescence markers were determined in the white blood cells of NSCL/P patients, their mothers, and in the lip and palatal tissues of patients at the time of cheiloplasty and palatoplasty. Total Alu methylation was not significantly different between groups. However, a decrease in Alu hypermethylation, increased partial Alu methylation, RAGE, and p16 expression were shown in CLP, the most severe cleft type. Alu methylation in tissues did not differ between groups. In mothers, an increase in Alu methylation was observed only in the CLP. Therefore, the pathogenesis of NSCL/P may be related to Alu methylation of the mother promoting loss of Alu methylation and subsequently senescence in the children.

Shared genetic risk between major orofacial cleft phenotypes in an African population.

Nonsyndromic orofacial clefts (NSOFCs) represent a large proportion (70%-80%) of all OFCs. They can be broadly categorized into nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Although NSCL/P and NSCPO are considered etiologically distinct, recent evidence suggests the presence of shared genetic risks. Thus, we investigated the genetic overlap between NSCL/P and NSCPO using African genome-wide association study (GWAS) data on NSOFCs. These data consist of 814 NSCL/P, 205 NSCPO cases, and 2159 unrelated controls. We generated common single-nucleotide variants (SNVs) association summary statistics separately for each phenotype (NSCL/P and NSCPO) under an additive genetic model. Subsequently, we employed the pleiotropic analysis under the composite null (PLACO) method to test for genetic overlap. Our analysis identified two loci with genome-wide significance (rs181737795 [p = 2.58E-08] and rs2221169 [p = 4.5E-08]) and one locus with marginal significance (rs187523265 [p = 5.22E-08]). Using mouse transcriptomics data and information from genetic phenotype databases, we identified MDN1, MAP3k7, KMT2A, ARCN1, and VADC2 as top candidate genes for the associated SNVs. These findings enhance our understanding of genetic variants associated with NSOFCs and identify potential candidate genes for further exploration.

Genetic associations and parent-of-origin effects of PVRL1 in non-syndromic cleft lip with or without cleft palate across multiple ethnic populations.

This study investigated the associations of PVRL1 gene variants with non-syndromic cleft lip with or without cleft palate (NSCL/P) by evaluating transmission distortion and parent-of-origin (POO) effects in multiple ethnic populations. We conducted allelic and genotypic transmission disequilibrium tests (TDT) on 10 single-nucleotide variants (SNVs) in PVRL1 using data from 142 Korean families with an affected child. POO effects were analyzed using the POO likelihood ratio test, comparing transmission rates of maternally and paternally inherited alleles. To assess generalizability and ethnic heterogeneity, we compared results from Korean families with data from the Center for Craniofacial and Dental Genetics, which included 2,226 individuals from 497 European and 245 Asian trios. TDT analysis identified significant over-transmission of the rs7940667 (G361V) C allele in Korean families (p=0.007), a finding replicated in both Asian (p=6.5×10-7) and European families (p=1.6×10-10). Eight SNVs showed strong TDT evidence in larger Asian and European datasets after multiple comparison corrections (p<0.0073). Of these, 4 SNVs (rs7940667, rs7103685, rs7129848, and rs4409845) showed particularly robust association (p<5×10-8). POO analysis revealed significant maternal over-transmission of the rs10790330-A allele in Korean families (p=0.044). This finding was replicated in European families (p=9.0×10-4). Additionally, 3 other SNVs, rs7129848 (p=0.001) and the linked SNVs rs3935406 and rs10892434 (p=0.025), exhibited maternal over-transmission in the validation datasets. Our findings provide robust evidence supporting the associations of PVRL1 variants with NSCL/P susceptibility. Further research is necessary to explore the potential clinical applications of these findings.

Role of MTHFR, IRF6, PAX7 and TP63 SNPs in susceptibility to non-syndromic orofacial cleft, a candidate gene study in a Portuguese population.

