Risk Of Non-syndromic Orofacial Clefts Research Articles

Genetic Variants in METTL16 Affect the Risk of Non-Syndromic Orofacial Clefts.

N6-methyladenosine (m6A) is the most prevalent modification of RNA in eukaryotes which is associated with many cellular processes and diseases. Here, our objective is to explore whether genetic variants in m6A modification genes are associated with the risk of non-syndrome orofacial clefts (NSOCs). The transmission disequilibrium test (TDT) was performed to calculate the association between single nucleotide polymorphisms (SNPs) in m6A modification genes and NSOCs risk in 944 case-parent trios. The function of SNP was predicted by HaploReg, RegulomeDB and histone enrichment data. The expression quantitative trait locus (eQTL) analysis was examined using Genotype-Tissue Expression (GTEx) and eQTLGen. The role of gene in the development of NSOCs was assessed with correlation and enrichment analysis based on gene expression data in mice craniofacial tissue and zebrafish embryo. We identified that rs8078195 (A > C) in METTL16 was suggestively associated with the increased risk of NSOCs (OR = 1.32, p = 1.80E - 03). The region surrounding rs8078195 was subjected to deoxyribonuclease hypersensitivity and enriched with multiple histone modifications. In addition, it had a significant eQTL effect with METTL16 in skin tissue and human peripheral blood, which played an important role in NSOCs development. Bioinformatic analysis indicated that METTL16 contributed to the development of NSOCs probably by regulating cell cycle process. Rs8078195 in METTL16 was associated with the occurrence of NSOCs.

Folate intake, markers of folate status and oral clefts: An updated set of systematic reviews and meta-analyses.

There has been a longstanding debate about the role of folate in the etiology of orofacial clefts (OFCs). Studies of different measures of nutritional intake or folate status have been done to investigate the possible role of folate in the prevention of OFC. Only one knowledge synthesis has attempted to bring together different types of evidence. The aim of the present work was to update it. Evidence for associations between OFC and dietary folate, supplement use, folic acid fortification, biomarkers of folate status, and variants of MTHFR (C677T and A1298C) were included. Potentially eligible articles were systematically identified from PubMed, Medline, Embase, and Web of Science (2007-2020) and combined using random-effects meta-analysis when appropriate. Quality assessments were conducted using the Newcastle-Ottawa scale and Cochrane's risk of bias tool. Sixty-four studies published since the previous knowledge synthesis were identified, with eight of these identified through a supplementary search from October, 2018 to August, 2020. There was an inverse association between folic acid-containing supplement use before or during pregnancy and cleft lip with or without cleft palate (CL/P) (OR 0.60, 95% CI 0.51-0.69), with considerable between-study heterogeneity. The prevalence of CL/P showed a small decline post-folic acid fortification in seven studies (OR 0.94, 95% CI 0.86-1.02). No association was found between OFC and genetic markers of folate status. The coronavirus-19 pandemic has threatened food availability globally and therefore there is a need to maintain and even enhance surveillance concerning maternal intake of folate and related vitamins. The risk of non-syndromic OFC was reduced among pregnant women with folic acid-containing supplements during the etiologically relevant period. However, high heterogeneity between included studies, incomplete reporting of population characteristics and variation in timing of exposure and supplement types mean that conclusions should be drawn with caution.

Nonsyndromic orofacial clefts in Chile: LINE-1 methylation and MTHFR variants.

Aim: To evaluate the risk of nonsyndromic orofacial clefts (NSOFCs) associated withLINE-1 methylation, as amarker of global DNA methylation, and the effect of MTHFR functional variants on this variable. Patients & methods: LINE-1 methylation was evaluated by bisulfite modification coupled to DNA pyrosequencing in 95 NSOFC cases and 95 controls. In these subjects, MTHFR genotypes for variants c.C677T (rs1801133) and c.A1298C (rs1801131) were obtained. Results: Middle levels (second tertile) of LINE-1 methylation increase the risk of NSOFCs. In addition, LINE-1 methylation depends on c.A1298C genotypes in controls but not in cases. Conclusion: A nonlinear association between global DNA methylation and NSOFCs was detected in this Chilean population, which appears to be influenced by MTHFR functional variants.

Variants in miRNA regulome and their association with the risk of nonsyndromic orofacial clefts.

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.

Maternal Folic Acid Intake and Risk of Nonsyndromic Orofacial Clefts: A Hospital-Based Case-Control Study in Bangalore, India.

