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.
Citation: Lu Y, Liu Q, Xu W, Li Z, Jiang M, Li X, et al. (2013) TGFA and IRF6 Contribute to the Risk of Nonsyndromic Cleft Lip with or without Cleft Palate in Northeast China. PLoS ONE 8(8): e70754. https://doi.org/10.1371/journal.pone.0070754
Editor: John G. Meara, Boston Children's Hospital, United States of America
Received: February 8, 2013; Accepted: June 23, 2013; Published: August 6, 2013
Copyright: © 2013 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The funders of Scientific and Technological Project of Liaoning Province (Grant No.2009225007-7) played an important role in study design, data analysis, preparation of the manuscript and decision to publish, the Natural Science Foundation of Liaoning Province (Grant No. 2011010192-401) played an important role in the sample collection and diagnosis of the NSCL/P.
Competing interests: The authors have declared that no competing interests exist.
Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects, affecting 1 in 500 to 1000 births worldwide . In China, this common malformation occurs at a rate of 1.6 per 1000 live births. NSCL/P presents a significant public health problem because treatment requires comprehensive surgical, orthodontic, phoniatric and psychological management .
Progress has been made toward defining genetic variations involved in oral-facial cleft by utilizing gene discovery techniques, including genome wide linkage, association mapping and candidate gene approaches. Positive associations between 14 interacting genes and NSCL/P have been reported . Carinci et al. reviewed genes and available loci in the literature and suggested that 6p24, 2p13, 19q13.2, 4q, Msx1, IRF6, PVRL1, TP73L, 13q33, 1q34 and SUMO1 are related to oral clefts . Single nucleotide polymorphism (SNP) genotyping using competitive allele-specific PCR were conducted in 12 candidate genes, including CLPTM1, CRISPLD2, FGFR2, GABRB3, GLI2, PTCH1, RARA, RYK, SATB2, SUMO1, TGFA, and IRF6, and reported that PTCH1, SUMO1, and TGFA contribute to nonsyndromic oral clefts . Through multiple stages involving positional cloning, candidate gene sequencing and developmental gene expression analysis, FOXE1 had been identified as a major disease gene for NSCL/P .
Most recently, several genome-wide association studies (GWAS) reported a major susceptibility locus for NSCL/P on 8q24.21 and other genes . A case-control genome-wide association study (GWAS) in Germany found significant evidence of association with markers in 8q24.21 , and a US case-control GWAS confirmed this region, with rs987525 being the most significant marker in both studies . Mangold and colleagues subsequently used an expanded dataset from Europe and identified additional loci at chromosomes 10q25 (VAX1, ventral anterior homeobox 1) and 17q22 (NOG, noggin) that achieved genome-wide significance . Beaty and colleagues performed a GWAS using a case-parent trio from Europe, the United States, China, Taiwan, Singapore, Korea and the Philippines and found that SNPs in two genes not previously associated with CL/P (ABCA4 on chromosome 1p22.1 and MAFB on 20q12) achieved genome-wide significance, and three potential candidate genes (PAX7 on 1p36, VAX1 on 10q25.3 and NTN1 on 17p13) had one or more SNPs near genome-wide significance . These novel loci have subsequently been replicated in independent studies –. Leslie et al. resequenced the ARHGAP29 and identified a nonsense variant, a frame shift variant, and fourteen missense variants were overrepresented . The GWAS Meta analysis study based on the two GWAS identifies six new risk loci .
Transforming growth factor alpha (TGFA) belongs to a large family of proteins that regulate cell proliferation, differentiation, migration and apoptosis. Ardinger et al. first reported an association between the Taq I variant in TGFA and NSCL/P . TGFA has since been extensively investigated for linkage, association and gene-environment interactions with inconsistent results. The mutations (C3827T, G3822A, and T3851C) in 3′untranslated conserved regions were reported to be associated with oral-facial clefts . Sull et al. recently tested 17 SNPs in a region surrounding TGFA on chromosome 2p13 and reported over-transmission of rs3771494 as the minor allele without considering the parent-of-origin in four populations .
