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.     

非综合征性唇腭裂候选基因的研究进展

非综合征性唇腭裂是人类常见的先天性畸形之一,分为非综合征性唇裂或伴腭裂和单纯性腭裂,是一种多基因多因素遗传性疾病,常见的非综合征性唇腭裂相关性候选基因有干扰素调节因子6、亚甲基四氢叶酸还原酶、转化生长因子、肌节同源盒基因1和视黄酸受体仅等。本文就最常见的候选基因位点与非综合征性唇腭亵关系的研究进展作一综述。     

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.     

Investigation of MYH14 as a candidate gene in cleft lip with or without cleft palate

Clefts of the orofacial region are among the most common facial defects and are caused by abnormal facial development during gestation. Cleft lip with or without cleft palate (CL/P) is a birth defect with a complex etiology resulting from a mixture of genetic and environmental factors. In the present study we considered myosin 14 ( MYH14 ) as a candidate gene for CL/P. This gene codes for the heavy chain of non-muscle myosin IIC (NMMHC-IIC), maps in the OFC3 region, and shares significant homology with myosin 9, a gene that our group has recently seen to be involved in CL/P. A linkage disequilibrium investigation was conducted with six single nucleotide polymorphisms in MYH14 and a sample of 239 CL/P nonsyndromic patients and their parents. Our family-based investigation provided no evidence of association between MYH14 and CL/P alleles. These data do not support the involvement of MYH14 in CL/P among the Italian population.     

染色体基因位点与非综合征性唇腭裂致病机制的关系

非综合征性唇腭裂是人类最常见的先天性畸形之一,是一种多基因多因素的遗传疾病.近二十年来,学者们先后在多条染色体上发现了与非综合征性唇腭裂有关的基因位点.本文就染色体基因位点与非综合征性唇腭裂致病机制的关系作一综述.     

非综合征性唇腭裂与MSX1基因传递不平衡研究

目的探讨MSX1基因与湖南汉族人群非综合征性唇腭裂（nonsyndromic cleft lip and palate，NSCLP）遗传易感性的关系。方法以MSX1基因内含子区的CA重复微卫星作为遗传标记，采用聚合酶链式反应（polymerase chain reaction，PCR）-变性聚丙烯酰胺凝胶（polyacrylamide gel electrophoresis，PAGE）基因分型技术对湖南汉族129个NSCLP核心家系387名成员进行基因型分析，并行传递不平衡检验（transmission disequilibrium test，TDT）及Logistic回归分析。结果TDT分析显示，MSX1基因CA4等位基因在唇裂伴（不伴）腭裂（cleft lip with or without palate，CL／P）和单纯性腭裂（cleft palate only，CPO）组均被优势传递给患病后代（P=0．018，P=0．041）。Logistic回归分析结果支持隐性遗传模式，CA4本身或其作为一致病基因的遗传标志以隐性遗传模式被遗传（P=0．009）。结论MSX1基因与湖南汉族人群NSCLP相关联，可能是其易感基因或与之存在连锁不平衡。     

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.     

Association study of single nucleotide polymorphisms of MAFB with non-syndromic cleft lip with or without cleft palate in a population in Heilongjiang Province,northern China

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common complex birth defect. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a new gene that may be involved in susceptibility to cleft lip with or without cleft palate (CL/P). To further assess its role in NSCLP, we investigated 3 identified single nucleotide polymorphisms in MAFB (rs13041247, rs6065259, and rs11696257) and examined them for association with NSCLP in 344 patients and 324 healthy controls in a northern Chinese Han population with a high incidence of the syndrome. Peripheral blood samples were taken when patients enrolled in the study and DNA samples were extracted from the blood. The 3 single nucleotide polymorphisms were genotyped using a mini-sequencing method (Snapshot^® Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK). We found that rs6065259 was the most important single nucleotide polymorphism in MAFB (OR_6065259-AA = 0.45; 95% CI: 0.28 to 0.71; p = 0.0027), followed by rs13041247; however, no association was found between rs11696257 and NSCLP. Our study provides further evidence regarding the role of MAFB variations in the development of NSCLP in this northern Chinese Han population.     

No evidence for linkage and association between 4q microsatellite markers and nonsyndromic cleft lip and palate in chilean case-parents trios.

