Speech Outcomes after Delayed Hard Palate Closure and Synchronous Secondary Alveolar Bone Grafting in Patients with Cleft Lip, Alveolus and Palate.

Background The best timing of closure of the hard palate in individuals with cleft lip, alveolus, and palate (CLAP) to reach the optimal speech outcomes and maxillary growth is still a subject of debate. This study evaluates changes in compensatory articulatory patterns and resonance in patients with unilateral and bilateral CLAP who underwent simultaneous closure of the hard palate and secondary alveolar bone grafting (ABG). Methods A retrospective study of patients with nonsyndromic unilateral and bilateral CLAP who underwent delayed hard palate closure (DHPC) simultaneously with ABG at 9 to 12 years of age from 2013 to 2018. The articulatory patterns, nasality, degree of hypernasality, facial grimacing, and speech intelligibility were assessed pre- and postoperatively. Results Forty-eight patients were included. DHPC and ABG were performed at the mean age of 10.5 years. Postoperatively hypernasal speech was still present in 54% of patients; however, the degree of hypernasality decreased in 67% ( p < 0.001). Grimacing decreased in 27% ( p = 0.015). Articulation disorders remained present in 85% ( p = 0.375). Intelligible speech (grade 1 or 2) was observed in 71 compared with 35% of patients preoperatively ( p < 0.001). Conclusion This study showed an improved resonance and intelligibility following DHPC at the mean age of 10.5 years, however compensatory articulation errors persisted. Sequential treatments such as speech therapy play a key role in improvement of speech and may reduce remaining compensatory mechanisms following DHPC.

[Gene-gene/gene-environment interaction of transforming growth factor-ß signaling pathway and the risk of non-syndromic oral clefts].

OBJECTIVE: To explore the association between polymorphisms of transforming growth factor-ß (TGF-ß) signaling pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) among Asian populations, while considering gene-gene interaction and gene-environment interaction. METHODS: A total of 1 038 Asian NSCL/P case-parent trios were ascertained from an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate the genes affec-ting risk to NSCL/P. After stringent quality control measures, 343 single nucleotide polymorphism (SNP) spanning across 10 pivotal genes in the TGF-ß signaling pathway were selected from the original genome-wide association study(GWAS) dataset for further analysis. The transmission disequilibrium test (TDT) was used to test for SNP effects. The conditional Logistic regression models were used to test for gene-gene interaction and gene-environment interaction. Environmental factors collected for the study included smoking during pregnancy, passive smoking during pregnancy, alcohol intake during pregnancy, and vitamin use during pregnancy. Due to the low rates of exposure to smoking during pregnancy and alcohol consumption during pregnancy (<3%), only the interaction between maternal smoking during pregnancy and multivitamin supplementation during pregnancy was analyzed. The threshold for statistical significance was rigorously set at P =1.46×10-4, applying Bonferroni correction to account for multiple testing. RESULTS: A total of 23 SNPs in 4 genes yielded nominal association with NSCL/P (P<0.05), but none of these associations was statistically significant after Bonferroni' s multiple test correction. However, there were 6 pairs of SNPs rs4939874 (SMAD2) and rs1864615 (TGFBR2), rs2796813 (TGFB2) and rs2132298 (TGFBR2), rs4147358 (SMAD3) and rs1346907 (TGFBR2), rs4939874 (SMAD2) and rs1019855 (TGFBR2), rs4939874 (SMAD2) and rs12490466 (TGFBR2), rs2009112 (TGFB2) and rs4075748 (TGFBR2) showed statistically significant SNP-SNP interaction (P<1.46×10-4). In contrast, the analysis of gene-environment interactions did not yield any significant results after being corrected by multiple testing. CONCLUSION: The comprehensive evaluation of SNP associations and interactions within the TGF-ß signaling pathway did not yield any direct associations with NSCL/P risk in Asian populations. However, the significant gene-gene interactions identified suggest that the genetic architecture influencing NSCL/P risk may involve interactions between genes within the TGF-ß signaling pathway. These findings underscore the necessity for further investigations to unravel these results and further explore the underlying biological mechanisms.

A meta-analysis of alveolar bone grafting using bone substitutes in cleft lip and palate patients.