The interferon regulatory factor 6 (IRF6) gene, located on chromosome 1q32.3-q41, has been frequently studied and is strongly associated with oral-facial cleft risk. This gene encodes interferon regulatory factor 6, which is a key element in oral and maxillofacial development. Scapoli et al. selected four SNPs based on Zucchero et al.  and reported that rs2013162 and rs2235375 have strong linkage disequilibrium with NSCL/P in an Italian family . Srichomthong et al. suggested that IRF6 rs2235371 (V274I) is responsible for 16.7% of the genetic contribution to CL/P . Large studies in different populations – have provided evidence that IRF6 is important in the etiology of NSCL/P.
Many studies have suggested that other genes and loci are associated with NSCL/P–. Due to NSCL/P genetic heterogeneity in different populations, we investigated the contribution of previously reported candidate genes TGFA, IRF6, BCL3, TGFB3, MTHFR, PVRL1, and SUMO1 for NSCL/P in northeast China (Table 1).
Erdogan et al. used a novel allele-specific primer elongation microarray to test SNPs in human mitochondrial DNA (mtDNA) . Jagomagi et al. performed SNP genotyping using an arrayed primer extension technique . In this research, we designed a similar microarray to test the risk of 12 SNPs in 7 genes that may contribute to NSCL/P in a population from northeast China. This microarray is a high throughout, efficient, accurate and convenient method for test SNPs, and the current study of northeast China populations was supplement for the previous association study between candidate genes and NSCL/P.
Materials and Methods
This study was approved by the ethics committee at the Liaoning Province Research Institute of Family Planning. Written informed consent was obtained from all study participants. Parents or legal guardians provided written consent on behalf of minors.
Patients and Families
We evaluated 236 unrelated patients with NSCL/P, their parents (185 mothers and 154 fathers, including 128 complete trios), and 400 control individuals. All participants were recruited from Stomatology Hospital of China Medical University in northeast China between 2008 and 2012. All patients underwent a pre-operation examination and questionnaire, and photographs were taken to record the shape of the lips, alveolar ridge, and hard and soft palates to diagnose cleft lip and palate. The patients had a physical examination to document the shape of the skull, eyes, nose, chin, neck, chest, feet, and hands to exclude syndromes that may be associated with cleft lip and palate, such as Albert syndrome, Edward syndrome, and Pierre Robin syndrome. All the controls were recruited from healthy volunteers. Controls with a family history of clefts and other anomalies were excluded based on a questionnaire that evaluated the medical history of the patients' family for three generations. A physical examination was also performed on the controls, particularly regarding movement of the lips and soft palate to detect occult deformities. A five milliliter peripheral blood sample was collected. DNA was obtained from blood samples with a TIANamp Blood DNA kit (TianGen) and used as the template for PCR amplification.
Allele Special Primer Extension Microarray Preparation
A total of 24 oligonucleotide tags used as probe captures were obtained from www.genome.wi.mit.edu. The 3′-amino modified tags (Table 2) were synthesized by Nanjing Genescript Biological Technology Company. Tags were suspended at a concentration of 30 µM in 0.3 M carbonate buffer (pH 8.0) and spotted onto slides in duplicate using a Nano-Plotter 2.1 arrayer (GeSiM Germany) at a rate of ten hits per spot in a humidified chamber (60% relative humidity). After spotting, the microarrays were incubated at room temperature overnight in a humid chamber and broiled at 60°C for 1 hour. Microarrays were rinsed in dH2O and dried.
Allele-specific Primer Design
Each allele-specific primer was composed of a reverse complement of the tag (com-tag) at forward and backward sequences. For each SNP, two allele-specific primers were designed to match the two SNP alleles (Table 2). In addition, a com-tag labeled with Cy3 was synthesized as a positive control.
Multiplex PCR and Allele-specific Primer Extension
PCR was amplified using the Multiplex PCR Assay Kit (Takara) in a 20 µL volume (Multiplex PCR mix2 10 µL, Multiplex PCR mix1 0.2 µL, 0.2 µM primers, and 50 ng DNA). Thermocycling was performed with an initial 60 second denaturation at 94°C followed by 35 cycles of 30 seconds at 94°C, 90 seconds at 57°C, 90 seconds at 72°C, and final extension at 72°C for 10 minutes. The PCR product was used as a template for multiplex allele-specific primer extension. To remove excess dNTPs and primers, 2 units of shrimp alkaline phosphatase (Takara) and 4 units of EXO I (Takara) were added to 10 µL of the PCR product, followed by incubation at 37°C for 30 min and 96°C for 10 min. A total of 10 µL of treated PCR products were added to 5 µL ASPE containing 1×Thermopol reaction buffer, 80 µM each of dATP, dGTP, and dTTP (Takara), 40 µM Cy3-dCTP (GE), 0.2 units of Vent exo-DNA polymerase (New England Biolabs), and 25 nM allele-specific primers. Extension conditions were as follows: initial 3 minutes at 94°C, 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and a final extension at 72°C for 5 minutes.
