Genetic factors define CPO and CLO subtypes of nonsyndromicorofacial cleft.

Nonsyndromic orofacial cleft (NSOFC) is a severe birth defect that occurs early in embryonic development and includes the subtypes cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP). Given a lack of specific genetic factor analysis for CPO and CLO, the present study aimed to dissect the landscape of genetic factors underlying the pathogenesis of these two subtypes using 6,986 cases and 10,165 controls. By combining a genome-wide association study (GWAS) for specific subtypes of CPO and CLO, as well as functional gene network and ontology pathway analysis, we identified 18 genes/loci that surpassed genome-wide significance (P < 5 × 10-8) responsible for NSOFC, including nine for CPO, seven for CLO, two for both conditions and four that contribute to the CLP subtype. Among these 18 genes/loci, 14 are novel and identified in this study and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. Interestingly, we observed an opposite effect of the genetic variants in the IRF6 gene for CPO and CLO. Moreover, the gene expression dosage effect of IRF6 with two different alleles at the same single-nucleotide polymorphism (SNP) plays important roles in driving CPO or CLO. In addition, PAX9 is a key TF for CPO. Our findings define subtypes of NSOFC using genetic factors and their functional ontologies and provide a clue to improve their diagnosis and treatment in the future.

Homozygote C/C at rs12543318 was risk factor for non-syndromic cleft lip only from Western Han Chinese population.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder, and it results from both of the genetic modifiers and environmental factors, with genetic modifiers contributes to it more than environmental factors. GWASs made great progress in identifying the candidate genes for NSCL/P, but the findings need to be replicated in other populations. In this study, we selected eleven SNPs from recent GWASs and GWAS meta-analysis to investigate their associations among 308 NSCL/P trios (134 non-syndromic cleft lip only (NSCLO) trios and 174 non-syndromic cleft lip with cleft palate (NSCLP) trios) from Han Chinese population. All SNPs were genotyped using SNPscan method and analyzed the data with FBAT, PLINK, and R package. Allelic TDT analysis showed that allele A at rs12543318 was associated with NSCLO trios (P = .0032, OR = 0.57, 95% CI: 0.39-0.83), and parent-of-origin effect analysis indicated that allele A at rs12543318 was significantly maternally undertransmitted among NSCLO (P = .0046), which implied the potential influence of genomic imprinting; global TDT further confirmed this association. Individual genotypic TDT showed homozygote C/C at rs12543318 was overtransmitted among NSCLO (Z = 3.79, P = .00015) and NSCL/P groups (Z = 3.83, P = .00013), which indicated that it could increase the risk to have cleft babies. Our findings indicated that rs12543318 was associated with NSCLO from Western Han Chinese population, which will give new scientific evidence for later researches in the etiology of NSOCs.

BMP7 Gene involved in nonsyndromic orofacial clefts in Western Han Chinese.

BACKGROUND: Nonsyndromic orofacial clefts (NSOCs) are the most common craniofacial birth defects with complex etiology in which multiple genes and environmental exposures are involved. Bone morphogenetic protein 7 (BMP7), as a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to play crucial roles in palate and other orofacial ectodermal appendages development in animal models. MATERIAL AND METHODS: This study was designed to investigate the possible associations between BMP7 gene and the NSOCs (221 case-parent trios) in Western Han Chinese. Five tagSNPs at BMP7, rs12438, rs6099486, rs6127973, rs230188 and rs6025469 were picked and tried to cover the entire gene. In order to identify the contribution of BMP7 gene to the etiology of NSOCs, we performed several statistical analysis from different aspects including transmission disequilibrium test (TDT), pairwise linkage disequilibrium (LD), parent-of-origin effect and Chi-squared/Fisher's exact tests. RESULTS: Rs6127973 G allele and G/G homozygotes were over-transmitted for both NSOCs (P=0.005 and P=0.011, respectively) and NSCL/P (P=0.0061 and P=0.011, respectively), rs6127973 G allele was also paternally over-transmitted for both NSOCs (P=0.0061) and NSCL/P (P=0.011). CONCLUSIONS: This study suggested that rs6127973 may be a risk factor of being NSOCs and confirmed the role of BMP7 gene in orofacial deformity from Western Han Chinese, which will also supply scientific evidence for future research and genetic counseling.

