Nonsyndromic Cleft Lip With Or Without Cleft Palate Research Articles

MTHFR rs2274976 polymorphism is a risk marker for nonsyndromic cleft lip with or without cleft palate in the Brazilian population

BackgroundPolymorphisms within the MTHFR (rs2274976) and MTHFD1 (rs2236225) genes were previously associated with maternal susceptibility for having an offspring with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in the Brazilian population. However, as the genotypes of the patients with NSCL/P were not evaluated, it is not clear whether the effects are associated with maternal or offspring genotypes. The aim of this study was to evaluate the association of rs2274976 and rs2236225 in the pathogenesis of NSCL/P.MethodsBy using the TaqMan 5′‐exonuclease allelic discrimination assay, the present study genotyped the rs2274976 and rs2236225 polymorphisms in 147 case–parent trios, 181 isolated samples of NSCL/P and 478 healthy controls of the Brazilian population. Transmission disequilibrium test and structured case–control analysis based on the individual ancestry proportions were performed.ResultsThe transmission disequilibrium test showed a significant overtransmission of the rs2274976 A allele (p = 0.004), but no preferential parent‐of‐origin transmission was detected. The structured case–control analysis supported those findings, revealing that the minor A allele of rs2274976 was significantly more frequent in NSCL/P group compared with control group (p = 0.001), yielding an odds ratio of 3.46 (95% confidence interval, 2.05–5.85). No association of rs2236225 polymorphism with NSCL/P was observed in both transmission disequilibrium test and case–control analysis.ConclusionThe results of the study revealed that the presence of the rs2274976 A allele is a risk marker for the development of NSCL/P in the Brazilian population. Birth Defects Research (Part A) 100:30–35, 2014. © 2013 Wiley Periodicals, Inc.

Analysis of susceptibility loci for nonsyndromic orofacial clefting in a European trio sample

Nonsyndromic cleft lip with or without cleft palate (NSCL/P), the most common type of orofacial clefting, is one of the most frequent congenital defects. Based on epidemiological data, NSCL/P can be distinguished from nonsyndromic cleft palate only (NSCPO). Both phenotypes have a complex etiology and environmental and genetic factors are involved in their development. To date, genome-wide association studies have identified 12 genetic factors that increase the risk for NSCL/P in Europeans. Six of them have been independently replicated in samples derived from the same population. The aim of the present study was to replicate the remaining six NSCL/P risk loci in chromosomal regions 1p22.1, 1p36, 3p11.1, 8q21.3, 15q22.2, and 20q12 in a family-based sample of European descent. Each of the top-associated SNPs (single nucleotide polymorphisms) was genotyped in 343 NSCL/P and 266 NSCPO nuclear trios. Single-marker association analysis in the NSCL/P sample showed a significant association with SNP rs742071 (1p36, Pcorrected = 3.74 × 10(-3) ), which is located in the intronic region of PAX7, a gene known to be functionally implicated in craniofacial development. Two additional loci, 1p22.1 and 20q12, were nominally significant, but did not withstand correction for multiple testing. There was no evidence that the NSCL/P risk alleles contribute to the etiology of NSCPO, further supporting that these two subtypes of orofacial clefting are primarily etiologically distinct.

SNPs and interaction analyses of IRF6, MSX1 and PAX9 genes in patients with non-syndromic cleft lip with or without palate

Non‑syndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital deformity, often associated with missing or deformed teeth. The genes interferon regulatory factor 6 (IRF6), muscle segment homeobox 1 (MSX1) and paired box gene 9 (PAX9) are important for the development of the maxillofacial region and dentition. The aim of this study was to explore how genetic variations in IRF6, MSX1 and PAX9, as well as gene‑gene interactions, are associated with NSCL/P. We investigated 9 IRF6 tag single nucleotide polymorphisms (SNPs), 2 MSX1 tag SNPs and 8 PAX9 tag SNPs selected from HapMap data from the Chinese population. The SNPs were examined for associations with NSCL/P in 204 patients and 226 controls. The results demonstrated a significant association between NSCL/P and rs2073485, rs2235371, rs2236909 and rs861020 in the IRF6 gene, and haplotype analysis supported these findings. A marginally significant difference was observed in the NSCL/P group for rs17176643 in the PAX9 gene compared to the control group. The results of gene‑gene interaction analyses also indicated that the combination of rs2073485, rs2235371 or rs2236909 in IRF6 and rs17176643 in PAX9, increased the risk of NSCL/P. Thus, our study provided additional understanding of IRF6 variations in patients with NSCL/P and suggested that interactions between the IRF6 and PAX9 genes are potentially important for susceptibility to NSCL/P.