Non-syndromic orofacial cleft (NSOC) is a complex phenotype, involving multiple genetic and environmental factors. Association studies exploring the genetic susceptibility to this prevalent oral malformation show variability of results in different populations. Using a candidate gene approach, we aimed to verify the role of four single-nucleotide polymorphisms (SNPs) in the susceptibility to NSOC in Portuguese patients. A total of 254 non-consanguineous individuals of Portuguese were recruited, including 120 patients with NSOC and 134 controls. About 92% of these patients had non-syndromic cleft lip with or without cleft palate (NSCL/P) and 8% had only non-syndromic cleft palate (NSCP). SNPs in the MTHFR (rs1801133), IRF6 (rs642961), PAX7 (rs742071) and TP63 (rs9332461) genes were studied, using a real-time approach with TaqMan probes. Allelic, genotypic, dominant, recessive and over-dominant models were explored using a chi-squared test. Adjusted p-value was calculated for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR). All SNPs were in Hardy-Weinberg equilibrium. For MTHFR, IRF6, and PAX7 SNPs, no statistically significant difference was highlighted for any of the evaluated models. For TP63 SNP, data fitted an over-dominant model, with a protective effect for heterozygotes (OR 1.897; CI 95% [1.144-3.147]; p < .016, when comparing controls vs. cases), but significance was lost when applying adjusted p-value for multiple comparisons (4 × 5 tests). In this Portuguese population, there was no evidence of an association between the evaluated SNPs and NSOC. For TP63 SNP, the possibility of a protective effect of heterozygotes should be further investigated.

Rare variants analyses suggest novel cleft genes in the African population

Non-syndromic orofacial clefts (NSOFCs) are common birth defects with a complex etiology. While over 60 common risk loci have been identified, they explain only a small proportion of the heritability for NSOFCs. Rare variants have been implicated in the missing heritability. Thus, our study aimed to identify genes enriched with nonsynonymous rare coding variants associated with NSOFCs. Our sample included 814 non-syndromic cleft lip with or without palate (NSCL/P), 205 non-syndromic cleft palate only (NSCPO), and 2150 unrelated control children from Nigeria, Ghana, and Ethiopia. We conducted a gene-based analysis separately for each phenotype using three rare-variants collapsing models: (1) protein-altering (PA), (2) missense variants only (MO); and (3) loss of function variants only (LOFO). Subsequently, we utilized relevant transcriptomics data to evaluate associated gene expression and examined their mutation constraint using the gnomeAD database. In total, 13 genes showed suggestive associations (p = E−04). Among them, eight genes (ABCB1, ALKBH8, CENPF, CSAD, EXPH5, PDZD8, SLC16A9, and TTC28) were consistently expressed in relevant mouse and human craniofacial tissues during the formation of the face, and three genes (ABCB1, TTC28, and PDZD8) showed statistically significant mutation constraint. These findings underscore the role of rare variants in identifying candidate genes for NSOFCs.

Genetic and functional research strategies of non-syndromic cleft lip with or without cleft palate in the post genome-wide association study era

The emergence of genome-wide association studies (GWAS) has greatly promoted the genetic research of non-syndromic cleft lip with or without cleft palate (NSCL/P). There have been more than 40 regions concerning NSCL/P identified by GWAS, whereas specific susceptible loci and their potential function remains unclear. In the post-GWAS era, precise localization of susceptible loci in candidate regions and exploration of underlying biological mechanism will contribute to further understanding of genetic etiology of NSCL/P. The present article reviewed the genetic and functional research strategies of NSCL/P in post-GWAS era.