Hybridization, Scanning of Microarrays and Data Analyses
To prepare Cy3-labeled single strand products, 10 µL allele-specific extension products were added to a 10 µL mixture containing 1.33×SSC, 0.067% SSC, 0.033 mg/ml salmon sperm DNA, and 50 nM Cy3-labeled com-tag. The mix was denatured at 94°C for 10 minutes and cooled for 5 minutes on ice. Hybridization was performed using a hybridization solution in a hybridization chamber at 60°C for 2 hours. Hybridization was followed by two washes in 2×SSC/0.1% SDS (preheated to 50°C) for 5 minutes, washes in 0.2×SSC/0.1% SDS for 1 minute, then rinsed in dH2O and dried by centrifugation for 3 minutes at 500 rpm.
Microarrays were scanned at 100-µm resolution using a GenePix 4000B scanner, and TIFF images were imported into GenePix 6.0 software (Axon Instruments, USA). For each allele, the mean pixel intensity was subtracted from the mean background intensity. Alleles with a mean intensity lower than the cutoff (mean background +1000) were excluded. SNP genotypes were identified by the allelic fraction (AF), which was calculated as follows: AF = allele B/allele A+allele B. AF values >0.6, <0.4, and between 0.4 and 0.6 represented allele B homozygotes, allele A homozygotes, and heterozygotes, respectively. Microarray results can be acquired from Gene Expression Omnibus (GSE45770 accession number).
Hardy-Weinberg equilibrium (HWE) was used to assess all SNPs. Case-control statistical analysis was performed using the SPSS 17.0 statistical software package (http://www-03.ibm.com/software/products/us/en/spss-stats-standard/). Odds ratios and 95% confidence intervals were calculated for the case and control groups. The Family Based Association Test (FBAT) package (http://www.biostat.harvard.edu/fbat/default.html) was used to test for over-transmission of the target alleles in case-parent trios. Linkage disequilibrium (LD) was estimated using haploview (http://www.broad.mit.edu/mpg/haploview) based on the genotypes of the control samples. TGFA and IRF6 haplotypes were calculated using the FBAT software package.
Genotype and Case-control Comparisons
Figure 1 shows the SNP microarray results. Each SNP AF was calculated as described, and the SNP genotypes were clearly identified.
a: Spot arrays printed on the slide. b: Results of allele-specific primer extension microarray after hybridization.
To ensure accuracy, 50 samples were run in duplicate, and 10% of all samples were sequenced directly. SNP genotypes were highly reproducible (99.5%), and direct sequenced demonstrated agreement with the microarray (99.6%).
Hardy-Weinberg equilibrium was assessed for the 12 SNPs. There was no evidence of deviation from HWE for any of the SNPs. Genotype distribution between cases was compared with controls (Table 3).
There was no evidence of genotypic association with the risk of NSCL/P in northeast China for the following SNPs: TGFA rs1058213 (C3827T), BCL3 rs1046881, TGFB3 rs3917201, MTHFR rs13306554 (C667T) and rs1801131 (A1298C), PVRL1 rs104894281 (G546A), and SUMO1 rs7580433. However, a statistically significant increased risk of NSCL/P was observed with TGFA rs3771494, rs3771523 (G3822A), and rs11466285 (T3851C) and IRF6 rs2235371 (V274I) and rs2013162.
