New evidence for the role of cystathionine beta-synthase in non-syndromic cleft lip with or without cleft palate

Orofacial clefts have a multifactorial aetiology encompassing both genetic and environmental components. While there is wide agreement on the importance of both genetic and nutritional factors, genetic influence in particular has not been well defined. As genetic variants in folate and homocysteine metabolism have been reported to influence the risk of orofacial clefts, an Italian cleft lip with or without cleft palate (CL/P) data set was enrolled for an analysis based on family association to test betaine-homocysteine methyltransferase (BHMT and BHMT2) and cystathionine beta-synthase (CBS) variants. No significant level of association was found between BHMT and BHMT2 variants, while evidence of an allelic association with CL/P was found for the single nucleotide polymorphism rs4920037, mapping at the CBS gene. A log-linear approach indicated that the best genetic model takes into account both mother and child genotypes. This suggests that human orofacial development is influenced by CBS genotypes that possibly operate through intergenerational fetal-maternal interaction.     

Maternal malnutrition, environmental exposure during pregnancy and the risk of non-syndromic orofacial clefts

Oral Diseases (2011) 17 , 584–589 Objective: To explore the risk factors of non‐syndromic orofacial clefts. Subjects and Methods: A case–control study was conducted in China, 537 infants born with non‐syndromic cleft lip with/without cleft palate, 176 infants born with cleft palate (CP), and 221 normal controls were recruited to participate in a questionnaire based study to identify risk factors related to maternal nutrition. Results: Single‐factor Chi‐square analysis identified 12 factors as significantly related to non‐syndromic orofacial clefts (P < 0.05). Multiple logistic regression showed five of these factors were associated with non‐syndromic orofacial clefts, male gender and maternal passive smoking during early pregnancy were risk factors for non‐syndromic orofacial clefts (OR = 1.86 and 11.42; 95%CI: 2.28–2.69 and 6.87–19.00, respectively), whereas maternal weight gain during pregnancy and folic acid supplementation during early pregnancy were protective (OR = 0.15 and 0.67; 95%CI: 0.034–0.63 and 0.44–1.00, respectively). Conclusions: Our data may provide references for cleft lip and CP prevention programs, and counseling programs in China.     

MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate

Jagomägi T, Nikopensius T, Krjut?kov K, Tammekivi V, Viltrop T, Saag M, Metspalu A. MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci 2010; 118: 213–220. © 2010 The Authors. Journal compilation©2010 Eur J Oral Sci Recent studies suggest that multiple interacting loci, with possible additional environmental factors, influence the risk for nonsyndromic oral clefts, one of the most common birth defects in humans. Advances in high‐throughput genotyping technology allow the testing of multiple markers, simultaneously, in many candidate genes. We tested for associations between 176 haplotype‐tagging single nucleotide polymorphisms (SNPs) in 18 candidate genes/loci and nonsyndromic clefts in a case–control study in an Estonian sample (153 patients, 205 controls). The most significant associations with nonsyndromic cleft lip with or without cleft palate (CL/P) were found for SNPs in MSX1, MTHFR, and PVRL2, including several common haplotypes in the MTHFR and MSX1 genes. The strongest association was observed for rs6446693 in the MSX1 region, which remained statistically significant after Bonferroni correction. The strongest association with nonsyndromic cleft palate (CP) was found for the SNP rs11624283 in the JAG2 gene. Epistatic interactions were observed for SNPs within PVRL2, between BCL3 and EDN1, and between IRF6 and MSX1 genes. This study provides further evidence implicating MSX1 and MTHFR in the etiology of nonsyndromic CL/P across different populations.     

CRISPLD2 polymorphisms are associated with non‐syndromic cleft lip with or without cleft palate in a northern Chinese population

Shi J, Jiao X, Song T, Zhang B, Qin C, Cao F. CRISPLD2 polymorphisms are associated with non‐syndromic cleft lip with or without cleft palate in a northern Chinese population. Eur J Oral Sci 2010; 118: 430–433. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Non‐syndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect. This complex genetic disorder results from interactions between genes and environmental factors. Numerous genes have been reported in studies demonstrating association between the cleft lip and palate phenotypes and the alleles at single‐nucleotide polymorphisms (SNPs) within specific genes. Recently, the cysteine‐rich secretory protein LCCL domain containing 2 (CRISPLD2) has been revealed to be a novel candidate gene for NSCLP. The SNPs rs1546124, rs4783099 and rs16974880 in CRISPLD2 were highly significant in Caucasian and Hispanic multiplex families but showed no association in Colombian and Irish populations. In the current study, we examined these three SNPs in a northern Chinese population and found an association between these polymorphisms and NSCLP in both single‐marker and haplotype analyses. Our data further strengthen the conclusion that altered CRISPLD2 is associated with NSCLP susceptibility.     