TGFA and IRF6 Contribute to the Risk of Nonsyndromic Cleft Lip with or without Cleft Palate in Northeast China

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) are common birth defects with a complex etiology. Multiple interacting loci and possible environmental factors influence the risk of NSCL/P. 12 single nucleotide polymorphisms (SNPs) in 7 candidate genes were tested using an allele-specific primer extension for case-control and case-parent analyses in northeast China in 236 unrelated patients, 185 mothers and 154 fathers, including 128 complete trios, and 400 control individuals. TGFA and IRF6 genes showed a significant association with NSCL/P. In IRF6, statistical evidence of an association between rs2235371 (p = 0.003), rs2013162 (p<0.0001) and NSCL/P was observed in case-control analyses. Family based association tests (FBATs) showed over-transmission of the C allele at the rs2235371 polymorphism (p = 0.007). In TGFA, associations between rs3771494, rs3771523 (G3822A), rs11466285 (T3851C) and NSCL/P were observed in case-control and FBAT analyses. Associations between other genes (BCL3, TGFB3, MTHFR, PVRL1 and SUMO1) and NSCL/P were not detected.

Endothelial nitric oxide synthase VNTR (intron 4 a/b) polymorphism association with nonsyndromic oral clefts

Objective: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with substantial clinical and social impact, and whose causes include both genetic and environmental factors. Folate and homocysteine metabolism have been indicated to play a role in the etiology of NSCLP. The aim of this study was to determine the prevalence of NOS3 27-bp VNTR and to evaluate whether this polymorphism contributed to the risk of NSCLP. Material and methods: We studied the 27 base pair tandem repeat polymorphism in intron 4 of the endothelial nitric oxide synthase (NOS3) gene in 230 unrelated individuals belong to 7 Indian populations along with 141 NSCLP cases and 142 unrelated controls. The genotyping was performed by polymerase chain reaction and electrophoresis. The data were statistically analysed using the χ2-test. Results: The NOS3 27-bp VNTR 4a allele is present in six of the seven populations analysed and allele frequencies range from 6.8% in Sugali to 23.5% in Madiga populations. NOS3 showed protective association with predisposition towards NSCLP for the heterozygous (4b/a) genotypes (4b/b vs. 4b/a: OR =0.58, 95% CI =0.34 to 0.99, p=0.044). Conclusions: The current study suggests significant differences in the frequency of the NOS3 VNTR allele across the populations. There is protective association between NOS3 27-bp VNTR polymorphism and NSCLP in the Indian population.

Association Between Betaine Homocysteine S-Methyl Transferase (BHMT) rs3797546 Gene Polymorphisms and the Risk of Nonsyndromic Cleft Lip and/or Cleft Palate in South-East Population of Iran

Background: Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common congenital anomalies and has a multifactorial etiology that involves both genetic and environmental factors. Objectives: The present study was aimed to evaluate the possible association between BHMT rs3797546 gene polymorphisms and NS-CL/P in a sample of Iranian population. Materials and Methods: This case-control study was performed on 100 NS-CL/P cases and 100 healthy controls. Tetra amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was used to detect the polymorphism. Analysis of data was done using χ2 test for categorical data, the associations between genotypes and NS-CL/P were estimated by computing the odds ratio (OR) and 95% confidence intervals (95% CI) from logistic regression analyses. Results: No significant difference was found between the groups regarding BMHT rs3797546 gene polymorphism. The C allele was not associated with NS-CL/P (OR = 1.27, 95% CI = 0.85-2.79, P = 0.690). Conclusions: Our data suggested that BMHT rs3797546 gene polymorphism was not associated with risk/ protection for NS-CL/P in a sample of Iranian population. Larger studies are required to validate our findings.