OBJECTIVE: Nonsyndromic cleft lip/palate (NSCLP) has the characteristics of a complex genetic trait. Linkage and association studies have suggested that one or more clefting loci may be located on chromosome 4q. The goal of this study was to evaluate the possible linkage and association due to linkage disequilibrium between five microsatellite markers located on 4q28 to 4q33 and NSCLP, using the case-parent trio design. SUBJECTS AND METHODS: A total of 56 Chilean families (32 simplex and 24 multiplex) were recruited. Microsatellite markers were analyzed using polymerase chain reaction with fluorescent-labeled forward primers, followed by electrophoresis on a laser-fluorescent sequencer. Case-parents trios were ascertained to assess linkage and linkage disequilibrium through a multistage procedure. Transmission disequilibrium tests for multiple alleles were carried out to assess the statistical significance of 4q28 to 4q33 microsatellite markers. RESULTS: Only weak evidence for linkage was obtained for the FGA marker (asymptotic uncorrected p value = .08 and empirical p value = .05). Only the FGA and UCP1 markers were selected for association analysis in trios, with unrelated cases achieving a nearly significant result for the UCP1 marker (asymptotic uncorrected p value = .07 and empirical p value = .19). CONCLUSION: Though the FGA and UCP1 markers showed nearly significant p values for linkage and association, respectively, the results of the present study provided insufficient evidence of the existence of a major susceptibility locus in the 4q region that was analyzed in the present study.     

Linkage disequilibrium between MSX1 and non-syndromic cleft lip/palate in the Chilean population

Non-syndromic cleft lip/palate (NSCLP) is a complex genetic trait. Linkage and association studies have suggested that a clefting locus could be located on chromosome 4p. Sixty Chilean families were recruited for this study; from these, we used unrelated trios to evaluate the possible linkage disequilibrium between MSX1 and NSCLP. An intragenic marker, MSX1-CA, and an extragenic marker, D4S432 at a distance of 0.8 cM from MSX1, were analyzed by means of polymerase chain-reaction with fluorescent-labeled forward primers, followed by electrophoresis on a laser-fluorescent sequencer. We carried out a transmission/disequilibrium test (TDT) for multiple alleles to evaluate the presence of linkage disequilibrium. Results showed a preferential transmission of the 169-bp allele of MSX1 (p = 0.03). Although there was no preferential transmission for the D4S432 marker, the overall extended TDT (ETDT) showed a significant result (p = 0.01). The authors' findings support the hypothesis of the contribution of MSX1 in the etiology of NSCLP in the Chilean population.     

Association of Wnt3A gene variants with non-syndromic cleft lip with or without cleft palate in Chinese population

Objective

Non-syndromic cleft lip with or without cleft palate (NSCLP) is one of the most common birth defects all over the world. Both genetic and environmental factors may contribute to NSCLP. Recent studies have demonstrated that Wnt/β-catenin signalling pathway is required for lip and palate formation. WNT family may play an important role in the development of NSCLP. This study aimed to evaluate the association between Wnt3A gene polymorphisms and NSCLP in Chinese population from Northwest China.

Design

216 patients with NSCLP and 233 normal controls were genotyped for two SNPs of Wnt3A by PCR-RFLP. Both SNPs genotype frequencies were analysed between cases group and controls group.

Results

The frequencies of rs752107 TT and rs3121310 AA were significantly higher in NSCLP cases group (7.4%, 15.3%) than that in controls group (2.1%, 9.5%) with p-value = 0.013, 0.014, corrected p value (p-corr) <0.05 and with odds ratio (OR) = 3.49, 95% confidence interval [CI]: 1.244–9.79, OR = 2.27, 95% CI: 1.17–4.38, respectively; the frequency of rs3121310 GA was also higher in NSCLP cases group (57.4%) than in controls group (52.0%) with p-value = 0.042 and OR = 1.56 (95% CI: 1.02–2.39). And the frequency of rs752107 TT of Wnt3A showed higher risk in female patients, while the frequency of A allele of rs3121310 showed stronger association in male patients.

Conclusions

This is the first report that two SNPs of Wnt3A (rs752107 and rs3121310) are significantly associated with NSCLP in Chinese population. These findings provide a context for understanding the genetic aetiology of NSCLP.     