Association of EPHA3 Gene Variation with Oral Hygiene in an Iranian Population

Background: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most prevalent congenital birth anomaly. The EPHA3 gene is suggested to play a pivotal role in the development of oral clefts. Objectives: This study aimed to evaluate the influence of EPHA3 gene polymorphisms on the risk of NSCL/P within an Iranian cohort. Methods: We performed genotyping of the EPHA3 gene polymorphisms rs7650466, rs1398197, rs17801309, rs1054750, and rs7632427 in 150 NSCL/P patients and 152 healthy controls using PCR-RFLP, T-ARMS-PCR, and ARMS-PCR methods. Results: The results indicated that the rs1398197 variation significantly reduced the risk of NSCL/P in a heterozygous codominant model (OR = 0.58, 95% CI = 0.36 - 0.94, P = 0.027, G/A vs. G/G), a dominant model (OR = 0.56, 95% CI = 0.35 - 0.89, P = 0.014, G/A + A/A vs. G/G), and at the allele level (OR = 0.62, 95% CI = 0.43 - 0.91, P = 0.014, A vs. G). The rs1054750 polymorphism showed a decreased risk of NSCL/P in codominant (OR = 0.62, 95% CI = 0.39 - 0.99, P = 0.047, T/C vs. T/T) and dominant models (OR = 0.62, 95% CI = 0.39 - 0.98, P = 0.042, T/C + C/C vs. T/T). The rs17801309 polymorphism was not associated with any risk or protection from NSCL/P. rs7650466 and rs7632427 were not polymorphic in the study sample. Conclusions: Our findings suggest that variants of the EPHA3 gene may be linked with a reduced risk of NSCL/P.

Integrative Multi-omics Analysis Identifies Genetic Variants Contributing to Non-syndromic Cleft Lip with or without Cleft Palate.

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. 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. 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. 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.

Genetic Associations of Oral Clefts in Arabs.

This study aims to investigate genetic association between Non-syndromic Cleft lip with or without palate (NCLP) and 14 specific Single Nucleotide Polymorphism (SNPs) reported to be associated with NCLP from previous Genome Wide Association Studies (GWAS). A prospective case-control study. Ministry of Health (MOH) Cleft and Craniofacial Clinic and Kuwait University. One hundred sixty-four NCLP patients were recruited from MOH Cleft and Craniofacial clinic, and 491 controls from the Kuwait DNA bank established at Kuwait University. Total gDNA was extracted from whole blood withdrawn from patients and genotyped by real time PCR. Hardy-Weinberg Equilibrium was tested, and the set p value for significance (p < 0.05) was adjusted using the Benjamini - Hoochberg procedure to achieve 5% false discovery rate. Logistic regression multivariate analysis was used to test statistically significant differences between cases and controls. Genotyping both groups for the variants was determined through the allele discrimination software program. There was statistically significant difference between cases and controls in relation to two SNPs; LOC102724968 (rs13041247) (MAF cases/control = C (0.28/0.39) OR Homozygous = 1.30; 95% CI = 1.09-1.56, p = 0.0041) and PVT1 (rs987525) (MAF cases/control = A (0.41/0.27) OR heterozygous = 1.48; 95% CI =1.12-1.95, p = 0.0073), increasing the susceptibility to NCLP. Genetic variations are associated with the occurrence of oral clefts. Customized Next Generation Sequencing (NGS) panel to the Arab ethnicity is encouraged. In Addition, national preconception genetic carrier screening tests should expand to include common craniofacial anomalies.

Unveiling dysregulated lncRNAs and networks in non-syndromic cleft lip with or without cleft palate pathogenesis