Orofacial clefts are the most common congenital anomaly worldwide. Cleft etiology appears to be multifactorial, with genetic and environmental components. Although periconceptional folic acid supplementation has been shown to be protective for neural tube defects, current evidence for its role in cleft prevention is mixed with few studies from low- and middle-income countries. To investigate the association between periconceptional folic acid intake and incidence of nonsyndromic orofacial clefts among infants in Bangalore, India. A hospital-based case-control study (106 cases, 212 controls) utilizing a questionnaire to collect data on prenatal supplements, dietary folate, and potentially confounding factors. Multivariate logistic regression analysis was used to assess relationships between folic acid supplementation and all nonsyndromic clefts, and in separate analyses for cleft lip and/or palate (CL/P) and cleft palate (CP), adjusting for statistically significant variables. A statistically significant protective association was found for separate folic acid supplements (not combined with iron or multivitamins) taken in the periconceptional period and all clefts combined (adjusted odds ratio [OR]: 0.62, 95% confidence interval [CI], 0.45-0.86) and CL/P (adjusted OR: 0.57; 95% CI, 0.38-0.86). Higher levels of dietary folate were found to be associated with a reduced risk for all clefts (adjusted OR: 0.98, 95% CI, 0.96-0.99), CL/P (adjusted OR: 0.98, 95% CI, 0.96-0.99), and CP (adjusted OR: 0.96, 95% CI, 0.93-0.99). This study provides limited evidence for a protective association of periconceptional folic acid supplementation with nonsyndromic orofacial clefts. The low proportion of mothers taking folic acid supplements in the periconceptional period highlights the need for increased education and awareness regarding prenatal nutrition.

Genetic Variant in lncRNA RP11-462G12.2 Confers the Risk of Non-Syndromic Orofacial Clefts

Background: Non-syndromic Orofacial Clefts (NSOCs) are one of the most common human birth defects. Here, we test the hypothesis that, lncRNA RP11-462G12.2 rs2262251, in high linkage disequilibrium (LD) with a newly NSOC single nucleotide polymorphism (SNP) rs8049367, was a casual SNP of NSOC. Methods: SNP rs2262251 was genotyped in a cohort of 1,321 NSOC cases and 1,274 controls, and the mechanism of rs2262251 in the development of NSOC was explored with a series of biochemical and cell molecular biology assays. Findings: We found that rs2262251 was significantly associated with the risk of NSOC and cleft lip with/without palate (CL/P), and the C allele presented a protective role against the occurrence of NSOC and CL/P. The SNP rs2262251 G allele was associated with increased expression of lncRNA RP11-462G12.2 in cells and human lip tissues. Over-expression of rs2262251 [G] contributed to reduced numbers of G0/G1 phase cells, inhibited cell apoptosis, and promoted cell proliferation. Furthermore, rs2262251 [C] could interact with miR-744-5p and regulate the expression of IQSEC2, both of which were expressed in human lip tissues and mouse craniofacial development. Interpretation: We report for the first time that lncRNA RP11-462G12.2 rs2262251 might participate in a 'lncRNA-miRNA-gene' regulatory axis during the development of NSOC. Funding Statement: National Natural Science Foundation of China (81570959, 81200808, 81230022, 81400546); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD-2014-37); Natural Science Foundation of Jiangsu Province (BL2014073, 15KJA320002); Qing Lan Project, six distinguished talent (2016-WSW-008), Jiangsu provincial medical talent (Yongchu pan); Jiangsu provincial key medical discipline (zdxka2016026). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Written informed consent was obtained from each subject at recruitment. This study was approved by the institutional review board of Nanjing Medical University (Approval number: NJMUERC [2008] No. 20).

MSH homeobox 1 polymorphisms and the risk of non-syndromic orofacial clefts: a meta-analysis.