Susceptibility locus for non‐syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients

Nikopensius T, Birnbaum S, Ludwig KU, Jagomägi T, Saag M, Herms S, Knapp M, Hoffmann P, Nöthen MM, Metspalu A, Mangold E. Susceptibility locus for non‐syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients. Eur J Oral Sci 2010; 118: 317–319. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental factors. Recently, two novel susceptibility loci and three suggestive loci for NSCL/P were identified by a genome‐wide association scan (GWAS) in a German population with subsequent independent replication in a mixed European population. The aim of the present study was to investigate whether these newly detected loci confer similar effects in the North‐East European Baltic population. A total of 101 NSCL/P patients and 254 controls from Estonia were included. A significant association was observed for rs7078160 (P = 0.0016) at chromosome 10q25, which confirms the association of this locus with NSCL/P in the Baltic population. No significant association was found for the other four loci, a result that may have been attributable to the limited power of the sample.     

Genetic and environmental risk factors for submucous cleft palate

A multifactorial aetiology with genetic and environmental factors is assumed for orofacial clefts. Submucous cleft palate (SMCP), a subgroup of cleft palates with insufficient median fusion of the muscles of the soft palate hidden under the mucosa, has a prevalence of 1:1,250-1:5,000. We described the prevalence of risk factors among 103 German patients with the subtype SMCP and genotyped 24 single nucleotide polymorphisms (SNPs) from 12 candidate genes for orofacial clefts. Analysis of risk factors yielded a positive history for maternal cigarette smoking during pregnancy in 25.2% of the patients, and this was significantly more frequent than in the normal population. The group of patients differed in allele frequencies at SNP rs3917192 of the gene TGFB3 (nominal P = 0.053) and at SNP rs5752638 of the gene MN1 (nominal P = 0.075) compared with 279 control individuals. Our results indicate a potential role of maternal smoking during pregnancy for the formation of SMCP. The analysis of genetic variants hints at the contribution of TGFB3 and MN1 in the aetiology of SMCPs.     

Genotype and haplotype analysis of WNT genes in non-syndromic cleft lip with or without cleft palate

The wingless-type MMTV integration site family (Wnt) signalling pathway plays a crucial role in craniofacial development. Recently, nucleotide variants in WNT genes have been shown to be associated with oral congenital anomalies, including facial clefts. Therefore, in the current study we decided to assay the association of nucleotide variants in selected WNT genes with the risk of non-syndromic cleft lip with or without cleft palate (NCL/P) in the Polish population. Fourteen polymorphisms in WNT3, WNT3A, WNT5A, WNT8A, WNT9B, and WNT11 were tested in a group of 210 patients with NCL/P and in a properly matched control group. The most significant results were found for the WNT3 rs3809857 variant, which, under the assumption of a recessive model, was associated with a two-fold decrease in the risk of NCL/P (OR(TT vs. GT + GG) = 0.492, 95% CI: 0.276-0.879, P = 0.015). Moreover, haplotype analysis revealed that WNT3 is significantly correlated with NCL/P. The global P-values for haplotypes of rs12452064_rs7207916 and rs3809857_rs12452064_rs7207916 were 0.0034 and 0.0014, respectively, and these results were statistically significant, even after the permutation test correction. In conclusion, our study confirmed the involvement of polymorphisms in the WNT3 gene in NCL/P aetiology in the tested population.     