Three polymorphisms in IRF6 and 8q24 are associated with nonsyndromic cleft lip with or without cleft palate: Evidence from 20 studies

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common craniofacial malformation in humans. Three polymorphisms, rs2235371 and rs642961 in interferon regulatory factor 6 (IRF6), rs987525 on 8q24, have been shown to be associated with NSCL/P risk in several studies. However, the magnitudes of the association varied between studies. We therefore performed a meta-analysis to investigate this relationship. Two authors independently extracted information on the characteristics of the eligible studies. Either a fixed- or a random-effects model was used to calculate the overall combined risk estimates. Overall, 20 published case-control studies were included in the meta-analysis. We found that rs2235371 A allele had a significantly decreased risk (OR: 0.73, 95% CI: 0.61-0.88), whereas rs642961 A allele had a significantly increased risk of NSCL/P (OR: 1.44, 95% CI: 1.30-1.59), compared with the G allele. For 8q24 rs987525, the A allele was associated with a significantly increased risk of NSCL/P, compared with the C allele (OR: 1.71, 95% CI: 1.40-2.09). Furthermore, in the stratified analysis by ethnicity and types of NSCL/P, significant associations were still observed in the subgroups of ethnicity and types. Taken together, the results suggest that the IRF6 rs2235371, rs642961, and 8q24 rs987525 polymorphisms are associated with NSCL/P risk.

Resequencing of VAX1 in patients with nonsyndromic cleft lip with or without cleft palate

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common of all congenital anomalies, and has a multifactorial etiology involving both environmental and genetic factors. Recent genome-wide association studies (GWAS) identified strong association between a locus on chromosome 10q25.3 and NSCL/P in European samples. One gene at 10q25.3, the ventral anterior homeobox 1 (VAX1) gene, is considered a strong candidate gene for craniofacial malformations. The purpose of the present study was to provide further evidence that VAX1 is the causal gene at the 10q25.3 locus through identification of an excess of rare mutations in patients with NSCL/P. The 5'UTR, complete coding regions, and adjacent splice sites of the two known VAX1 isoforms were sequenced in 384 patients with NSCL/P and 384 controls of Central European descent. Observed variants were investigated with respect to familial cosegregation or de novo occurrence, and in silico analyses were performed to identify putative effects on the transcript or protein level. Eighteen single-base variants were found, 15 of them rare and previously unreported. In the long VAX1 isoform, predicted functionally relevant variants were observed more often in NSCL/P cases, although this difference was not significant (p = 0.17). Analysis of family members demonstrated incomplete cosegregation in most pedigrees. Our data do not support the hypothesis that highly penetrant rare variants in VAX1 are a cause of NSCL/P. To determine whether VAX1 is the causative gene at 10q25.3 further research, in particular into the biologic function of its long isoform, is warranted. Birth Defects Research (Part A), 2012.

‘Location, Location, Location’: a spatial approach for rare variant analysis and an application to a study on non-syndromic cleft lip with or without cleft palate

Motivation: For the analysis of rare variants in sequence data, numerous approaches have been suggested. Fixed and flexible threshold approaches collapse the rare variant information of a genomic region into a test statistic with reduced dimensionality. Alternatively, the rare variant information can be combined in statistical frameworks that are based on suitable regression models, machine learning, etc. Although the existing approaches provide powerful tests that can incorporate information on allele frequencies and prior biological knowledge, differences in the spatial clustering of rare variants between cases and controls cannot be incorporated. Based on the assumption that deleterious variants and protective variants cluster or occur in different parts of the genomic region of interest, we propose a testing strategy for rare variants that builds on spatial cluster methodology and that guides the identification of the biological relevant segments of the region. Our approach does not require any assumption about the directions of the genetic effects.Results: In simulation studies, we assess the power of the clustering approach and compare it with existing methodology. Our simulation results suggest that the clustering approach for rare variants is well powered, even in situations that are ideal for standard methods. The efficiency of our spatial clustering approach is not affected by the presence of rare variants that have opposite effect size directions. An application to a sequencing study for non-syndromic cleft lip with or without cleft palate (NSCL/P) demonstrates its practical relevance. The proposed testing strategy is applied to a genomic region on chromosome 15q13.3 that was implicated in NSCL/P etiology in a previous genome-wide association study, and its results are compared with standard approaches.Availability: Source code and documentation for the implementation in R will be provided online. Currently, the R-implementation only supports genotype data. We currently are working on an extension for VCF files.Contact: heide.fier@googlemail.com