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital facial malformation with a complex, incompletely understood origin. Long noncoding RNAs (lncRNAs) have emerged as pivotal regulators of gene expression, potentially shedding light on NSCL/P's etiology. This study aimed to identify critical lncRNAs and construct regulatory networks to unveil NSCL/P's underlying molecular mechanisms. Integrating gene expression profiles from the Gene Expression Omnibus (GEO) database, we pinpointed 30 dysregulated NSCL/P-associated lncRNAs. Subsequent analyses enabled the creation of competing endogenous RNA (ceRNA) networks, lncRNA-RNA binding protein (RBP) interaction networks, and lncRNA cis and trans regulation networks. RT-qPCR was used to examine the regulatory networks of lncRNA in vivo and in vitro. Furthermore, protein levels of lncRNA target genes were validated in human NSCL/P tissue samples and murine palatal shelves. Consequently, two lncRNAs and three mRNAs: FENDRR (log2FC = − 0.671, P = 0.040), TPT1-AS1 (log2FC = 0.854, P = 0.003), EIF3H (log2FC = − 1.081, P = 0.041), RBBP6 (log2FC = 0.914, P = 0.037), and SRSF1 (log2FC = 0.763, P = 0.026) emerged as potential contributors to NSCL/P pathogenesis. Functional enrichment analyses illuminated the biological functions and pathways associated with these lncRNA-related networks in NSCL/P. In summary, this study comprehensively delineates the dysregulated transcriptional landscape, identifies associated lncRNAs, and reveals pivotal sub-networks relevant to NSCL/P development, aiding our understanding of its molecular progression and setting the stage for further exploration of lncRNA and mRNA regulation in NSCL/P.

Genetic variants in BCL-2 family genes influence the risk of non-syndromic cleft lip with or without cleft palate.

The BCL-2 family is crucial for cell death regulation and is involved in development, tissue homeostasis, and immunity. This study aimed to investigate the association between genetic variants in BCL-2 family genes and non-syndromic cleft lip with or without cleft palate (NSCL/P) risk. A two-stage case-control study was conducted in this association study. Gene-based analysis using Multi-marker Analysis of GenoMic Annotation was performed in the first stage cohort, which included 565 cases and 1269 controls. A logistic regression model was employed to assess the effect of single nucleotide polymorphisms (SNPs) on susceptibility to NSCL/P. Candidate SNPs were replicated by extra dbGaP case-parent trios. Haploreg, RegulomeDB, and UCSC Genome Browser were used to identify enhancer effects of promising SNPs. Bulk RNA sequencing data obtained from the Gene Expression Omnibus was used to identify co-expressed genes. Single-cell RNA sequencing dataset was used to infer the cell population of the candidate gene. The "Monocle" package was used to analyze the pseudotime cell trajectories. Rs3943258 located in the enhancer region was associated with the risk of NSCL/P (Pmeta = 5.66 × 10-04 ) and exhibited an eQTL effect for BCL2 (P = 3.96 × 10-02 ). Co-expression and pathway enrichment analysis revealed that genes related to Bcl2 were significantly enriched in the PI3K-Akt signaling pathway, MAPK signaling pathway, and Wnt signaling pathway. Five cell clusters were identified in single-cell RNA sequencing, and Bcl2 was mainly located in the mesenchyme. The rs3943258 located within BCL2 was probably related to NSCL/P susceptibility.

Genetic Inheritance Models of Non-Syndromic Cleft Lip with or without Palate: From Monogenic to Polygenic.

Non-syndromic cleft lip with or without palate (NSCL/P) is a prevalent birth defect that affects 1/500-1/1400 live births globally. The genetic basis of NSCL/P is intricate and involves both genetic and environmental factors. In the past few years, various genetic inheritance models have been proposed to elucidate the underlying mechanisms of NSCL/P. These models range from simple monogenic inheritance to more complex polygenic inheritance. Here, we present a comprehensive overview of the genetic inheritance model of NSCL/P exemplified by representative genes and regions from both monogenic and polygenic perspectives. We also summarize existing association studies and corresponding loci of NSCL/P within the Chinese population and highlight the potential of utilizing polygenic risk scores for risk stratification of NSCL/P. The potential application of polygenic models offers promising avenues for improved risk assessment and personalized approaches in the prevention and management of NSCL/P individuals.