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.

Short-term nasal symmetry repaired by force-balanced muscular reconstruction technique.

OBJECTIVES: This study aimed to compare the postoperative outcome of the new and classical muscular reconstruction technique combined with nasal internal-fixation method for secondary deformity post unilateral cleft lip repair. A rationale is provided for the further surgical improvement of secondary deformities. METHODS: Sixty patients aged 4-18 years with secondary unilateral cleft lip-nose deformity were involved in this research. The deformities of 28 patients were repaired using the muscular force balance technique through nasal internal fixation method, and 32 were repaired using classical muscular reconstruction technique. Two-dimensional analysis was used to evaluate the nose-lip morphology of pre- and post-operative patients through standardized photographs seven days after surgery. RESULTS: Compared with preoperative nasal morphology in the muscular force balance technique group, the 7-days postoperative results of this group showed the significantly improved short-term outcomes in the correction of columellar deflection, alar rim angle, nasal shape, and the symmetry of alar base width, nostril width, nostril height, alar rim angle (P<0.05). CONCLUSIONS: The new muscular reconstruction technique with nasal internal-fixation method has a significant effect on nasal repair.

[Palatal fistula rate after Huaxi Sommerlad-Furlow palatoplasty].

OBJECTIVE: This study aimed to determine the palatal fistula rate, explore the influencing factors of Huaxi Sommerlad-Furlow (SF) palatoplasty. METHODS: A retrospective review of 385 consecutive cleft-palate cases was performed to determine the incidence of postoperative fistula and assess the possible contributing factors, such as sex, weight, age, cleft type, operator skills, preoperative white blood cell, preventive antibiotic use, and postoperative temperature. RESULTS: Fistulas occurred in 15/385 patients (3.9%). Among them, 1 fistula was located at the junction of the hard and soft palates, 12 fistulas in hard palate, and 2 fistulas in alveolar near the hard palate. No evidence suggested that sex, weight, age, preoperative white blood cell, preventive antibiotic use, and postoperative temperature are associated with fistula formation. The incidences of cleft palate fistulas as encountered by senior professors (3.03%) and associate senior professors (2.23%) were significantly lower than those by attending doctors (14.29%, P<0.05). The incidences of cleft palate fistulas in bilateral completely cleft palate cases (20.6%) were significantly higher than those in hard and soft (3.6%) and unilateral cleft palate cases (2.6%, P<0.05). CONCLUSIONS: Huaxi SF palatoplasty can avoid the inhibited maxillary growth without requiring lateral relaxing incision, which poses an acceptable risk of fistula formation. The palatal fistula rate is not related to the sex, weight, age of operation, prophylactic use of antibiotics before operation, infection before operation, temperature after operation and other factors. The occurrence of the fistula is related mainly to cleft type and experience level of the surgeon.

VAX1 gene associated non-syndromic cleft lip with or without palate in Western Han Chinese.

OBJECTIVE: Non-syndromic cleft lip with or without palate (NSCL/P) is one of the most common human birth defects, it results from multiple genetic and environmental risk factors. Recently, GWA studies identified associations between NSCL/P and two genetic risk loci, rs7078160 and rs4752028, at VAX1. DESIGN: Currently, we tried to investigate the roles of the two loci among 302 NSCL/P trios (129 non-syndromic cleft lip only (NSCLO) trios and 173 non-syndromic cleft lip and cleft palate (NSCLP) trios) from Western Han Chinese. The two SNPs were genotyped by SNPscan method; Hardy-Weinberg equilibrium test, allelic TDT and parent-of-origin effect were performed by PLINK software, and genotypic TDT and haplotype by FBAT software. RESULTS: Allelic TDT analysis revealed allele A at rs7078160 was over-transmitted among NSCL/P group (P = 0.0086, ORtransmission = 1.36, 95%CI: 1.08-1.72). Parent-of-origin effect analysis revealed a paternal special over-transmission of allele A at rs708260 in NSCL/P group (P = 0.0079). Haplotype AC of rs7078160-rs4752028 was significant over-transmitted in the NSCL/P group. CONCLUSIONS: Our study firstly confirmed that allele A at rs7078160 at VAX1 gene was a risk factor for NSCL/P in Western Han Chinese population.