For TGFA rs3771494, ORs for cases with TC+CC, TC and CC genotypes compared with TT homozygotes were 1.601 (95% CI, 1.122–2.286, p = 0.009), 3.568 (95% CI, 1.94–6.564, p<0.0001), and 1.886 (95% CI, 1.356–2.624, p = 0.001), respectively. For rs3771523, the OR of the GA heterozygote compared with the GG homozygote was 1.673 (95% CI, 1.155–2.425, p = 0.006). Although the OR of the AA genotype compared with GG was not statistically significant (p = 0.076), the OR of GA+AA compared with the GG homozygote was significant (p = 0.002). For rs11466285, a significant association was observed between patients and controls (p<0.05).
For IRF6 rs2235371 and rs2013162, a risk of NSCL/P was observed in all of the genetic models evaluated (Table 3). The ORs of CT, TT, and CT+TT compared with the CC genotype were 0.664 (95% CI, 0.471–0.937, p = 0.02), 0.33 (95% CI, 0.18–0.584, p = 0.001), and 0.618 (95% CI, 0.448–0.852, p = 0.003), respectively. For IRF6 rs2013162, the ORs of CA, AA, and CA+AA compared with the CC genotype were 1.806 (95% CI, 1.242–2.627, p = 0.002), 2.923 (95% CI, 1.754–4.873, p<0.0001), and 2.008 (95% CI, 1.402–2.876, p<0.0001), respectively.
FBAT showed strong associations between TGFA rs3771494, rs3771523 (G3822A), and rs11466285 (T3851C) and IRF6 rs2235371 (V274I) and rs2013162 and NSCL/P (Table 4). These SNPs showed an over-transmission of the minor frequency alleles, with the exception of IRF6 rs2235371, which showed under-transmission of the T allele and over-transmission of the common C allele.
LD and Haplotype Analysis
Pairwise LD was measured for TGFA and IRF6 (Table 5). LD analysis showed tight linkage between the SNPs in IRF6 and TGFA.
Haplotypes of the four TGFA SNPs (rs3771494, rs1058213, rs11466285, and rs3771523) and two IRF6 SNPs (rs2013162 and rs2235371) were also determined. The C-G-C-C (order: rs3771494-rs3771523-rs11466285-rs1058213) and C-A (order: rs2235371-rs2013162) haplotypes showed significant associations with NSCL/P (Table 6).
We used a microarray with allele-specific primer extension to test 12 SNPs in 7 genes reported to be associated with NSCL/P and observed that SNPs in TGFA and IRF6 had statistically significant associations with NSCL/P based on case-control and case-parent analyses consisting of 236 patients and 400 controls from northeast China.
The TGFA gene has been well studied since Ardinger et al. first reported an association between the Taq I variant and NSCL/P . However, there have been conflicting results reported for different population due to study design, sample size, and TGFA variants used . Machida et al tested SNPs in the 3′ UTR of TGFA and reported that there were no associations with NSCL/P , but Shiang et al. reported significant associations between those SNPs and cleft palate . Letra et al. recently calculated the attributable fraction of high-risk alleles at IRF6 rs2235371 and TGFA rs1058213 in a Brazilian population and estimated the contribution of interactions between the two genes to be approximately 1% . They also evaluated IRF6-TGFA interactions in 142 case-parent trios and detected significant over-transmission of high-risk alleles to the affected child (p = 0.001). Sull et al. examined associations between TGFA markers and NSCL/P in case-parent trios from four populations . The authors genotyped 17 SNPs and reported significant transmission of the minor allele in rs3771494 (OR = 1.59, p = 0.004).
In this study, a significant association with rs3771494 (OR = 1.88, p<0.0001) was observed in the case-control analysis. FBAT analysis also showed over-transmission of the C allele in rs3771494 (p = 0.016). These consistent results suggest that TGFA is strongly associated with NSCL/P in populations in northeast China. We also observed an association between rs3771523 (OR = 1.73, p = 0.002) and rs11466285 (OR = 1.81, p = 0.001) and over-transmission of the A allele in rs3771523 (p = 0.01) and the C allele in rs11466285 (p = 0.03) based on FBAT analysis. However, a significant association was not observed between rs1058213 and NSCL/P in the present study (OR = 1.14, p = 0.427), which is in accordance with previous studies. Using HBAT analysis, we observed an over-transmission of the C-G-C-C haplotype (order: rs3771494-rs3771523-rs11466285-rs1058213).