MSH homebox 1 (MSX1) is a susceptibility gene for non-syndromic orofacial clefts (NSOCs). Here, a meta-analysis was conducted to assess their associations. A systematic search of PubMed to 1 September 2017, was performed to retrieve all eligible studies. Odds ratios (ORs) were used to calculate the associations. The stability of the results was evaluated by sensitivity analysis. Publication bias was assessed using Begg's funnel plots and the Egger test. In silico Msx1 expression during early mouse craniofacial development was evaluated by the Gene Expression Omnibus. In the overall analysis, MSX1 rs12532 (G>A) contributed to a decreased risk of NSOC. In an analysis stratified according to disease type, rs12532 was associated with the risk of cleft palate only (CPO) but not with the risk of cleft lip with or without cleft palate (CL/P). The association of rs12532 with the occurrence of NSOC in Asian and Caucasian populations but not South American populations was observed in an analysis stratified according to ethnicity. However, no significant associations were detected between any of the other MSX1 SNPs and the risk of NSOC in either the overall or subgroup analysis. The Msx1 gene was widely expressed in mouse craniofacial structures from embryonic day (E)8.5-E10.5. Taken together, the study indicates that MSX1 rs12532 is associated with the risk of NSOC.

Genetic variants of microRNA processing genes and risk of non-syndromic orofacial clefts.

MicroRNA (miRNA) processing genes play important roles in the craniofacial development. The aim of this study was to explore the associations between single nucleotide polymorphisms (SNPs) of miRNA processing genes with the risk of non-syndromic orofacial clefts (NSOC). We genotyped 12 potentially functional SNPs from seven miRNA processing genes (GEMIN3, DROSHA, DGCR8, GEMIN4, PIWIL1, XPO5, and DICER) in a case-control study of 602 NSOC cases and 605 controls. Two SNPs were associated with the susceptibility of CL/P: rs10719 in DROSHA led to an increased risk of cleft lip with or without palate (CL/P) (GA/AA: p=.024, OR=1.33, 95% CI=[1.04, 1.70]; GG+GA/AA: p=.037, OR=1.29, 95% CI=[1.02, 1.63]), while rs493760 in DROSHA (CC/TT: p=.049, OR=0.58, 95% CI=[0.34, 0.99]) could reduce the risk of CL/P. In addition, rs10719 (A)-rs493760 (C) haplotype contributed to a decreased risk of CL/P (OR=0.77, 95% CI=[0.63, 0.94]), whereas the rs10719 (G)-rs493760 (C) haplotype contributed to the increased risk of cleft palate only (CPO) (OR=2.70, 95% CI=[1.15, 6.35]). However, there was no difference observed in these SNPs after the Bonferroni correction. Taken together, our results provided the potential evidence that rs10719 and rs493760 might contribute to the risk of CL/P and suggested potential genetic basis and mechanisms of CL/P. The lack of association between these SNPs and CPO might be due to the limited sample size of CPO subgroup.

Genetic variants of 20q12 contributed to non-syndromic orofacial clefts susceptibility.

Previous genomewide association studies (GWAS) identified a region near MAFB at chr20q12 associated with non-syndromic orofacial clefts (NSOC) susceptibility. However, whether other SNPs in this area could independently contribute to non-syndromic orofacial clefts in Chinese populations remained obscure. We selected 24 SNPs based on a haplotype-tagging SNP strategy and evaluated their associations with risk of non-syndromic orofacial clefts in a large-scale two-stage case-control study with 1278 cases and 1295 controls. Genotyping was performed with Sequenom and TaqMan assay. Associations between the SNPs and risk of non-syndromic orofacial clefts were estimated from unconditional logistic regression analyses. Overall, six SNPs were found to be the susceptible factors of non-syndromic orofacial clefts. The most significant and independent SNP was rs6129653 (additive model of P value=1.4E-06). In subgroup analysis, its significant associations with cleft lip only (CLO) and cleft lip and palate (CLP) were observed. Furthermore, in silico bioinformatics analysis indicated that rs6129653 was located in the transcriptionally active region and associated with MAFB expression in human brain tissues. Rs6129653 was an independent locus of non-syndromic orofacial clefts among Chinese populations possibly due to its potential of distal transcriptional regulation of MAFB expression.

FOXE1 polymorphisms and non-syndromic orofacial cleft susceptibility in a Chinese Han population.