Identification of a Novel Variant of PDGFC Associated with Nonsyndromic Cleft Lip and Palate in a Chinese Family.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) accounts for 70% of the total number of patients with cleft lip with or without cleft palate (CL/P) and is the most common type of congenital deformity of the craniomaxillofacial region. In this study, whole exome sequencing (WES) and Sanger sequencing were performed on affected members of a Han Chinese family, and a missense variant in the platelet-derived growth factor C (PDGFC) gene (NM_016205: c.G93T: p.Q31H) was identified to be associated with NSCL/P. Bioinformatic studies demonstrated that the amino acid corresponding to this variation is highly conserved in many mammals and leads to a glutamine-to-histidine substitution in an evolutionarily conserved DNA-binding domain. It was found that the expression of PDGFC was significantly decreased in the dental pulp stem cells (DPSCs) of NSCL/P cases, compared to the controls, and that the variant (NM_016205: c.G93T) reduced the expression of PDGFC. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that Pdgfc deficiency disrupted NSCL/P-related signaling pathways such as the MAPK signaling pathway and cell adhesion molecules. In conclusion, our study identified a missense variant (NM_016205: c.G93T) in exon 1 of PDGFC potentially associated with susceptibility to NSCL/P.

TGFA gene polymorphisms are not associated with the aetiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) in south Indian population

IntroductionTransforming Growth Factor Alpha (TGFA) plays a role in the development of the facial structures, including the lip and palate. MethodologyAbout 148 case/parent trios were genotyped for rs1058213, rs2166975, rs3771494, rs3821261 and rs426081 polymorphisms in TGFA gene. Transmission/disequilibrium test (TDT) and the parent-of-origin effects (POE) statistics were implemented. FAMHAP and Haploview packages were used to test the effect of multi-SNP haplotypes and linkage disequilibrium respectively. ResultsAt individual SNP level, TDT did not reveal significant excess transmission of parental alleles to their affected child. None of the analysed markers showed significant POE. Significant LD between SNPs of rs1058213, rs2166975 and rs377149, rs3821261 were observed. Analysis multi –SNP haplotypes did not reveal significant excess transmission of haplotypes to the affected child. ConclusionAnalysis of TDT and POE of five SNPs in TGFA gene indicated that the TGFA is not associated with the aetiology of NSCL/P.

Genome-wide meta-analyses identify five new risk loci for nonsyndromic orofacial clefts in the Chinese Han population.

Nonsyndromic orofacial clefts (NSOFCs) are the most common craniofacial birth malformations in humans and are generally classified as nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Genome-wide association studies (GWASs) of NSOFCs have demonstrated multiple risk loci and candidate genes; however, published risk factors are able to explain only a small fraction of the observed NSOFCs heritability. Here, we performed GWASs of 1615 NSCPO cases and 2340 controls, and then conducted genome-wide meta-analyses of NSOFCs, totaling 6812 NSCL/P cases, 2614 NSCPO cases, and 19,165 controls from the Chinese Han population. We identify 47 risk loci with genome-wide pmeta -value <5.0 × 10-8 , 5 risk loci (1p32.1, 3p14.1, 3p14.3, 3p21.31, and 13q22.1) of which are new. All of the 47 susceptibility loci conjointly account for 44.12% of the NSOFCs' heritability in the Chinese Han population. Our results improve the comprehending of genetic susceptibility to NSOFCs and provide new views into the genetic etiology of craniofacial anomalies.