IRF6 is the most frequently studied gene related to NSCL/P. Research has shown common alleles in IRF6 that are associated with NSCL/P, which has been independently replicated in genome-wide association (GWA) ,  and candidate gene studies , . Animal models have also supported the role of IRF6 in NSCL/P , . Birnbaum et al. reported that the T allele in rs2235371 (V274I) coding for isoleucine is under-representation in NSCL/P patients compared with controls and may have a protective effect with respect to NSCL/P . However, significant associations were not found between rs2013162 and NSCL/P, which differs from previous findings , , , . Letra et al. demonstrated a significant association between the V274I polymorphism (rs2235371) with complete left cleft lip/palate (p = 0.001) in a Brazilian Caucasian population . Huang et al. found a significant association between rs2235371 and rs2235375 and NSCL/P in west China, but not rs2013162 . In the present study, both rs2235371 (p = 0.003) and rs2013162 (p<0.0001) showed significant differences between patients and controls. rs2013162 results are in contrast to the Huang et al. study, which may be due to population heterogeneity between west and northeast China. FBAT analysis of rs2235371 showed under-representation T allele and over-transmission of the C allele from parent to child, indicating that the minor T allele may be a protective factor in NSCL/P, which is consistent with a previous study . In rs2013162, we observed an over-transmission of the A allele (minor allele) and under-transmission of the C allele (common allele). In haplotype analyses of NSCL/P trios, the C-A (order: rs2235371-rs2013162) haplotype showed significant over-transmission (p = 0.03). These results suggest that the IRF6 gene is strongly associated with an increased risk of NSCL/P in populations in northeast China.
Small ubiquitin-related modifier (SUMO1) is strongly expressed in the upper lip, primary palate and MEE of the secondary palate . Several studies ,  have shown associations between gene variations in SUMO1 and NSCL/P. Jia et al. reported that the C allele in rs7580433 is overtransmitted from parents to affected individuals in west China . However, we did not observe an association between rs7580433 in SUMO1 and cases and controls (p = 0.561). FBAT analysis of trios did not detect an association between rs7580433 and NSCL/P (p = 0.68).
We failed to replicate an association between candidate gene SNPs, including BCL3, TGFB3, MTHFR and PVRL1, and NSCL/P in our patient sample. This could be the result of (1) locus and allelic heterogeneity in NSCL/P, (2) selection of too few SNPs that did not span the entire gene, (3) a small number of analyzed patients and controls, and (4) lack of analysis of gene-gene and gene-environmental interactions, which can play an important role in NSCL/P etiology.
In this study, we tested 12 SNPs in 7 candidate genes using a microarray technique. This study examined associations between SNPs and NSCL/P in northeast China. We confirmed that polymorphic variants of TGFA and IRF6 are strongly associated with NSCL/P in a population in northeast China. This is a supplement research for the previous studies. To understand the fully genetic architecture of these genes, additional SNP-based and resequencing studies with a large sample of patients were still needed in further study.
The authors thank all of the participants who donated blood samples for use in this study and the staff at the School of Stomatology at China Medical University who assisted in collecting blood samples.
Conceived and designed the experiments: JXL WTH QL YPL. Performed the experiments: YPL WX. Analyzed the data: YPL WX XFL MJ ZJL. Contributed reagents/materials/analysis tools: MJ NZ YS WL CM. Wrote the paper: YPL WHF WTH JXL.
- 1. Murray JC (1995) Face facts: genes, environment, and clefts. Am J Hum Genet 57: 227–232.
- 2. Mostowska A, Hozyasz KK, Wojcicki P, Biedziak B, Paradowska P, et al. (2010) Association between genetic variants of reported candidate genes or regions and risk of cleft lip with or without cleft palate in the polish population. Birth Defects Res A Clin Mol Teratol 88: 538–545.
- 3. Schliekelman P, Slatkin M (2002) Multiplex relative risk and estimation of the number of loci underlying an inherited disease. Am J Hum Genet 71: 1369–1385.
- 4. Carinci F, Scapoli L, Palmieri A, Zollino I, Pezzetti F (2007) Human genetic factors in nonsyndromic cleft lip and palate: an update. Int J Pediatr Otorhinolaryngol 71: 1509–1519.
- 5. Carter TC, Molloy AM, Pangilinan F, Troendle JF, Kirke PN, et al. (2010) Testing reported associations of genetic risk factors for oral clefts in a large Irish study population. Birth Defects Res A Clin Mol Teratol 88: 84–93.