FOXE1 plays an important role in craniofacial development. The aim of this study was to investigate associations between genetic variants of FOXE1 and risk of non-syndromic orofacial clefts in a Chinese population. Three potentially functional SNPs of FOXE1 (rs3758250 and rs907577 in the 5' upstream and rs7043516 in the 3'-UTR) were selected and their associations with non-syndromic orofacial cleft susceptibility were investigated in a case-control study from a Chinese population (602 cases and 605 controls). Genotyping was performed with double ligation and multiplex fluorescence PCR. Associations between the SNPs and risk of non-syndromic orofacial clefts and its subgroups were estimated from unconditional logistic regression analysis. Luciferase reporter assay was conducted to assess SNP function. Overall, we did not find any of the individual SNP or haplotype was associated with NSOC susceptibility. Nevertheless, in stratified analysis, we found rs7043516, locating in the 3'-UTR of FOXE1, was associated with risk of cleft lip only. Further invitro luciferase assay indicated that this SNP could contribute to differential binding ability with miRNA. Taken together, this study showed that rs7043516 may be considered as a potentially susceptible marker of cleft lip only among Chinese Han populations.

Association of Parental Environmental Exposures and Supplementation Intake with Risk of Nonsyndromic Orofacial Clefts: A Case-Control Study in Heilongjiang Province, China.

The aim of present study was to check the possible association of potential parental environmental exposures and maternal supplementation intake with the risk of nonsyndromic orofacial clefting (NSOC). A retrospective study comprised 499 cases and 480 controls was conducted in Heilongjiang Province. Chi-square analysis and unconditional multiple logistic regression were used in the study. The results showed that maternal history of fever and the common cold without fever (ORCL/P = 3.11 and 5.56, 95%CI: 1.67–5.82 and 2.96–10.47, ORCPO = 3.31 and 8.23, 95%CI: 1.58–6.94 and 4.08–16.95), paternal smoking and alcohol consumption (ORCL/P = 2.15 and 5.04, 95%CI: 1.37–3.38 and 3.00–8.46, ORCPO = 1.82 and 4.40, 95%CI: 1.06–3.13 and 2.50–7.74), maternal exposure to organic solvents, heavy metals, or pesticides (ORCL/P = 6.07, 5.67 and 5.97, 95%CI: 1.49–24.76, 1.34–24.09 and 2.10–16.98, ORCPO = 10.65, 7.28 and 3.48, 95%CI: 2.54–44.67, 1.41–37.63 and 1.06–11.46) and multivitamin use during the preconception period (ORCL/P = 0.06, 95%CI: 0.02–0.23, ORCPO = 0.06, 95%CI: 0.01–0.30) were associated with cleft lip or without cleft palate (CL/P) and cleft palate only (CPO). Maternal history of skin disease and negative life events (ORCL/P = 12.07 and 1.67, 95%CI: 1.81–80.05 and 1.95–2.67) were associated with CL/P. Some potential parental hazardous exposures during the periconception period and maternal use of multivitamins during the preconception period were associated with risk of NSOC.

Passive smoking in the etiology of non-syndromic orofacial clefts: a systematic review and meta-analysis.

BackgroundStudies have found a consistent positive association between maternal smoking and non-syndromic orofacial clefts (NSOFC). However, no comprehensive assessment of the association between NSOFC and passive smoking has been undertaken. This systematic review and meta-analysis explores the relationship between maternal passive smoking and NSOFC, and compares the associations between passive and active smoking.Methods and FindingsSearch strategy, inclusion / exclusion criteria, and data extraction from studies reporting maternal passive smoking and NSOFC was implemented without language restrictions. Risks of bias in the identified studies were assessed and this information was used in sensitivity analyses to explain heterogeneity. Meta-analysis and meta-regression of the extracted data were performed. Egger's test was used to test for small study effects. Fourteen eligible articles were identified. Maternal passive smoking exposure was associated with a twofold increase in risk of NSOFC (odds ratio: 2.11, 95% confidence interval: 1.54–2.89); this was apparent for both cleft lip with and without palate (OR: 2.05, 95% CI: 1.27–3.3) and cleft palate (OR: 2.11, 95% CI: 1.23–3.62). There was substantial heterogeneity between studies. In the studies that provided data enabling crude and adjusted odd ratios to be compared, adjustment for potential confounders attenuated the magnitude of association to about a 1.5-fold increase in risk.ConclusionOverall, maternal passive smoking exposure results in a 1.5 fold increase in risk of NSOFC, similar to the magnitude of risk reported for active smoking, but there is marked heterogeneity between studies. This heterogeneity is not explained by differences in the distribution of cleft types, adjustment for covariates, broad geographic region, or study bias/quality. This thorough meta-analysis provides further evidence to minimize exposure to environmental tobacco smoke in policy making fora and in health promotion initiatives.