Further evidence for the involvement of MYH9 in the etiology of 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 birth defects and has a multifactorial etiology that includes both genetic and environmental components. MYH9 , the gene coding for the heavy chain of non-muscle myosin II, has been considered as a good candidate gene in NSCL/P on the basis of its expression profile during craniofacial morphogenesis. Reports in an Italian sample, as well as in an ethnically mixed North American sample, of a positive association between single-nucleotide polymorphisms in the MYH9 gene and NSCL/P have provided further support for the role of MYH9 in the development of NSCL/P. In the present study, we aimed to replicate these findings by conducting a family-based association study with seven single nucleotide polymorphisms in MYH9 using a sample of 248 NSCL/P patients and their parents. Single marker analysis resulted in a highly significant association for rs7078. In haplotype analysis, the most significant result was obtained for the SNP combination (rs7078; rs2071731; rs739097; rs5995288). Our results thus confirm the potential involvement of MYH9 in the etiology of NSCL/P in our patients of Central European origin, although further studies are warranted to determine its exact pathogenetic role.     

Association between BMP4 gene polymorphisms and cleft lip with or without cleft palate in a population from South China

ObjectivePrevious studies have suggested an association between several polymorphisms of the BMP4 gene and susceptibility to non-syndromic cleft lip with or without cleft palate (NSCL/P) in various populations. However, this association may vary according to ethnic group and the form of NSCL/P. This study analyzed the association between the BMP4 gene polymorphisms rs762642, rs17563, and rs10130587 with the risk of cleft lip only (CLO), cleft palate only (CPO), and cleft lip with palate (CLP) in a population from South China.MethodsThis case-control study included 165 patients with NSCL/P (53 patients with CPO, 52 with CLO, and 60 with CLP) and 52 healthy volunteers. Peripheral blood samples were collected from all subjects to genotype the rs762642, rs17563, and rs10130587 polymorphisms by direct sequencing. Genotype and allelic frequencies of these polymorphisms were compared between healthy volunteers and patients with various forms of NSCL/P.ResultsThe genotype and allelic frequencies of rs762642 differed significantly between subgroups (CPO and CLP) and normal controls, whereas a significant difference was observed only in the CLO subgroup for the rs17563 polymorphism and in the CLO and CLP groups for the rs10130587 polymorphism. In addition, we identified a novel association of a BMP4 gene polymorphism, which was in linkage disequilibrium with the rs10130587 polymorphism, with CLO and CLP.ConclusionThe BMP4 gene polymorphisms rs762642, rs17563, and rs10130587 exhibit different associations with different forms of NSCL/P, suggesting that different forms of NSCL/P may have different etiologies.     

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.     

Nonsyndromic cleft lip with or without cleft palate in China: assessment of candidate regions.

OBJECTIVE: Although Asians have the highest birth prevalence of oral-facial clefts, the majority of gene mapping studies of cleft lip with or without cleft palate (CL/P) have been in European or American Caucasians. Therefore, the objective of this study of Chinese families was to evaluate linkage and association between CL/P and 10 genetic markers in five chromosomal regions that have shown positive results in Caucasians. SETTING: Families were ascertained through nonsyndromic CL/P surgical probands from hospitals throughout Shanghai, China. PARTICIPANTS: Study participants included 671 individuals from 60 families with two or more members affected with oral-facial clefts. Of the 671 total individuals, 145 were affected. RESULTS: Ten markers from chromosomes 2, 4, 6, 17, and 19 were assessed (TGFA, MSX1, D4S194, D4S175, F13A1, GATA185H, D17S250, D17S579, D19S49, APOC2). LOD scores were calculated between each of the 10 markers and CL/P as well as model-free statistics of linkage (SimIBD) and association (TDT). None of the markers showed significantly positive LOD scores with CL/P. A significantly positive result (p =.01) was seen using SimIBD for APOC2 on chromosome 19, and a positive TDT result (p =.004) was obtained for D19S49, near APOC2. CONCLUSIONS: This is the first gene mapping study of CL/P in China. These results indicate that most of the genetic regions with positive results in Caucasian families may not be involved in CL/P found in China, although there is some positive evidence for the candidate region on chromosome 19.     