PRDM16基因及母亲孕期环境暴露因素与非综合征型唇腭裂相关性研究

目的 探究PRDM16基因rs7525173、rs2236518、rs2493264及母亲孕早期吸烟饮酒与非综合征型唇腭裂（NSCL/P）发生的相关性。方法 收集157个患者-父母核心家系,采用连接酶检测反应（LDR）和直接测序两种方法进行基因分型,使用传递不平衡检验（TDT）、连锁不平衡检验（LD）等对数据进行统计分析。收集1 710例唇腭裂患者及956例健康新生儿,填写《唇腭裂患者流行病学调查问卷》,对孕期父母吸烟饮酒暴露因素进行分析。结果 rs2236518位点C等位基因在腭裂组中存在过传递（P<0.05）,其余位点在各组中均无明显统计学意义。母亲吸烟、母亲被动吸烟及母亲饮酒3个因素存在统计学差异（P<0.05）。结论 PRDM16基因rs2236518多态性与NSCL/P存在相关性,母亲吸烟、母亲被动吸烟及母亲饮酒与唇腭裂的发生存在密切联系。     

Genes causing clefting syndromes as candidates for non‐syndromic cleft lip with or without cleft palate: a family‐based association study

Clefts of the orofacial region are among the most common congenital defects, caused by abnormal facial development during gestation. Non‐syndromic cleft lip with or without cleft palate (NSCLP) is a complex trait most probably caused by multiple interacting loci, with possible additional environmental factors. As facial clefts form part of more than 300 syndromes, one strategy for identifying the genetic causes of NSCLP could be to study candidate genes responsible for clefting syndromes. Three genes were selected for this investigation: TP63, which codes for the tumour protein p63 and causes Ectrodactyly‐Ectodermal dysplasia‐orofacial Cleft syndrome; JAG2, a downstream gene of TP63; and MID1, which is responsible for Opitz syndrome. A linkage disequilibrium investigation was performed with intragenic single nucleotide polymorphisms on each of these genes in a sample study of 239 patients/parents trios. Evidence which suggests that JAG2 and MID1 may play a role in NSCLP was obtained.     

Studies of genes involved in craniofacial development and tumorigenesis: FGF3 contributes to isolated oral clefts and may interact with PAX9

Objective. Previous studies suggest individuals born with oral clefts and their families have a higher susceptibility for cancer, which raises the hypothesis that these two conditions share common molecular pathways. This study evaluated the association between oral clefts and polymorphisms in genes that play a role in craniofacial and tumor development. Materials and methods. Four hundred and ninety-seven subjects born with oral clefts and 823 unaffected subjects were recruited. Twenty-nine markers in 13 genes were genotyped by the Taqman method. Chi-square was used to compare allele and genotype frequencies. Bonferroni correction for multiple testing was used and the established alpha was 0.0003. This study also used logistic regression to test if genetic variants were associated with oral clefts using positive family history of cancer and age as covariates. Results. There was no association between family history of cancer and oral clefts (p = 0.51). None of the 1320 study participants had a diagnosis of cancer at the time of participation in the study. The marker rs4980700 in FGF3 was associated with oral clefts (p = 0.0002). Logistic regression analysis also provided evidence for gene–gene interaction between FGF3 (rs4980700) and PAX9 (rs2073242), increasing the risk for isolated oral clefts (p = 0.0003). Conclusion. FGF3 is associated with oral clefts and may interact with PAX9.     

Family‐based genome‐wide association study in Patagonia confirms the association of the DMD locus and cleft lip and palate

The etiology of cleft lip with or without cleft palate (CL±P) is complex and heterogeneous, and multiple genetic and environmental factors are involved. Some candidate genes reported to be associated with oral clefts are located on the X chromosome. At least three genes causing X‐linked syndromes [midline 1 (MID1), oral‐facial‐digital syndrome 1 (OFD1), and dystrophin (DMD)] were previously found to be associated with isolated CL±P. We attempted to confirm the role of X‐linked genes in the etiology of isolated CL±P in a South American population through a family‐based genome‐wide scan. We studied 27 affected children and their mothers, from 26 families, in a Patagonian population with a high prevalence of CL±P. We conducted an exploratory analysis of the X chromosome to identify candidate regions associated with CL±P. Four genomic segments were identified, two of which showed a statistically significant association with CL±P. One is an 11‐kb region of Xp21.1 containing the DMD gene, and the other is an intergenic region (8.7 kb; Xp11.4). Our results are consistent with recent data on the involvement of the DMD gene in the etiology of CL±P. The MID1 and OFD1 genes were not included in the four potential CL±P‐associated X‐chromosome genomic segments.     