A miRNA-binding-site SNP of MSX1 is Associated with NSOC Susceptibility

MSX1 is a favorable candidate gene for susceptibility to non-syndromic orofacial clefts (NSOCs). However, the roles of MSX1 genetic variants in the development of NSOC are controversial and vary among human populations. In the present study, the roles of 4 potentially functional single-nucleotide polymorphisms (SNPs) of MSX1 (rs12532 in 3′-untranslated region [UTR], and rs3821947, rs3821949, and rs4464513 in 5′ upstream) were investigated in a case-control study of 602 NSOC cases and 605 healthy controls. The findings showed that rs12532 located within 3′-UTR of MSX1 could influence the risk of developing NSOC. Individuals who carried the variant genotype (rs12532AA genotype) showed a decreased possibility of developing NSOC (AA vs. GG: OR = 0.69, 95% CI = [0.49, 0.98]). Interestingly, similar effects were also observed on cleft lip with palate (CLP), in a stratified analysis (allelic comparison-12532A allele vs. 12532G allele, OR = 0.80, 95% CI = [0.66, 0.99]; genotypic comparison-AA vs. GG, OR = 0.58 95% CI = [0.37, 0.91]). Sequence analysis indicated that this SNP might alter the binding ability of miR-3649, confirmed by luciferase activity assay showing a lower expression level of rs12532 A allele compared with that of the G allele (p < .001 for 293A and COS7 cell lines). Furthermore, an in vivo study showed that MSX1 expression among individuals carrying the AA genotype of rs12532 was markedly lower than that in those with the GG genotype, while the inverse correlation was observed for miR-3649, thus providing a possible interaction between MSX1 and miR-3649 in the etiology of NSOC. Taken together, these findings indicate that SNPs in the miRNA-binding sites might play an important role in the development of NSOCs. Furthermore, if confirmed in subsequent studies, the polymorphisms may be considered as additional markers for the evaluation of infants’ risk of NSOCs.

The axis inhibition protein 2 polymorphisms and non‐syndromic orofacial clefts susceptibility in a Chinese Han population

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. 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. 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. 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.

MTHFR C677T and A1298C polymorphisms and risk of nonsyndromic orofacial clefts in a south Indian population

ObjectivesSeveral lines of evidence suggest that the decrease in folate in periconceptional period or maternal use of folate antagonists has been associated with a higher risk of orofacial clefts (OFCs). MTHFR is a critical enzyme in folate metabolism that catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus playing a vital role in DNA synthesis and DNA methylation. The aim of our study was to determine whether there is any association between the susceptibility to Nonsyndromic cleft lip with or without cleft palate (NSCL/P) amongst the variations of MTHFR genotypes in South Indian population. MethodsOur sample comprised 123 cases with NSCL/P and 141 controls without clefts or family history of clefting. The most common polymorphisms C677T (rs1801133) and A1298C (rs1801131) on the MTHFR gene were screened for the genotypes using PCR-RFLP. ResultsBoth C677T and A1298C are polymorphic with minor allele frequencies of 0.131 and 0.429, respectively, for controls. Genotype data in control and cleft groups are following the Hardy Weinberg Equilibrium. There were no significant differences in genotypes of both polymorphisms between controls and NSCL/P. The pairwise LD values (D′ and r2) between C677T and A1298C are 1.0 and 0.096 respectively indicating no significant LD between these two SNPs. Haplotype phenotype analysis did not show the evidence for association. Gene–gene interaction showed the distribution of the observed combinations of the two MTHFR polymorphisms was not different between NSCL/P and controls (p=0.887). ConclusionsOur results do not support the hypothesis, that variants in the MTHFR gene confer a risk for NSCL/P in the South Indian population.