Transforming growth factor-β3 intron 5 polymorphism as a screening marker for non-syndromic cleft lip with or without cleft palate

In this study, we evaluated the effect of transforming growth factor β3 intron 5 position +104 A➝G (TGF-β3 IVS5+104AG) transition in patients with a non-syndromic cleft lip with or without cleft palate (NSCL/P). A total of 68 patients and 114 controls were recruited for the study. A genotyping procedure was carried out using the PCR-RFLP method. For statistical analysis, the Chi-square test was used to compare data between the patient and control groups. The frequencies of the AA, AG and GG genotypes were 24, 29 and 47%, respectively, for the patients and 54, 36 and 10%, respectively, for the control group. The GG genotype and G allele were significantly different in the patient group compared with the control (p=0.0001). We conclude that SfaN1 polymorphism in TGF-β3 may be a good screening marker for the prediction of NSCL/P in patients. However, more studies with extended sample numbers should be carried out to clarify the effect of the examined gene region on NSCL/P.

Expression and mutation analyses implicate ARHGAP29 as the etiologic gene for the cleft lip with or without cleft palate locus identified by genome‐wide association on chromosome 1p22

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and environmental factors. Genome-wide association studies have successfully identified five novel loci associated with NSCL/P, including a locus on 1p22.1 near the ABCA4 gene. Because neither expression analysis nor mutation screening support a role for ABCA4 in NSCL/P, we investigated the adjacent gene ARHGAP29. Mutation screening for ARHGAP29 protein coding exons was conducted in 180 individuals with NSCL/P and controls from the United States and the Philippines. Nine exons with variants in ARHGAP29 were then screened in an independent set of 872 cases and 802 controls. Arhgap29 expression was evaluated using in situ hybridization in murine embryos. Sequencing of ARHGAP29 revealed eight potentially deleterious variants in cases including a frameshift and a nonsense variant. Arhgap29 showed craniofacial expression and was reduced in a mouse deficient for Irf6, a gene previously shown to have a critical role in craniofacial development. The combination of genome-wide association, rare coding sequence variants, craniofacial specific expression, and interactions with IRF6 support a role for ARHGAP29 in NSCL/P and as the etiologic gene at the 1p22 genome-wide association study locus for NSCL/P. This work suggests a novel pathway in which the IRF6 gene regulatory network interacts with the Rho pathway via ARHGAP29. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

Association Between PAX9 Single-Nucleotide Polymorphisms and Nonsyndromic Cleft Lip With or Without Cleft Palate

The purpose of this study was to investigate the contribution of PAX9 gene to the risk of nonsyndromic cleft lip with or without cleft palate (NS-CL/P). The samples consisted of 142 Korean NS-CL/P families (90 males and 52 females; 9 cleft lip, 26 cleft lip and alveolus, and 107 cleft lip and palate; 76 trios and 66 dyads). A total of 10 single-nucleotide polymorphisms (SNPs) were tested for association with Korean CL/P case-parent trios using transmission disequilibrium test (TDT) and conditional logistic regression models. The minor allele frequency, heterozygosity, and a χ test for Hardy-Weinberg equilibrium at each SNP were computed between parents. Pairwise linkage disequilibrium was computed as both D' and r2 for all SNPs. Both allelic and genotypic TDTs were performed for individual SNPs using family-based association test program. Sliding windows of haplotypes consisting of 2 to 8 SNPs were tested using haplotype-based association test program. Genotypic odd ratios were obtained from conditional logistic regression models using STATA software. The family-based TDT using individual SNPs and 2- to 8-SNP haplotypes of the gene indicated a significant association at rs17104928 (P = 0.014). The haplotype analysis revealed that the association was most significant for the haplotype consisting of 3 SNPs (rs2073247, rs17104928, and rs17176643; P = 0.007). G/A heterozygote at rs17104928 had a significantly increased association with NS-CL/P (genotypic odd ratio, 2.88; 95% confidence interval, 1.42-5.84; P = 0.0014, dominant model). The high-risk SNP and genotype may provide a better understanding of the etiologic role of PAX9 gene in NS-CL/P and potential options for genetic counseling.