Lack of association between IRF6 polymorphisms (rs2235371 and rs642961) and non‐syndromic cleft lip and/or palate in a Brazilian population

Oral Diseases (2010) 16 , 193–197 Background: Interferon regulatory factor 6 (IRF6) gene has emerged as a potential susceptibility gene for non‐syndromic cleft lip and/or palate (NSCL/P) in different populations. The aim of this study was to determine the association of IRF6 rs2235371 and rs642961 polymorphisms with NSCL/P in a Brazilian population. Methods: Two hundred and twenty‐eight patients affected by NSCL/P and 126 healthy individuals were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay. Results: Overall genotype distributions of rs2235371 and rs642961 polymorphisms were as expected by Hardy‐Weinberg equilibrium test. The rs2235371 polymorphic genotype GA was identified in 10.1% of the patients with NSCL/P and in 10.3% of the control group, revealing no statistical difference. Similarly, the frequency of rs642961 minor genotypes (GA and AA) was quite similar between control group (28.6%) and NSCL/P group (25.4%), without significant difference. Conclusion: Our findings are consistent with a lack of involvement of IRF6 rs2235371 and rs642961 polymorphisms in the NSCL/P pathogenesis in the Brazilian population.     

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. Key words: Single nucleotide polymorphisms, nonsyndromic orofacial clefts, BMP7.     

Replication of two novel susceptibility loci for non‐syndromic orofacial clefts in a Chinese population

Oral Diseases (2011) 17 , 304–308 Objectives: Non‐syndromic orofacial clefts (NSOC) are the most common developmental disorders in human beings. Recently, two genome‐wide association studies in European Caucasians identified three novel NSOC susceptibility loci: rs987525 on 8q24, rs7078160 on 10q25.3, and rs223371 on 17q22. The aim of this study was to determine the association of these polymorphisms with NSOC susceptibility and its subgroups in a Chinese Han population. Material and methods: In this study, 199 NSOC patients and 210 healthy individuals were recruited. SNP rs987525 was not genotyped because of its low frequency in the study subjects. The other two polymorphisms (rs7078160 and rs223371) were respectively genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and Taqman‐MGB assay. Results: Overall genotype distributions of rs7078160 and rs223371 polymorphisms were consistent with Hardy–Weinberg equilibrium test. The allele and genotype frequencies of the two polymorphisms were not significantly different between cases and controls. Further analysis indicated that none of the genotypes was associated with increased risk of NSOC. Similar results were also found when all cleft cases were stratified by cleft types. Conclusion: Our findings are consistent with a lack of involvement of the rs7078160 and rs223371 polymorphisms in the development of NSOC in the Chinese Han population.     

IRF6 gene variants in Central European patients with non‐syndromic cleft lip with or without cleft palate

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non‐syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case–control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non‐synonymous coding variant V274I (rs2235371) and five IRF6‐haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 × 10^?6) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38–2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21–3.10) for the homozygous genotype, values that are similar to those reported in a previously published family‐based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP‐based and resequencing studies using large samples of patients.     

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.     

Evaluating SKI as a candidate gene for non‐syndromic cleft lip with or without cleft palate

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common of all congenital malformations and has a multifactorial etiology. Findings in mice suggest that the v‐ski sarcoma viral oncogene homolog (SKI) gene is a candidate gene for orofacial clefting. In humans, a significant association between rs2843159 within SKI and NSCL/P has been reported in patients from the Philippines and South America. In the South American patients, the association was driven by the subgroup of patients with non‐syndromic cleft lip only (NSCLO). Here we investigated the association with rs2843159 in a Mayan Mesoamerican population (172 NSCL/P patients and 366 controls). In addition, we analyzed the phenotypic subgroups NSCLO and non‐syndromic cleft of lip and palate (NSCLP). A trend towards association between rs2843159 and NSCL/P was observed in the Mayan cohort (P = 0.097), and we found a stronger association in the NSCLP subgroup (P = 0.072) despite a limited sample size. To investigate whether other common variants within the SKI gene contribute to NSCL/P susceptibility in European and Asian populations, we also analyzed genotypic data from two recent genome‐wide association studies using set‐based statistical approaches. These analyses detected a trend toward association in the European population. Our data provide limited support for the hypothesis that common SKI variants are susceptibility factors for NSCL/P.