Infants’ MTHFR Polymorphisms and Nonsyndromic Orofacial Clefts Susceptibility

Nonsyndromic orofacial clefts (NSOCs) are among the most common birth defects and occur in 1 per 700 to 1000 live births worldwide, although the incidence varies with geographic origin, ethnicity, and socioeconomic status. Non-syndromic orofacial clefts can be divided into the lip only (CLO), both the lip and palate (CLP), and the palate only (CPO). Nonsyndromic orofacial clefts carry large public health burdens because of the immediate and long-term medical costs, increased overall mortality, and social impacts on patients and their families. Any disturbance from genetic background or environmental teratogenic factors during critical stages of embryonic development can lead to failure of the orofacial closure mechanism. Maternal folic acid supplementation during early pregnancy may decrease the risk of orofacial clefts. The mechanism that controls low plasma folate levels is still unknown, but one possibility is the polymorphisms in genes encoding enzymes of the folate pathway. One enzyme is methylene-tetrahydrofolate reductase (MTHFR), located at chromosome 1p36.3 and composed of 2.2 kb with 11 exons. It has 2 functional polymorphisms, C677T (rs1801133) and A1298C (rs1801131). The C677T polymorphism controls altered enzyme activity, thermolability, and mild-to-moderate hyperhomocysteinemia. The A1298C variant also decreases enzymatic activity but does not affect homocysteine plasma levels. This meta-analysis was performed, using 17 case-control studies, to determine the effects of the 2 variants on the risk of NSOC. PubMed and EMBASE databases were searched to identify articles on infants’ MTHFR C677T or A1298C polymorphisms and NSOC risk. The selected case-control studies contained detailed distributions of the infants’ MTHFR C677T or A1298C genotypes among cases and controls and reported the risk of NSOC or its subgroups. The Hardy-Weinberg equilibrium was calculated based on the genotypes among controls. For studies with subjects from different ethnic groups, data were extracted separately, with ethnicity categorized as white, Asian, and African. The difference in frequency of MTHFR C677T and A1298C polymorphisms in cases and controls was assessed by the χ2 test. The strength of the association between the polymorphisms and NSOC risk was assessed by odds ratios (ORs) with 95% confidence intervals (CIs). Eight of the 17 studies examined only C677T; the other 9 examined both variants. Eleven studies focused on CL/P, 4 on both CL/P and CPO, and 2 studies on all cases. The meta-analysis of the associations between the C677T polymorphism and the risk of NSOC included 3439 cases and 5109 controls. Among Asians, the CT heterozygote and TT homozygote resulted in elevated risks of NSOC compared with CC homozygote (OR, 1.741; 95% CI, 1.043–2.907). The frequency of MTHFR 677T allele was significantly higher in Asian cases than that in Asian controls, suggesting an increased susceptibility to NSOC (OR, 1.420; 95% CI, 1.191–1.693, for T allele vs C allele). For the A1298C polymorphism, 9 studies with 1572 cases and 2665 controls were assessed. The frequency of MTHFR 1298C allele in the case group of whites was significantly lower than that in the control group (0.271 vs 0.343, P < 0.001), suggesting a protective effect against NSOC in whites (OR, 0.711; 95% CI, 0.641–0.790). However, logistic regression revealed that MTHFR A1298C was not associated with NSOC susceptibility under any of the genetic models. Funnel plots and the Egger test did not indicate any significant publication bias. This meta-analysis provides additional evidence that MTHFR C677T and A1298C polymorphisms are involved in the development of NSOC. These results may increase the understanding of MTHFR in the etiology of NSOCs. Future well-designed studies with different ethnic populations are necessary to verify these findings.

Different roles of two novel susceptibility loci for nonsyndromic orofacial clefts in a Chinese Han population.

Nonsyndromic orofacial clefts (NSOC) are the most common developmental anomalies in human beings. Recently, a large-scale genome-wide association study identified two novel NSOC susceptibility loci: rs13041247 near MAFB and rs560426 near ABCA4. In the present study, we recruited 396 NSOC cases and 384 healthy controls to replicate their associations with risk of NSOC as well as their subgroups in a Chinese Han population. We found the overall genotype and allele frequencies of rs13041247, but not rs560426 were significantly different between cases and controls. Further logistic regression analysis showed rs13041247 CT, CC, and CT/CC were associated with decreased NSOC susceptibility, compared with rs13041247 TT wide-type homozygote. Moreover, the apparent protection against cleft lip with or without cleft palate (CL/P), cleft lip with cleft palate (CLP), and cleft lip only (CLO) was also identified in stratified analysis. However, none of any rs560426 genotypes or alleles was associated with risk of NSOC or their subgroups. Taken together, our findings confirmed the contribution of MAFB in the etiology of NSOC in a Chinese Han population.