Family-based study of association between MAFB gene polymorphisms and NSCL/P among Western Han Chinese population.

BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) are the most common human congenital birth defects with a complex etiology. MAFB has been reported as a candidate gene involved in the pathogenesis of NSCL/P from genome-wide association study (GWAS) findings, and no replication studies have been performed in Western Han Chinese. OBJECTIVES: The aim of this study was to investigate the associations of MAFB among NSCL/P trios in Western Han Chinese. MATERIAL AND METHODS: We selected 6 single nucleotide polymorphisms (SNPs) (rs6072081, rs6065259, rs17820943, rs13041247, rs11698025 and rs6102085) near MAFB based on previous GWAS findings and recruited 298 case-parents trios with NSCL/P from Western Han Chinese population, while genotypes were done by SNPscan technology. RESULTS: Strong evidence of an association was found at rs17820943 (p = 0.0023; odds ratio - ORtranmission = 0.7 and 95% confidence interval [CI]: 0.55-0.88) and rs13041247 (p = 0.0023; ORtranmission = 0.7 and 95% CI: 0.55-0.88) among NSCL/P; genotypic transmission-disequilibrium test (TDT) analysis further confirmed this. C/C homozygote at rs17820943 (z = 3.44 and p = 0.00058) and T/T homozygote at rs13041247 (z = 3.14 and p = 0.0017) was over-transmitted among NSCL/P, which indicated they could increase the risk of having an affected baby. Sliding window haplotype analysis showed that haplotypes consisting of C allele at rs17820943 and T allele at rs13041247 were still over-transmitted among NSCL/P (lowest p = 0.0021). CONCLUSIONS: This study further confirmed that the targeted SNPs at MAFB were associated with NSCL/P trios from Western Han Chinese population, which provides more scientific evidence for the future research and genetic counseling.

Rs12941170 at SOX9 gene associated with orofacial clefts in Chinese.

OBJECTIVE: Non-syndromic orofacial cleftings (NSOCs) are considered as complex trait, which results from genetic and/or environmental modifiers. Current findings could only explain small portion of the NSOCs. SOX9 gene plays an important role during craniofacial development in animal models and the Pierre Robin sequence (PRS). However, its role in non-syndromic clefts remains unknown. DESIGN: In this study, we selected eight SNPs in and around SOX9 gene to make maximum coverage, and genotyped them by using RFLP-PCR and ligase detection reaction (LDR) methods to test its associations among 151 NSOCs (53 NSCLP, 52 NSCLO and 46 NSCPO) from Western Han Chinese population. RESULTS: Allelic TDT results showed that G allele at rs12941170 of SOX9 was under-transmitted among NSOCs (p=0.00014, OR=0.55 and 95%CI: 0.40-0.75), which could indicate that the G allele is protective against NSOCs; parent-of-origin effect analysis showed that G allele at rs12941170 was maternally under-transmitted (p=0.002), while there was no statistically difference between the maternal and paternal transmission of it. To test if the adjacent SNPs travel together from parents to the affected individual, we carried out the sliding window haplotype analysis, it is interesting to find that the haplotypes carrying the G allele at rs12941170 also was under-transmitted for NSOCs, NSCL/P, NSCLP and NSCPO (lowest p=0.00033). CONCLUSIONS: This study suggested that G allele at rs12941170 was protective, which could decrease the risk for NSOCs from Western Han Chinese population, and it will provide new reference for future research and genetic counseling in NSOCs.