In individuals with cleft lip and palate (CLP), an alveolar bone graft (ABG) is carried out for alveolar cleft closure. Several sources for ABG include autologous bone, xenologous bone, and alloplastic substitutes. Autologous bone has been the preferred source for ABG. Alloplastic substitutes might serve as an alternative. This study aimed to compare the outcomes between autologous and alloplastic as sources for ABG. This study made use of eight web databases. Randomized control trials (RCTs) and non-RCTs were included. CLP patients with alveolar cleft with imaging studies, computed tomography (CT scan) and/or cone beam CT scan, and bone graft volume within 6-12 months postintervention were selected. Bone graft volume within 6-12 months postintervention was assessed. Three studies met the inclusion criteria. After 6-12 months of follow-up, there were no statistically significant differences in bone graft volume between autologous and alloplastic bone grafts (fixed-effect model estimate value = 0.21; confidence interval - 0.301-0.730; P = 0.414). The limitations include small research sample sizes, a high likelihood of bias among included studies, and different alloplastic materials from each included study. Autologous and alloplastic bone grafts showed similar effectiveness in alveolar bone grafting. Further clinical trial studies with bigger sample sizes and similar interventions are needed as evidence for future reviews.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The rs3943258 located within BCL2 was probably related to NSCL/P susceptibility.

Association between IRF6, TP63, GREM1 Gene Polymorphisms and Non-Syndromic Orofacial Cleft Phenotypes in Vietnamese Population: A Case-Control and Family-Based Study.

This study aims to identify potential variants in the TP63-IRF6 pathway and GREM1 for the etiology of non-syndromic orofacial cleft (NSOFC) among the Vietnamese population. By collecting 527 case-parent trios and 527 control samples, we conducted a stratified analysis based on different NSOFC phenotypes, using allelic, dominant, recessive and over-dominant models for case-control analyses, and family-based association tests for case-parent trios. Haplotype and linkage disequilibrium analyses were also conducted. IRF6 rs2235375 showed a significant association with an increased risk for non-syndromic cleft lip and palate (NSCLP) and cleft lip with or without cleft palate (NSCL/P) in the G allele, with pallele values of 0.0018 and 0.0003, respectively. Due to the recessive model (p = 0.0011) for the NSCL/P group, the reduced frequency of the GG genotype of rs2235375 was associated with a protective effect against NSCL/P. Additionally, offspring who inherited the G allele at rs2235375 had a 1.34-fold increased risk of NSCL/P compared to the C allele holders. IRF6 rs846810 and a G-G haplotype at rs2235375-rs846810 of IRF6 impacted NSCL/P, with p-values of 0.0015 and 0.0003, respectively. In conclusion, our study provided additional evidence for the association of IRF6 rs2235375 with NSCLP and NSCL/P. We also identified IRF6 rs846810 as a novel marker associated with NSCL/P, and haplotypes G-G and C-A at rs2235375-rs846810 of IRF6 associated with NSOFC.

Analysis of candidate genes for cleft lip ± cleft palate using murine single-cell expression data.

Introduction: Cleft lip ± cleft palate (CL/P) is one of the most common birth defects. Although research has identified multiple genetic risk loci for different types of CL/P (i.e., syndromic or non-syndromic forms), determining the respective causal genes and understanding the relevant functional networks remain challenging. The recent introduction of single-cell RNA sequencing (scRNA-seq) has provided novel opportunities to study gene expression patterns at cellular resolution. The aims of our study were to: (i) aggregate available scRNA-seq data from embryonic mice and provide this as a resource for the craniofacial community; and (ii) demonstrate the value of these data in terms of the investigation of the gene expression patterns of CL/P candidate genes. Methods and Results: First, two published scRNA-seq data sets from embryonic mice were re-processed, i.e., data representing the murine time period of craniofacial development: (i) facial data from embryonic day (E) E11.5; and (ii) whole embryo data from E9.5-E13.5 from the Mouse Organogenesis Cell Atlas (MOCA). Marker gene expression analyses demonstrated that at E11.5, the facial data were a high-resolution representation of the MOCA data. Using CL/P candidate gene lists, distinct groups of genes with specific expression patterns were identified. Among others we identified that a co-expression network including Irf6, Grhl3 and Tfap2a in the periderm, while it was limited to Irf6 and Tfap2a in palatal epithelia, cells of the ectodermal surface, and basal cells at the fusion zone. The analyses also demonstrated that additional CL/P candidate genes (e.g., Tpm1, Arid3b, Ctnnd1, and Wnt3) were exclusively expressed in Irf6+ facial epithelial cells (i.e., as opposed to Irf6- epithelial cells). The MOCA data set was finally used to investigate differences in expression profiles for candidate genes underlying different types of CL/P. These analyses showed that syndromic CL/P genes (syCL/P) were expressed in significantly more cell types than non-syndromic CL/P candidate genes (nsCL/P). Discussion: The present study illustrates how scRNA-seq data can empower research on craniofacial development and disease.