LncRNA MIR31HG Regulates Proliferation and Migration by Targeting Matrix Gla Protein in Nonsyndromic Cleft Lip With or Without Cleft Palate.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common craniofacial birth defect with complex etiologies. Recently, the dysregulation of long noncoding RNAs (lncRNAs) has been implicated in many developmental diseases, including NSCL/P. However, the functions and mechanisms of lncRNAs in NSCL/P have not been fully elucidated. In this study, we found that lncRNA MIR31HG in NSCL/P patients was significantly downregulated than that in healthy individuals (GSE42589, GSE183527). In addition, single nucleotide polymorphism rs58751040 in MIR31HG was nominally associated with NSCL/P susceptibility (odds ratio: 1.29, 95% confidence interval: 1.03-1.54, p = 4.93 × 10-2) through a case-control study (504 NSCL/P cases and 455 controls). Luciferase activity assay showed that the C allele of rs58751040 revealed a decreased transcription activity of MIR31HG than the G allele. Moreover, knockdown of MIR31HG promoted cell proliferation and migration in human oral keratinocytes and human embryonic palate mesenchyme. Bioinformatic analysis and cellular studies suggested that MIR31HG may confer susceptibility to risk of NSCL/P through matrix Gla protein (MGP) signaling. In summary, we identified a novel lncRNA involved in the development of NSCL/P.

Integrating transcriptomics and genomics to identify fibroblast growth factor/receptor candidate genes for non‐syndromic orofacial cleft in Chinese

ObjectivesThe objective of this study was to explore the relationship between fibroblast growth factor/receptor (FGF/FGFR) and non-syndromic orofacial cleft (NSOC) in individuals of Han Chinese. DesignInitially, we performed RNA-Seq between non-syndromic cleft lip only (NSCLO) or non-syndromic cleft palate only (NSCPO) and control groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out to evaluate the functions of differentially expressed genes (DEGs) of FGF/FGFR. Then, we selected the most significant DEG FGFR2 and performed an association analysis in Chinese. Linkage disequilibrium (LD) and haplotype analyses were performed with HaploView and PLINK. Additional bioinformatics functional prediction for the notable single nucleotide polymorphisms was performed with HaploReg V4.1 and 3DSNP. ResultsFinally, we identified 32 mRNAs related to FGF/FGFR via RNA-Seq and chose FGFR2 in the subsequent association analysis. Results indicated that the single nucleotide polymorphism (SNP) rs2288336 in FGFR2 contributed significantly to both non-syndromic cleft lip with or without cleft palate (NSCL/P) and NSCLO, with p values of 5.00E-05 (OR = 0.79, 95% CI: 0.70–0.88) and 1.38E-04 (OR = 0.76, 95% CI: 0.65–0.87), respectively. In addition, rs3793893 in FGFR2 was found to be associated with NSCLO, with a p value of 1.02E-04 (OR = 0.67, 95% CI: 0.55–0.82). ConclusionsOur research demonstrated that FGFR2 is significantly more involved in NSOC than other FGF/FGFRs in Chinese and further identified rs2288336 and rs3793893 in FGFR2 associated with NSOC subtypes, which provide further evidence for the genetic etiology of NSOC in Han Chinese.

Association of TGFB3 and FGFs gene polymorphisms with cleft lip with or without cleft palate a systematic review

Objectives: The objective of this study was to conduct a systematic review of the possible association between transforming growth factor B3 (TGFB3) and fibroblast growth factors (FGFs) gene polymorphisms and nonsyndromic cleft lip with or without cleft palate (NSCL/P). Material and Methods: Two reviewers independently screened studies by examining all titles and abstracts. Studies were included if they met the following criteria: The outcome of interest was NSCL/P; the polymorphisms studied were TGFB3 and FGF; they presented sufficient data, that is, allele/genotype frequency between cases and controls; or their odds ratio with 95% confidence interval. Study quality was independently assessed by a risk of bias assessment for genetic association studies. Results: Based on the inclusion criteria, we have selected a total of six articles (four for TGFB and two for FGF). Particularly for the TGFB gene, we have found significant results in exon 4 in the variant g.15812T&gt;G, and in the single-nucleotide polymorphisms rs2300607 A/T, in the distribution between cases and controls. On the other hand, for the FGF gene, we observed a statistically significant in the genotype rs34010 CA. Conclusion: None of the genetic variations that show the association is verified in different populations; therefore, there is not enough scientific validation regarding the association between TGFB and FGF polymorphism and NSCL/P. The findings of the different studies suggest the need for further investigations with samples composed of a larger number of individuals in different populations, which should be performed with all the standards for genetic studies, thus allowing an understanding of the molecular basis of the disease.

