Genetic association and functional validation of ZFP36L2 in non-syndromic orofacial cleft subtypes.

BACKGROUND: Non-syndromic orofacial cleft (NSOC) is one of the most common craniofacial malformations with complex etiology. This study aimed to explore the role of specific SNPs in ZFP36L2 and its functional relevance in zebrafish models. METHODS: We analyzed genetic data of the Chinese Han population from two previous GWAS, comprising of 2512 cases and 2255 controls. Based on the Hardy-Weinberg Equilibrium (HWE) and minor allele frequency (MAF), SNPs in the ZFP36L2 were selected for association analysis. In addition, zebrafish models were used to clarify the in-situ expression pattern of zfp36l2 and the impact of its Morpholino-induced knockdown. RESULTS: Via association analysis, rs7933 in ZFP36L2 was significantly associated with various non-syndromic cleft lip-only subtypes, potentially conferring a protective effect. Zebrafish embryos showed elevated expression of zfp36l2 in the craniofacial region during critical stages of oral cavity formation. Furthermore, Morpholino-induced knockdown of zfp36l2 led to craniofacial abnormalities, including cleft lip, which was partially rescued by the addition of zfp36l2 mRNA. CONCLUSION: Our findings highlight the significance of ZFP36L2 in the etiology of NSOC, supported by both human genetic association data and functional studies in zebrafish. These results pave the way for further exploration of targeted interventions for craniofacial malformations.

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

OBJECTIVES: The 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. DESIGN: Initially, 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. RESULTS: Finally, 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). CONCLUSIONS: Our 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.

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

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

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

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

Causal Variations at IRF6 Gene Identified in Van der Woude Syndrome Pedigrees.

The purpose of this study is to analyze the clinical characteristics of patients with Van der Woude syndrome (VWS) and to detect variations in each patient. Finally, the combination of genotype and phenotype can make a clear diagnosis of VWS patients with different phenotype penetrance.Five Chinese VWS pedigree were enrolled. Whole exome sequencing of the proband was performed, and the potential pathogenic variation was further verified by Sanger sequencing in the patient and their parents. The human mutant IRF6 coding sequence was generated from the human full-length IRF6 plasmid by site-directed mutagenesis and cloned into the GV658 vector, RT-qPCR and Western blot were used to detect the expression of IRF6.We found one de novo nonsense variation (p. Gln118Ter) and three novel missense variations (p. Gly301Glu, p. Gly267Ala, and p. Glu404Gly) co-segregated with VWS. RT-qPCR analysis revealed that p. Glu404Gly significantly reduced the expression level of IRF6 mRNA. Western blot of cell lysates confirmed that IRF6 p. Glu404Gly abundance levels were lower than those for IRF6 wild type.This discovery of the novel variation (IRF6 p. Glu404Gly) expands the spectrum of known variations in VWS in Chinese humans. Genetic results combined with clinical phenotypes and differential diagnosis points from other diseases can make a definitive diagnosis and provide genetic counseling for families.

Evidence of the folate-mediated one-carbon metabolism pathway genes in controlling the non-syndromic oral clefts risks.

The folate-mediated one-carbon metabolism pathway is thought to play an important role in the etiology of non-syndromic oral clefts (NSOFC), although none of the genes in this pathway has shown significant signals in genome-wide association studies (GWAS). Recent evidence indicated that enhanced understanding could be gained by aggregating multiple SNPs effect simultaneously into polygenic risk score (PRS) to assess its association with disease risks. This study is aimed to assess the association between the genetic effect of folate-mediated one-carbon metabolism pathway and NSOFC risks using PRS based on a case-parent trio design. A total of 297 SNPs mapped from 18 genes in the folate-mediated one-carbon metabolism pathway were aggregated from a GWAS of 2458 case-parent trios recruited from an international consortium. We found a PRS based on the folate-mediated one-carbon metabolism pathway was significant among all NSOFC trios (OR = 1.95, 95% CI: 1.66-2.28, p = 2.39 × 10-16 ), as well as two major subtypes, non-syndromic cleft lip with or without cleft palate (NSCL/P) trios (OR = 1.71, 95% CI: 1.50-1.96, p = 7.66 × 10-15 ) and non-syndromic cleft palate only (NSCPO) trios (OR = 1.51, 95% CI: 1.36-1.68, p = 2.1 × 10-14 ). Similar results were also observed in further subgroup analyses stratified into Asian and European trios. The averaged PRS of the folate-mediated one-carbon metabolism pathway varied between the NSOFC case group and its comparison group (p < 0.05) with higher average PRS in the cases. Moreover, the top 5% pathway PRS group had 2.25 (95% CI: 1.85-2.73) times increased NSOFC risk, also 3.09 (95% CI: 2.50-3.81) and 2.06 (95% CI: 1.39-3.02) times increased risk of NSCL/P and NSCPO compared to the remainder of the distribution. The results of our study confirmed the folate-mediated one-carbon metabolism pathway was important in controlling risk to NSOFC and this study enhanced evidence towards understanding the genetic risks of NSOFC.