EvaluatingSKIas 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 of DVL2 and AXIN2 gene polymorphisms with cleft lip with or without cleft palate in a polish population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital anomalies, with a complex and still not fully understood etiology. Given the important role of the Wnt/β-catenin pathway during craniofacial development, we decided to test the hypothesis that common polymorphic variants of the genes encoding crucial components of this signaling pathway might contribute to the risk of NSCL/P in the Polish population. A set of 19 single nucleotide polymorphisms (SNPs) in the APC, AXIN1, AXIN2, CTNNB1, DVL2, and GSK-3β genes were analyzed using restriction fragment length polymorphism and high-resolution melting curve methods in a group of 280 patients with NSCL/P and a properly matched control group (n = 330). Both single-marker and haplotype analyses showed an association between SNPs in the DVL2 gene and the risk for NSCL/P. The strongest association was found under an overdominant model for the rs35594616 variant located in the exonic sequence of DVL2 (odds ratio [OR], 1.90; 95% confidence interval [CI], 1.37-2.62; p < 0.0001). Moreover, the gene-gene interaction analysis revealed a significant epistatic interaction between DVL2 gene SNPs in the susceptibility to orofacial clefts. Borderline association with a decreased risk of NSCL/P was also observed for the AXIN2 rs3923087 variant (dominant model OR, 0.69; 95% CI, 0.50-0.95; p = 0.03). This study suggests that polymorphic variants of the Wnt/β-catenin pathway genes have a role in the susceptibility to orofacial clefts. The DVL2 and AXIN2 genes might be candidate genes for this craniofacial anomaly in the Polish population. Birth Defects Research (Part A), 2012.

IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5' and 3' of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P=0.009; odds ratio (OR) for AA genotype=1.83 [95% Confidence interval (CI), 0.64-5.31]; OR for AG genotype=1.72 [95% CI, 1.03-2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H2=0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis.

Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci

We have conducted the first meta-analyses for nonsyndromic cleft lip with or without cleft palate (NSCL/P) using data from the two largest genome-wide association studies published to date. We confirmed associations with all previously identified loci and identified six additional susceptibility regions (1p36, 2p21, 3p11.1, 8q21.3, 13q31.1 and 15q22). Analysis of phenotypic variability identified the first specific genetic risk factor for NSCLP (nonsyndromic cleft lip plus palate) (rs8001641; P(NSCLP) = 6.51 × 10(-11); homozygote relative risk = 2.41, 95% confidence interval (CI) 1.84-3.16).

Association between parental MTHFR gene polymorphism 677C/T and nonsyndromic cleft lip and palate in offspring

To explore the association between parental genetic polymorphism of methylenetetrahydrofolate reductase (MTHFR) 677C/T and occurrence of nonsyndromic cleft lip with or without cleft palate (NSCL/P) in offspring in Shandong Province. MTHFR genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Parents of 89 NSCL/P patients treated at Qilu Hospital from August, 2006 to August, 2008 and those of 64 healthy children were recruited in this case-control study. Frequencies of T and C alleles in mothers of patients and healthy children were 65.73% and 46.09%, and 34.27% and 53.91%, respectively (Chi-square=13.663, P<0.01). Offspring whose mothers had T alleles were 2.243 times more likely to develop NSCL/P (95%CI: 1.408-3.572). Frequencies of T and C alleles in fathers of patients and healthy children were 62.92% and 55.47%, and 37.08% and 44.53%, respectively (Chi-square=2.222, P>0.05). The chance for parents of the patient and control groups to bear an affected fetus carrying homozygous mutations were 43% and 29%, respectively (P>0.05). In Shandong Province, maternal genotype for the MTHFR 677C/T polymorphism has a significant impact on the occurrence of NSCL/P in their offspring, whilst paternal genotype for this polymorphism may not be a risk factor for NSCL/P in their offspring.