A Variant in the IRF6 Promoter Associated with the Risk for Orofacial Clefting

The single-nucleotide polymorphism (SNP) rs2235371 (IRF6 V274I) is associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Han Chinese and other populations but appears to be without a functional effect. To find the common etiologic variant or variants within the haplotype tagged by rs2235371, we carried out targeted sequencing of an interval containing IRF6 in 159 Han Chinese with NSCL/P. This study revealed that the SNP rs12403599, within the IRF6 promoter, is associated with all phenotypes of NSCL/P, especially nonsyndromic cleft lip (NSCLO) and a subphenotype of it, microform cleft lip (MCL). This association was replicated in 2 additional much larger cohorts of cases and controls from the Han Chinese. Conditional logistic analysis indicated that association of rs2235371 with NSCL/P was lost if rs12403599 was excluded. rs12403599 contributes the most risk to MCL: its G allele is responsible for 38.47% of the genetic contribution to MCL, and the odds ratios of G/C and G/G genotypes were 2.91 and 6.58, respectively, for MCL. To test if rs12403599 is functional, we carried out reporter assays in a fetal oral epithelium cells (GMSM-K). Unexpectedly, the risk allele G yielded higher promoter activity in GMSM-K. Consistent with the reporter studies, expression of IRF6 in lip tissues from NSCLO and MCL patients with the G/G phenotype was higher than in those from patients with the C/C phenotype. These results indicate that rs12403599 is tagging the risk haplotype for NSCL/P better than rs2235371 in Han Chinese and supports investigation of the mechanisms by which the allele of rs12403599 affects IRF6 expression and tests of this association in different populations.

Genetic variants in Mammalian STE20-like protein kinase 2 were associated with risk of NSCL/P

AimMammalian STE20-like protein kinase 2 (MST2) plays an important role in apoptosis and the development of many disorders. Here, we aim to explore if genetic variants in MST2 are associated with the risk of non-syndromic cleft lip with or without palate (NSCL/P). Materials and methodsThe association study was performed in a two-stage study of 1,069 cases and 1,724 controls to evaluate the association between genetic variants in the MST2 and NSCL/P risk. The potential function of the candidate single nucleotide polymorphism (SNP) was predicted using HaploReg, RegulomeDB, and public craniofacial histone chromatin immunoprecipitation sequencing (ChIP-seq) data. Haploview was used to perform the haplotype of risk alleles. The expression quantitative trait loci (eQTL) effect was assessed using the Genotype-Tissue Expression (GTEx) project. Gene expression in mouse embryo tissue was performed using data downloaded from GSE67985. The potential role of candidate gene in the development of NSCL/P was assessed by correlation and enrichment analysis. ResultsAmong SNPs in MST2, rs2922070 C allele (Pmeta = 2.93E-04) and rs6988087 T allele (Pmeta = 1.57E-03) were linked with significantly increased risk of NSCL/P. Rs2922070, rs6988087 and their high linkage disequilibrium (LD) SNPs constituted a risk haplotype of NSCL/P. Individuals carrying 3–4 risk alleles had an elevated risk of NSCL/P compared to those who carried less risk alleles (P = 2.00E-04). The eQTL analysis revealed a significant association between these two variants and MST2 in muscle tissue of the body. The MST2 expressed during mouse craniofacial development and over-expressed in the human orbicularis oris muscle (OOM) of NSCL/P patients compared to controls. MST2 was involved in the development of NSCL/P by regulating the mRNA surveillance pathway, the MAPK signaling pathway, the neurotrophin signaling pathway, the FoxO signaling pathway and the VEGF signaling pathway. ConclusionMST2 was associated with the development of NSCL/P.