Association between variants around IRF6 and non-syndromic orofacial cleft in Western Han Chinese.

OBJECTIVES: Considering limitations of previous studies and differences across populations and subtypes, this study aimed to identify new potential SNPs around IRF6 associated with non-syndromic orofacial cleft (NSOC) in Western Han Chinese. MATERIALS AND METHODS: We recruited 376 NSOC case-parent trios, including 125 non-syndromic cleft lip only (NSCLO) trios, 151 non-syndromic cleft lip and palate (NSCLP) trios, and 100 non-syndromic cleft palate only (NSCPO) trios. Twenty-two single-nucleotide polymorphisms (SNPs) were genotyped using MassARRAY method. Hardy-Weinberg equilibrium test, allelic transmission disequilibrium test (TDT) analysis, sliding-window haplotype TDT analysis, and tests for parent-of-origin effect were performed using the PLINK software. Pairwise linkage disequilibrium (LD) was computed using the Haploview program. RESULTS: In TDT analysis, allele A at rs17015217 (p = 0.00011, OR = 0.61 and 95% CI: 0.47-0.78) and allele T at rs12080691 (p = 0.00011, OR = 0.61 and 95% CI: 0.47-0.78) were under-transmitted among NSCLO trios but over-transmitted among NSCPO trios. Haplotypes showing evidence of under-transmission in NSCLO trios were over-transmitted in NSCPO trios. In tests for parent-of-origin effects, T allele at rs12080691 presented paternal under-transmission among NSCLO trios but over-transmission among NSCPO trios. CONCLUSIONS: Allele A at rs17015217 and allele T at rs12080691 are associated with NSCLO and NSCPO with potential to have opposite effects on two subtypes in this sample from Western Han Chinese.

Target sequencing reveals the association between variants in VAX1 and NSCL/P in Chinese population.

OBJECTIVE: A significant genetic association between rs7078160 in VAX1 and NSCL/P has been established through genome-wide association studies (GWAS), and we previously replicated the association in the Chinese population. The critical issue in the post-GWAS era is to identify functional variations that have a real impact on disease in the susceptible regions highlighted by GWAS. This study aimed to elucidate functional variants in VAX1 fully. MATERIALS AND METHODS: Firstly, target sequencing was performed on 159 NSCL/P patients, followed by association analysis to discover disease-associated single-nucleotide polymorphisms (SNPs); we then replicated the findings using a larger sample (1626 cases, 2255 controls) and investigated how candidate SNPs affect disease occurrence using extensive annotation databases. Additionally, we compared the genetic profiles of NSCL/P subtypes. RESULTS: In this study, 6 SNPs in VAX1 were identified to be associated with NSCL/P in the Western Han Chinese population. Five of them were predicted to influence transcriptional factor-biding ability and were expression quantitative trait loci (eQTLs) of nearby genes in multiple tissues. CONCLUSION: The previously reported association between rs7078160 and NSCL/P was successfully replicated. Moreover, our findings firstly revealed that 5 SNPs in VAX1 are associated with NSCL/P in the Western Han Chinese population. LEVEL OF EVIDENCE: Original Reports.

MMP16 as NSCL ± P Susceptible Gene in Western Han Chinese.