Association of BRCA2 Gene Functional Polymorphisms with Nonsyndromic Cleft Lip With or Without Cleft Palate in a Chinese Population.

Background: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex congenital disease affected by genetic and environmental factors however, the specific pathogenic alleles and regulatory mechanisms remain unclear in many cases. Here, we aimed to study the association between eight potentially functional single nucleotide polymorphisms (SNPs) of the BRCA2 and MGMT genes and NSCL/P in a Chinese population through a case-control study. Materials and Methods: To investigate the relationship between potentially functional SNPs of the BRCA2 and MGMT genes and NSCL/P, we selected 200 affected patients and 200 unrelated normal controls in a Chinese population. The BRCA2 gene SNPs (rs11571836, rs144848, rs7334543, rs15869, rs766173 and rs206118) and MGMT gene SNPs (rs12917 and rs7896488) were genotyped using the SNaPshot technique and the resulting data were subjected to statistical and bioinformatic analyses. Results: Our study identified for the first time that alleles of the BRCA2 are associated with NSCL/P in a Chinese population and that the s11571836 G allele was protective against NSCL/P. Under four genetic models, rs11571836 had a significant correlation with NSCL/P. Preliminary bioinformatic analyses revealed four potential miRNA matching sites (miR-1244, miR-1323, miR-562, and miR-633) associated with the rs11571836 which is located in the 3' untranslated region of BRCA2. Conclusions: These results support the role of polymorphisms of BRCA2 gene in affecting susceptibility to NSCL/P and its progression, but further research is necessary to elucidate the mechanism by which the BRCA2 gene polymorphisms affect the penetrance of NSCL/P.

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

OBJECTIVES: To 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. DESIGN: A 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. RESULTS: The 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. CONCLUSION: PTCH1 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.

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.

Mechanical dissection and culture of mouse cranial neural crest cells.

Owing to the contribution of cranial neural crest cells (CNCCs) to the majority of craniofacial structures, they have been studied extensively for the pathogenesis of craniofacial diseases. To investigate and summarize how to isolate and culture the CNCCs from wild-type mice, a literature search was performed in online databases (PubMed and Web of Science) using optimized keywords "mouse," "cranial neural crest cell" and "culture." The literature was checked by two investigators according to the screening and exclusion criteria. Initially, 197 studies were retrieved from PubMed and 169 from Web of Science, and after excluding replicate studies, 293 articles were considered. Finally, 17 studies met all the criteria and were included in this review. The results showed that obtaining purified stem cells and balancing the need to promote cell growth and prevent unwanted early cell differentiation were the two key points in the isolation and culture of CNCCs. However, no standard criteria are available for answering these questions. Thus, it is important to emphasize the necessity for standardization of CNCC isolation, culture, and identification in research on craniofacial diseases.

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

OBJECTIVES: This 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. DESIGN: Initially, 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. RESULTS: In 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. CONCLUSION: This 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.

Association of genetic polymorphisms of VAX1, MAFB, and NTN1 with nonsyndromic cleft lip with or without cleft palate in Chinese population.

Nonsyndromic cleft lip with or without palate (NSCL/P) is a common birth defect involving genetic factors. We conducted this case-control study to verify the association of ten single-nucleotide polymorphisms (SNPs) of six genes (VAX1, MAFB, PAX7, ABCA4, NTN1, and NOG) with NSCL/P in the Chinese population. The study included 249 NSCL/P patients, 62 nonsyndromic cleft palate only (NSCPO) patients and 480 controls. Three loci, namely, VAX1 rs7078160, MAFB rs11696257, and NTN1 rs4791774, were associated with NSCL/P (Bonferroni method adjusted p values were 0.020, 0.00031, and 0.030, respectively). We also found that the disease risk of individuals carrying both VAX1 rs7078160 and NTN1 rs4791774 was higher than those carrying only one of them (p = 4.50 × 10-4 and 6.03 × 10-3, respectively). SNPs of genes VAX1 rs7078160, MAFB rs11696257, and NTN1 rs4791774 increased NSCL/P risk in the Chinese population.

Advances in research regarding the roles of non-coding RNAs in non-syndromic cleft lip with or without cleft palate: A systematic review.