Genetic variation of IRF6 and TGFA genes in an HIV‐exposed newborn with non‐syndromic cleft lip palate

We present here the first reported case of a non‐syndromic cleft lip and palate (NSCLP) in an HIV‐exposed newborn of a mother on antiretroviral therapy (ART) in Indonesia. Genetic testing was performed to confirm a suspected genetic condition. Genomic DNA was extracted from the blood, and genetic variations of the interferon regulatory factor 6 (IRF6) rs642961 (Mspl) (G>A) and transforming growth factor alpha (TGFA) BamHI (rs11466297, A>C) and RsaI (rs3732248, C>T) were performed by PCR‐RFLP and IRF6 gene analysis by PCR sequencing. Genotyping of DNA sequence variants in the IRF6 gene showed both parents had genotype GA, while the child had genotype GG (genotype wild type). There was no difference observed in the TGFA BamHI gene variant between the child and her mother and father that were wild‐type polymorphisms (normal), while the Rsa1 polymorphisms of them were heterozygotes. A genetic variant of IRF6 might be a protective factor for NSCLP, while Rsa1 gene variant (A) allele can be considered to be the risk factor associated with NSCLP development. This case report also highlights the possible etiologic role of ART in NSCLP; therefore, early control of adverse effects of ART might be an important factor in decreasing the incidence of the congenital anomalies in HIV‐infected children.     

Chromosome 17: gene mapping studies of cleft lip with or without cleft palate in Chinese families.

OBJECTIVE: Involvement of loci on chromosome 17, including retinoic acid receptor alpha (RARA) in nonsyndromic oral clefts has been reported in Caucasian populations, although never investigated in Asian populations. The purpose of the present study was to investigate several loci on chromosome 17, including RARA, in Chinese families. PARTICIPANTS: Thirty-six multiplex families (310 individuals), ascertained through nonsyndromic cleft lip with or without cleft palate surgical probands from hospitals in Shanghai, China, participated in the present study. There were 23 families whose probands had cleft lip and cleft palate (CLP) and 13 with cleft lip alone (CL). RESULTS: Seventeen markers, spanning chromosome 17 and about 10 cM apart were assessed. Logarithm of odds ratio (LOD) scores (two point and multipoint), model-free linkage analyses, and allelic association tests (transmission/disequilibrium, Fisher's exact tests, and chi-square) were performed on the total family sample, families with CLP probands (CLP subgroup), and families with CL probands (CL subgroup). LOD scores from the two-point analyses were inconclusive. Multipoint analyses rejected linkage except for a few regions in the CL subgroup. However, positive results were found using the model-free linkage and association methods (p < .05). The markers with positive results varied across the CL and CLP subgroups. However, the RARA region and loci nearby yielded consistently positive results. CONCLUSION: Genetic variation within the RARA locus or nearby appears to be involved in the pathogenesis of nonsyndromic oral clefts in this population. Furthermore, based on the differing pattern of results in the CL versus CLP subgroups, it appears that the formation of CL and CLP is because of either differing alleles at the same genetic locus or different but related (and/or linked) genes that modify the severity and expression of oral clefting.     

RFC1 and non-syndromic cleft lip with or without cleft palate: An association based study in Italy

The molecular basis of orofacial development is largely unknown and needs to be unravelled. Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial malformation, with an incidence of about 1/700 live births, although variable according to ethnicity. Being a multifactorial disease, it arises as a result of an interplay between genetic and environmental factors. Several approaches have been developed to identify susceptibility genes. Genes belonging to the folate/homocysteine pathway are attracting increasing interest because folate supplementation before and during early pregnancy can reduce the risk of NSCL/P. We performed a family based association study in order to assess if a genetic variant of RFC1 could be involved in NSCL/P onset.We genotyped 404 unrelated probands and their relatives for three biallelic polymorphic variants (rs1051266, rs4818789 and rs3788205), that were selected because they produced conflicting results on previous investigations.Evidence of association was found between the investigated polymorphisms and NSCL/P in our sample of the Italian population, albeit with weak significance levels.Results from this investigation provided a support of previous studies suggesting a role of RFC1 in NSCL/P aetiology, reinforcing the concept that genetic predisposition to NSCL/P varies enormously within different ethnic groups.     

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

ObjectiveNon-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.DesignCurrently, 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.ResultsAllelic TDT analysis revealed allele A at rs7078160 was over-transmitted among NSCL/P group (P = 0.0086, OR_transmission = 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.ConclusionsOur study firstly confirmed that allele A at rs7078160 at VAX1 gene was a risk factor for NSCL/P in Western Han Chinese 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.