OBJECTIVE: The role of MMP16 in lip development is unclear. This study aimed to identify nonsyndromic cleft lip with or without palate (NSCL ± P) susceptible loci of MMP16 in western Han Chinese. DESIGN: We performed targeted sequencing around MMP16 combined with a 2-phase association analysis on common variants. Phase 2 association analysis was performed with NSCL ± P specific subphenotypes (NSCL and NSCLP). Then we used rare variants burden analysis and genotyping, accompanied by motif analysis. SETTING: This study was completed in a tertiary medical center. PATIENTS, PARTICIPANTS: Phase 1 targeted sequencing included 159 patients with NSCL ± P and 542 normal controls; phase 2 included 1626 patients with NSCL ± P (1047 NSCL and 579 NSCLP) and 2255 normal controls. INTERVENTIONS: Venous blood samples were collected from patients and used to extract DNA. MAIN OUTCOME MEASURES: After Bonferroni correction, phase 1 significant threshold of p-value was 4.28 × 10-5 (0.05/1167 single nucleotide polymorphisms [SNPs]), and phase 2 was .00025 (0.05/200 SNPs). Burden analysis significant threshold p-value was .05. RESULTS: Common variants phase 1 association analysis identified 11 statistically significant SNPs (lowest p = 1.90 × 10-9, odds ratio (OR) = 0.27, 95% CI: 0.17-0.44), phase 2 replication identified 16 SNPs in NSCL ± P (lowest p = 6.26 × 10-6, OR = 0.77, 95% CI: 0.69-0.86) and 9 in NSCL (lowest p = 8.44 × 10-5, OR = 0.76, 95% CI: 0.66-0.87). Rare variants burden analysis showed no significant results, genotyping results showed they were maternally inherited. CONCLUSIONS: Our study identified MMP16 susceptible SNPs in NSCL ± P and NSCL, emphasizing its potential role in lip development. Our study also highlighted the importance to perform association analysis with subphenotypes divided.

A Novel Missense Variant in the TCOF1 Gene in one Chinese Case With Treacher Collins Syndrome.

The purpose of this study is to analyze the clinical characteristics of a Treacher Collins syndrome (TCS) patient carrying a de novo variant of TCOF1, and briefly analyze the correlation between genetic results and clinical features. Also, the pathogenesis and clinical treatment of TCS are reviewed.A Chinese pedigree with TCS containing 8 members was enrolled. Phenotype of the proband was evaluated by a surgeon, then whole exome sequencing of the proband was performed. Then we verified the proband-derived variants by Sanger sequencing in the pedigree. Correlation between genotype and phenotype was analyzed.The study was conducted in a stomatological hospital.A Chinese pedigree with TCS containing 8 members.To ascertain the genetic variants in the Chinese pedigree with TCS.Blood samples were collected.We reported a case of typical TCS with a de novo missense variant (NM_001371623.1:c.38T>G, p.(Leu13Arg)) in exon 1 of TCOF1, who presented asymmetrical facial abnormalities, including downward slanting of the palpebral fissures, sparse eyebrows, lateral tilt of the eyeballs, bilateral external ears deformities, hypoplasia of midface, reduction of the zygomatic body, bilateral orbital invagination, right external auditory canal atresia, mandibular ramus short deformity, cleft palate and the whole face was convex.This research found a novel variant of TCS in Chinese, expanding the spectrum of TCS pathogenic variants. Genetic results combined with clinical phenotype can make a definite diagnosis and provide genetic counseling for the family.

Targeted re-sequencing on 1p22 among non-syndromic orofacial clefts from Han Chinese population.

Rs560426 at 1p22 was proved to be associated with NSCL/P (non-syndromic cleft lip with or without the palate) in several populations, including Han Chinese population. Here, we conducted a deep sequencing around rs560426 to locate more susceptibility variants in this region. In total, 2,293 NSCL/P cases and 3,235 normal controls were recruited. After sequencing, association analysis was performed. Western blot, RT-qPCR, HE, immunofluorescence staining, and RNA sequencing were conducted for functional analyses of the selected variants. Association analysis indicated that rs77179923 was the only SNP associated with NSCLP specifically (p = 4.70E-04, OR = 1.84), and rs12071152 was uniquely associated with LCLO (p = 4.00E-04, OR = 1.30, 95%CI: 1.12-1.51). Moreover, de novo harmful rare variant NM_004815.3, NP_004806.3; c.1652G>C, p.R551T in ARHGAP29 resulted in a decreased expression level of ARHGAP29, which in turn affected NSCL/P-related biological processes; however, no overt cleft palate (CP) phenotype was observed. In conclusion, rs12071152 was a new susceptible variant, which is specifically associated with LCLO among the Han Chinese population. Allele A of it could increase the risk of having a cleft baby. Rs77179923 and rare variant NM_004815.3, NP_004806.3; c.1652G>C, p.R551T at 1p22 were both associated with NSCLP among the Han Chinese population. However, this missense variation contributes to no overt CP phenotype due to dosage insufficiency or compensation from other genes.