OBJECTIVE: To investigate by means of a literature review, what non-coding RNAs engage in non-syndromic cleft lip with or without cleft palate (NSCL/P) and how they lead to the occurrence of this malformation. DESIGN: A literature search of online databases (Medline via PubMed, Web of Science, Scopus, and Embase) was performed using appropriate keywords (e.g. non-coding RNA, miRNA, lncRNA, NSCL/P, non-syndromic cleft lip only, and non-syndromic orofacial cleft). The risk of bias in the included studies was then assessed, and a comprehensive review of reported non-coding RNAs associated with NSCL/P was performed. RESULTS: The initial search retrieved 133 studies reporting non-coding RNAs associated with NSCL/P; after excluding 18 replicates and 77 ineligible studies, 35 remained. Of these, 16 studies fulfilled all the criteria and were included in the systematic review. These studies established the roles of non-coding RNAs in the development of craniofacial structures. The differential expression of these non-coding RNAs could lead to orofacial clefts, indicating their significance in NSCL/P and their profound research value. CONCLUSION: There is evidence that non-coding RNAs are involved in the formation of NSCL/P. Specifically, they play significant roles in the regulation of genes and signalling pathways related to NSCL/P.

Association Between SNPs in 1q32.2 and NSCL ± P in Han Chinese Population.

OBJECTIVES: Non-syndromic cleft lip with or without cleft palate (NSCL ± P) is one of the most common birth malformations. Currently, numerous susceptibility SNPs have been reported by GWA studies, however, the replications of them among NSCL ± P from Han Chinese were very limited. DESIGN: In this study, we selected 16 SNPs around 1q32.2 based on the published GWA studies and replicated them among 302 trios with NSCL ± P from Han Chinese Population. The genotypic data was analyzed with FBAT, PLINK and R package. SETTING: The study was conducted in a tertiary medical center. PATIENTS, PARTICIPANTS: 302 patients with CL ± P and their parents. MAIN OUTCOME MEASURES: To ascertain the genetic variants in 1q32.2 in patients with CL ± P in Han Chinese Population. INTERVENTIONS: Blood samples were collected. RESULTS: We found T allele (Z = 4.26, p = 0.00002) and T/T homozygotes (Z = 4.4, p = 0.000011) at rs12063989 was significantly over-transmitted among non-syndromic cleft lip with or without cleft palate (NSCL ± P). CONCLUSIONS: We found rs12063989 exhibited significant association with the occurrence of NSCL ± P, which would provide new evidence for the future study in the etiology of NSCL ± P.

Transcriptomic analysis of the upper lip and primary palate development in mice.

Background: Normal fusion of the upper lip and primary palate is a complex process involving a series of characteristic and orderly regulated cellular events. Cleft lip with or without palate (CL/P), one of the most common congenital malformations, may be induced by abnormalities in any of these events. However, less is known about the precise regulatory process in the fusion of the upper lip and primary palate. Methods: Lambdoidal junction tissues of mice from embryonic days 10.5, 11.5, and 12.5- three key fusion stages-were acquired for RNA sequencing. Results: Gene expression profiles in distinct fusion stages of mice were identified. Some of the differentially expressed genes (DEGs) have been reported to affect upper lip and primary palate development. However, other DEGs, such as Krt5, Pax1, Ambn, Hey2, and Tnmd, have not previously been investigated. Gene set enrichment analysis (GSEA) of these DEGs revealed the sequential intensification of Wnt, PI3K-Akt, MAPK, Hippo, and TGF-beta signaling pathways and identified relatively highly expressed genes including Tnn, Wnt3a, and Wnt16. We also observed substantial alternative splicing events during the fusion of the upper lip and primary palate and identified potentially important genes including Gtpbp8, Armcx1, Tle3, and Numa1. Protein-protein interaction (PPI) network analysis identified a series of hub genes, including Col1a2, Fos, Bmp2, Shh, Col1a1, Wnt3a, Anxa1, Gem, etc. Conclusion: Overall, the results of this study provided a comprehensive analysis of the development of the upper lip and primary palate. Our work provides insight into future studies of normal upper lip and primary palate development and the etiology of CL/P.