Parental exposures and risk of nonsyndromic orofacial clefts in offspring: A case-control study in Greece

Objective To investigate the association between cleft lip and/or palate and nongenetic factors in Greece. Methods We designed a case-control study including 35 patients with nonsyndromic cleft lip and/or palate, retrospectively selected, and 35 control patients matched for prefecture of residence, prospectively selected from pediatric population hospitalized for abdominal pain or injury. Parents were interviewed about drug uptake, diseases, habits, non-occupational exposure to pollutants, and occupation. Questions covered the period from one year before until three months after conception. Results High-risk paternal occupations (mostly farmers) were significantly more frequent in cleft lip and/or palate than in controls ( p = 0.039) and increased significantly the cleft lip and/or palate risk in offspring (OR: 3.00; 95% CI: 1.03–8.70). Maternal occupation did not correlate with cleft lip and/or palate. Parental disease, drugs uptake, hazardous habits, maternal folate supplementation and non-occupational exposure to pollutants did not correlate with cleft lip and/or palate. There was a suggestion of increased risk with maternal passive exposure to tobacco (OR: 1.81; 95% CI: 0.69–4.74) and with residential proximity to industries (OR: 1.70; 95% CI: 0.61–4.74). Conclusions Paternal high-risk occupations probably exert a teratogenic effect on spermatogenesis or result in maternal contamination, and deserve specific preventive policies. The relation of smoking and residential proximity to industries with occurrence of cleft lip and/or palate deserves further study.

Association of CDH1 promoter polymorphism and the risk of non-syndromic orofacial clefts in a Chinese Han population

Objective CDH1 plays a key role in cell adhesion, which is vital to normal craniofacial morphogenesis and palatal fusion. It is therefore attractive as a candidate gene for non-syndromic orofacial clefts (NSOC). The SNP rs16260 (C > A), locating upstream of the transcriptional start site of CDH1 promoter, has a significant effect on transcriptional activity and CDH1 expression. The aim of this study was to determine the association of rs16260 with risk of NSOC and its subgroups in a Chinese Han population. Materials and methods 140 NSOC cases and 107 healthy individuals were recruited in the present study. The SNP rs16260 was genotyped by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). Results The genotype distributions of rs16260 in controls were consistent with Hardy–Weinberg equilibrium test. Lack of association was found between rs16260 and risk of NSOC. When all cleft cases were subsequently stratified into four groups (i.e. cleft lip with or without cleft palate, cleft lip only, cleft lip with cleft palate and cleft palate only), interestingly, we found that rs16260 overall genotype frequencies in cleft palate only (CPO) groups were significantly different with those in the controls ( P = 0.004) and rs16260 AA genotype significantly increased the risk of CPO by 5.90-fold (OR = 6.90, 95% CI = 1.47–32.40), providing the first evidence of CDH1 genetic variation in the etiology of CPO. Conclusions CDH1 promoter SNP rs16260 may contribute to risk of CPO in a Chinese Han population.

Significant Evidence of Association Between Polymorphisms inZNF533, Environmental Factors, and Nonsyndromic Orofacial Clefts in the Western Han Chinese Population

The etiology of nonsyndromic orofacial clefts (NSOC) has been considered "complex" or "multifactorial." Etiologic heterogeneity induces disparities in the results among different populations. The zinc finger protein 533 (ZNF533) and several environmental factors have been revealed to be associated with NSOC in several populations. We investigated three single-nucleotide polymorphisms (SNPs) and 10 environmental factors in 211 case-parent trios and 188 control individuals in the Western Han Chinese population to confirm the relationship between ZNF533, environmental factors, and the etiology of NSOC in the Western Han Chinese population. The transmission disequilibrium test, case-control analysis, multiple logistic regression, log-linear model, and conditional logistic regression were tested to confirm the contribution of the ZNF533 gene and environmental factors to the etiology of NSOC. Strong statistically significant evidence of association was found between the rs6757845 and rs1139 markers and NSOC. The haplotype G-G for rs6757845-rs1139 showed significant overtransmission among cleft lip with or without cleft palate (CL/P) trios and among cleft palate only trios. Additional 11 and 5 haplotypes were significantly overtransmitted and undertransmitted among CL/P and among cleft palate only trios, respectively. Maternal disease, use of medication, and passive smoking during the first trimester of pregnancy may increase the risk of NSOC. Maternal folic acid supplementation during the first trimester of pregnancy showed a protective effect on the etiology of NSOC. Genotype-environment interaction test showed a significant evidence of interaction effects between the genotypes at rs6757845 and maternal passive smoking during the first trimester among CL/P trios. These results confirm the effects of the ZNF533 gene and environmental factors on the etiology of NSOC.