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.

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

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

NELL-1 in Genome-Wide Association Studies across Human Diseases.

Neural epidermal growth factor-like (EGFL)-like protein (NELL)-1 is a potent and key osteogenic factor in the development and regeneration of skeletal tissues. Intriguingly, accumulative data from genome-wide association studies (GWASs) have started unveiling potential broader roles of NELL-1 beyond its functions in bone and cartilage. With exploration of the genetic variants of the entire genome in large-scale disease cohorts, GWASs have been used for establishing the connection between specific single-nucleotide polymorphisms of NELL1, in addition to osteoporosis, metabolic diseases, inflammatory conditions, neuropsychiatric diseases, neurodegenerative disorders, and malignant tumors. This review summarizes the findings from GWASs on the manifestation, significance level, implications on function, and correlation of specific NELL1 single-nucleotide polymorphisms in various disorders in humans. By offering a unique and comprehensive correlation between genetic variants and plausible functions of NELL1 in GWASs, this review illustrates the wide range of potential effects of a single gene on the pathogenesis of multiple disorders in humans.

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.

Association analysis of SNPs in GRHL3, FAF1, and KCNJ2 with NSCPO sub-phenotypes in Han Chinese.

OBJECTIVES: Non-syndromic cleft palate only (NSCPO) is a common congenital deformity with complex etiologies. GRHL3, FAF1, and KCNJ2 have been reported to be involved in the pathogenesis of NSCPO. Up till now, there have been no replication studies based on large Han Chinese. Therefore, this study aimed to investigate associations between GRHL3, FAF1, KCNJ2, and NSCPO sub-phenotypes patients in Han Chinese. MATERIALS AND METHODS: Firstly, we selected 2 SNPs based on previous literatures: FAF1 (rs3827730) and GRHL3 (rs41268753). Also, we selected 8 tagSNPs in GRHL3 (rs557811, rs609352, rs10903078, rs6659209, rs12401714, rs12568599, rs3887581, rs12024148) and 2 tagSNPs in KCNJ2 (rs75855040 and rs236514). Afterward, we evaluated these SNPs among 1668 NSCPO patients and 1811 normal controls from Han Chinese. Following data were analyzed by PLINK and Haploview program. RESULTS: Association analysis under additive model showed that allele A at rs12568599 in GRHL3 gene is significantly associated with NSCPO (p = 0.0034, OR = 1.38 and 95%CI: 1.11-1.72) and its sub-phenotype incomplete cleft palate (ICP) (p = 0.0039, OR = 1.4 and 95%CI: 1.11-1.75), and it could increase the risk of both NSCPO and ICP. CONCLUSIONS: This study firstly found that rs12568599 in GRHL3 is associated with NSCPO and ICP in Han Chinese, indicating that sub-phenotypes of NSCPO have different etiologies.

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.

SP1-Mediated Upregulation of Long Noncoding RNA ZFAS1 Involved in Non-syndromic Cleft Lip and Palate via Inactivating WNT/ß-Catenin Signaling Pathway.

Non-syndromic cleft lip and palate (NSCLP) is one of the most common congenital malformations with multifactorial etiology. Although long non-coding RNAs (lncRNAs) have been implicated in the development of lip and palate, their roles in NSCLP are not fully elucidated. This study aimed to investigate how dysregulated lncRNAs contribute to NSCLP. Using lncRNA sequencing, bioinformatics analysis, and clinical tissue sample detection, we identified that lncRNA ZFAS1 was significantly upregulated in NSCLP. The upregulation of ZFAS1 mediated by SP1 transcription factor (SP1) inhibited expression levels of Wnt family member 4 (WNT4) through the binding with CCCTC-binding factor (CTCF), subsequently inactivating the WNT/ß-catenin signaling pathway, which has been reported to play a significant role on the development of lip and palate. Moreover, in vitro, the overexpression of ZFAS1 inhibited cell proliferation and migration in human oral keratinocytes and human umbilical cord mesenchymal stem cells (HUC-MSCs) and also repressed chondrogenic differentiation of HUC-MSCs. In vivo, ZFAS1 suppressed cell proliferation and numbers of chondrocyte in the zebrafish ethmoid plate. In summary, these results indicated that ZFAS1 may be involved in NSCLP by affecting cell proliferation, migration, and chondrogenic differentiation through inactivating the WNT/ß-catenin signaling pathway.