Research progress on the regulation of craniofacial development and malformation by fibroblast growth factor 8 / 口腔疾病防治

@#Fibroblast growth factor 8 (FGF8) is a kind of secretory polypeptide that has crucial roles in the development of various tissues and organs. Current studies have found that FGF8 can regulate the differentiation of cranial neural crest cells by activating the mitogen-activated protein kinase (MAPK) signaling pathway and affect the establishment of mandibular arch polarity and the development of craniofacial symmetry by regulating the expression of target genes. Cleft lip with or without cleft palate, ciliopathies, macrostomia and agnathia are four developmental malformations involving the craniofacial region that seriously affect the quality of life of patients. The abnormal FGF8 signal caused by gene mutation, abnormal protein conformation or expression is closely related to the occurrence of craniofacial malformations, but the molecular mechanism and signaling pathway underlying these malformations have not been fully elucidated. Craniofacial development is a complex process mediated by a variety of signaling molecules. In the future, the role of various signaling molecules in craniofacial development and malformations need to be explored to provide a new perspective and vision for the prevention and treatment of these craniofacial malformations.

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.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Composición y función salival en niños con labio con/sin paladar hendido no sindrómico / Salivary composition and function in children with non-syndromic cl w/wo p / Composição e função salivar em crianças com fissuras labiais e/ou palatinas não sindrômicas

Resumen Introducción y Objetivo: La saliva cumple una importante función en la homeostasis oral, aunque no es evaluada rutinariamente en pacientes con labio con/sin paladar hendido (LH±PH). Este estudio pretendió comparar las características salivares de niños con LH±PH no sindrómico vs. niños sin esta diferencia craneofacial. Materiales y métodos: Se incluyeron 17 niños con LH±PH no sindrómico y 25 niños sin LH±PH, de los cuales se colectaron muestras de saliva estimulada (SE) y no estimulada (SNE). Se realizaron pruebas fisicoquímicas, cuantificación de iones (Ca, Mg y P), proteína total y recuento de streptococcus mutans en ambos grupos para ambas muestras de saliva. Para establecer comparaciones entre grupos se realizó la prueba no paramétrica de Mann-Whitney. Resultados: Hubo diferencias estadísticamente significativas en el pH de SE y SNE, así como en proteína total y capacidad amortiguadora de SNE. El pH (p=0,002) y capacidad amortiguadora (p=0,023) de SNE de los niños con LH±PH fueron menores, mientras que la concentración de proteína total (p=0,003) fue mayor con respecto al grupo control. Se encontró mayor proporción de estreptococo mutans en SNE de niños con LH±PH (6,39x104 UFC mL-1) respecto al grupo control (2,21x104 UFC mL-1), aunque sin detectar diferencias estadísticamente significativas (p=0,243). Conclusiones: Estos hallazgos sugieren una alteración en la función y composición de la SNE en pacientes con LH±PH no sindrómico, que correspondería a una alteración en la formación y función de las glándulas salivales en estos individuos que requiere ser investigada.

Abstract Introduction and objective: Saliva plays an important role in oral homeostasis, although it is not routinely evaluated in patients with CL±P. This study aimed to compare these characteristics in children with non-syndromic CL±P vs. children without this craniofacial difference. Materials and methods: This descriptive study included 17 children with non-syndromic CL±P from Fundación Clínica Noel and 25 children without CL±P. Stimulated (SS) and non-stimulated saliva (NSS) from both groups were collected for physicochemical tests, quantification of ions (Ca, Mg and P), total protein and streptococcus mutans count. Results: There were statistically significant differences in pH of SS and NSS, as well as in total protein concentration and buffer capacity in NSS. The pH (p = 0.002) and buffering capacity (p = 0.023) of NSS of CL±P children decreased, while total protein concentration (p = 0.003) increased compared to the control group. A higher streptococcus mutans count was found in NSS of CL±P children (6.39x104 CFU UFC mL-1) compared to the control group (2.21x104 UFC mL-1), although without detecting statistically significant differences (p = 0.243). Conclusions: These findings suggest an alteration in the function and composition of NSS in patients with non-syndromic CL±P; which could possibly be explained from the genetic/biological point of view by an alteration in the salivary glands formation and function in these individuals that requires further investigation.