Association of candidate genes with nonsyndromic clefts in Honduran and Colombian populations

Orofacial clefts are the most common craniofacial birth defects in humans, with the majority of orofacial clefts occurring as nonsyndromic cleft lip with or without cleft palate (NSCLP). We previously demonstrated associations between single-nucleotide polymorphisms (SNPs) in the IRF6 gene and NSCLP in the Honduran population. Here we investigated other candidate genes and chromosomal regions associated with NSCLP identified from genome-wide association studies (GWAS), including MAFB, ABCA4, 8q24, 9q22, 10q25, and 17q22 in two independent Hispanic populations. Case-control and family-based association testing. Honduran families with two or more members with NSCLP (multiplex) were identified. DNA was collected from affected and unaffected family members (488) and 99 gender-matched controls. NSCLP Colombian families were identified; DNA was collected from 26 proband-parent trios. All participants were genotyped for 17 SNPs in six chromosomal regions. Case-control association and family-based association testing (FBAT) analyses were conducted. Seven SNPs demonstrated association in at least one model in the Honduran population. In the Colombian families, five SNPs demonstrated significance in FBAT when patients with isolated cleft palate (CP) were included; four overlapped with SNPs demonstrating significance in the Honduran population, two with the same allele. One SNP retained significance with CP excluded. This study supports the previous GWAS findings and is the first to suggest a role for FOXE1, ABCA4, and MAFB in orofacial clefting in two separate Hispanic populations.

Association between single‐nucleotide polymorphisms on chromosome 1p22 and 20q12 and nonsyndromic cleft lip with or without cleft palate: New data in Han Chinese and meta‐analysis

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation associated with genetic and environmental risk factors. A recent genome-wide association study identified two novel susceptibility loci on chromosomes 1p22 and 20q12; however, conflicting results, especially for 1p22, have been reported in Han Chinese population. The aims of this study were to replicate this association with risk of NSCL/P in the southern Han Chinese population and to discern the effect of these loci by a meta-analysis. To this end, 305 patients with NSCL/P, 356 phenotypically normal controls, and an additional 176 case-parent trios were recruited. Four of the previously associated single nucleotide polymorphisms (SNPs) were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, two published datasets were combined with the present results to determine the precise roles of the loci. SNPs (rs6072081, rs13041247, and rs6102085) on 20q12 were found to be strongly associated with NSCL/P (Bonferroni-corrected and χ(2) test; p values < 0.05). Subsequent analysis of the case-parent trio provided similar results. However, neither the association study nor the trio analysis supported a causative role for SNP rs560426 on 1p22 in NSCL/P susceptibility. Stratified meta- analysis combining Chinese samples supported our findings. This cross-validation study confirmed the previous findings that SNPs in 20q12 are associated with NSCL/P in Han Chinese population. We further conclude that rs560426 on 1p22 might not have a major influence on susceptibility to NSCL/P in southern Han Chinese, but future studies with other Han Chinese populations are needed.

BMP4 was associated with NSCL/P in an Asian population.

BackgroundThe Bone Morphogenetic Protein 4 gene (BMP4) is located in chromosome 14q22-q23 which has shown evidence of linkage for isolated nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a genome wide linkage analysis of human multiplex families. BMP4 has been shown to play crucial roles in lip and palatal development in animal models. Several candidate gene association analyses also supported its potential risk for NSCL/P, however, results across these association studies have been inconsistent. The aim of the current study was to test for possible association between markers in and around the BMP4 gene and NSCL/P in Asian and Maryland trios.Methodology/Principal FindingsFamily Based Association Test was used to test for deviation from Mendelian assortment for 12 SNPs in and around BMP4. Nominal significant evidence of linkage and association was seen for three SNPs (rs10130587, rs2738265 and rs2761887) in 221 Asian trios and for one SNP (rs762642) in 76 Maryland trios. Statistical significance still held for rs10130587 after Bonferroni correction (corrected p = 0.019) among the Asian group. Estimated odds ratio for carrying the apparent high risk allele at this SNP was 1.61 (95%CI = 1.20, 2.18).ConclusionsOur results provided further evidence of association between BMP4 and NSCL/P.