Resumo Introdução e objetivo: A saliva desempenha um papel fundamental na homeostase oral, não é avaliada rotineiramente em pacientes com FL/P. Portanto, este estudo objetivou indagar essas características em crianças com FL/P não sindrômico. Materiais e métodos: 17 crianças com FL/P não sindrômico da Fundación Clínica Noel, e 25 crianças sem FL/P foram incluídas neste estudo. Amostras de saliva estimulada (SE) e saliva não estimulada (SNE) foram coletadas para realização de testes físico-químicos, quantificação de íons (Ca, Mg e P), proteína total e contagem de streptococcus mutans. Resultados: Houve diferenças estatisticamente significativas no pH de SE e SNE, bem como na concentração de proteína total e capacidade tampão de SNE. O pH (p=0,002) e a capacidade tampão (p=0,023) de SNE de crianças com FL/P foram menores, enquanto a concentração de proteína total (p=0,003) foi maior, quando comparado ao grupo controle. Um contagem maior de streptococcus mutans foi encontrada na saliva não estimulada de crianças com FL/P (6,39x104 UFC mL-1) em comparação ao grupo controle (2,21x104 UFC mL-1), embora sem detectar diferenças estatisticamente significativas (p=0,243). Conclusão: Esses achados sugerem uma alteração na função e composição da SNE em pacientes com FL/P não sindrômico; isso possivelmente poderia ser explicado do ponto de vista genético/biológico por uma alteração na formação e função das glândulas salivares nesses indivíduos que requer investigação.

Exosomes-a potential indicator and mediator of cleft lip and palate: a narrative review.

OBJECTIVE: This article summarizes the recent literature on noncoding ribonucleic acids (ncRNAs) in relation to cleft lip with or without palate and exosomes and their usage in craniofacial diseases. BACKGROUND: Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with genetic and environmental risk factors that affects numerous children and families. Surgical procedures can correct deformations; however, residual sequelae remain after surgery. Studies exploring the pathogenesis of CL/P are crucial for its early diagnosis and treatment and can inform treatment strategy decisions, etiology searches, and treatment during pregnancy. Recently, research has shown that most disease-related genes are ncRNAs, which are important transcripts in the human transcriptome. ncRNAs include microRNAs, long noncoding RNAs, and circular RNAs. These ncRNAs play essential roles in various pathophysiological processes, including cell proliferation, migration, apoptosis, and epithelial-mesenchymal transition. Previous studies on protein-coding genes have identified a number of genes related to CL/P; however, the pathogenesis of CL/P has not yet been thoroughly explained. Exosomes are vehicles that transfer various bioactive molecules between cells and represent a new method of intercellular communication. Research has shown that exosomes are related to some craniofacial diseases. METHODS: We searched the PubMed database for recently published English-language articles using the following keywords: "cleft lip with or without palate," "noncoding RNA," "exosomes," and "craniofacial diseases". We then reviewed the retrieved articles. CONCLUSIONS: As exosomes serve as cellular communicators and the palate consists of epithelial and mesenchymal cells, communication between the two cell types may affect its formation. Thus, exosomes could represent a new indicator and mediator of CL/P.

MTHFR promoter methylation might mitigate the effect of smoking at the level of LINE-1 in cleft lip tissues: A preliminary study.

BACKGROUND: The medial and maxillary aspects of the upper lip originate at separate embryonic stages and therefore may experience different maternal exposure patterns which may affect methylation. Based on this hypothesis, we investigated the level of methylation of the methylene tetrahydrofolate reductase promoter gene (mMTHFR) in tissues from cleft lip, and mMTHFR levels by MTHFR c.677C > T genotype. We further investigated whether mMTHFR mitigates the effect of smoking on long interspersed nuclear element (LINE-1) methylation in these tissues. METHODS: DNA extracted from medial and lateral tissues of 26 infants with nonsyndromic cleft lip with or without cleft palate (nsCL/P) was bisulfite converted and mMTHFR was measured on a pyrosequenser. LINE-1 methylation and MTHFR c.677C > T genotype data were obtained in our previous study. RESULTS: There was no substantial difference in mMTHFR (p = .733) and LINE-1 (p = .148) between the two tissues. mMTHFR was not influenced by MTHFR c.677C > T genotype, but there was suggestive evidence that the difference was larger among infants exposed to maternal smoking compared to nonexposed. LINE-1 methylation differences were significant (p = .025) in infants born to nonsmoking mothers, but this was not apparent (p = .872) in infants born to mothers who smoked. Our Pearson's correlation analysis suggested a weak inverse association between mMTHFR and LINE-1 (r = -.179, p = .381). CONCLUSION: Our preliminary observation of differences in patterns of mMTHFR levels in lip tissue suggests the interplay of gene and environment in the establishment of methylation in tissues at both sides of cleft lip. This requires investigation in a larger cohort, integrated with metabolic assessment.