- 6. Moreno LM, Mansilla MA, Bullard SA, Cooper ME, Busch TD, et al. (2009) FOXE1 association with both isolated cleft lip with or without cleft palate, and isolated cleft palate. Hum Mol Genet 18: 4879–4896.
- 7. Dixon MJ, Marazita ML, Beaty TH, Murray JC (2011) Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet 12: 167–178.
- 8. Birnbaum S, Ludwig KU, Reutter H, Herms S, Steffens M, et al. (2009) Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet 41: 473–477.
- 9. Grant SF, Wang K, Zhang H, Glaberson W, Annaiah K, et al. (2009) A genome-wide association study identifies a locus for nonsyndromic cleft lip with or without cleft palate on 8q24. J Pediatr 155: 909–913.
- 10. Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, et al. (2010) Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet 42: 24–26.
- 11. Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, et al. (2010) A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet 42: 525–529.
- 12. Rojas-Martinez A, Reutter H, Chacon-Camacho O, Leon-Cachon RB, Munoz-Jimenez SG, et al. (2010) Genetic risk factors for nonsyndromic cleft lip with or without cleft palate in a Mesoamerican population: Evidence for IRF6 and variants at 8q24 and 10q25. Birth Defects Res A Clin Mol Teratol 88: 535–537.
- 13. Nikopensius T, Birnbaum S, Ludwig KU, Jagomagi T, Saag M, et al. (2010) Susceptibility locus for non-syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients. Eur J Oral Sci 118: 317–319.
- 14. Butali A, Suzuki S, Cooper ME, Mansilla AM, Cuenco K, et al. (2013) Replication of Genome Wide Association Identified Candidate Genes Confirm the Role of Common and Rare Variants in PAX7 and VAX1 in the Etiology of Nonsyndromic CL(P). Am J Med Genet A 161: 965–972.
- 15. Leslie EJ, Mansilla MA, Biggs LC, Schuette K, Bullard S, et al. (2012) 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. Birth Defects Res A Clin Mol Teratol 94: 934–942.
- 16. Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, et al. (2012) Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet 44: 968–971.
- 17. Ardinger HH, Buetow KH, Bell GI, Bardach J, VanDemark DR, et al. (1989) Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. Am J Hum Genet 45: 348–353.
- 18. Machida J, Yoshiura K, Funkhauser CD, Natsume N, Kawai T, et al. (1999) Transforming growth factor-alpha (TGFA): genomic structure, boundary sequences, and mutation analysis in nonsyndromic cleft lip/palate and cleft palate only. Genomics 61: 237–242.
- 19. Sull JW, Liang KY, Hetmanski JB, Wu T, Fallin MD, et al. (2009) Evidence that TGFA influences risk to cleft lip with/without cleft palate through unconventional genetic mechanisms. Hum Genet 126: 385–394.
- 20. Zucchero TM, Cooper ME, Maher BS, Daack-Hirsch S, Nepomuceno B, et al. (2004) Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med 351: 769–780.
- 21. Scapoli L, Palmieri A, Martinelli M, Pezzetti F, Carinci P, et al. (2005) Strong evidence of linkage disequilibrium between polymorphisms at the IRF6 locus and nonsyndromic cleft lip with or without cleft palate, in an Italian population. Am J Hum Genet 76: 180–183.
- 22. Srichomthong C, Siriwan P, Shotelersuk V (2005) Significant association between IRF6 820G->A and non-syndromic cleft lip with or without cleft palate in the Thai population. J Med Genet 42: e46.
- 23. Park JW, McIntosh I, Hetmanski JB, Jabs EW, Vander Kolk CA, et al. (2007) Association between IRF6 and nonsyndromic cleft lip with or without cleft palate in four populations. Genet Med 9: 219–227.
- 24. Jugessur A, Rahimov F, Lie RT, Wilcox AJ, Gjessing HK, et al. (2008) Genetic variants in IRF6 and the risk of facial clefts: single-marker and haplotype-based analyses in a population-based case-control study of facial clefts in Norway. Genet Epidemiol 32: 413–424.
- 25. Wu T, Liang KY, Hetmanski JB, Ruczinski I, Fallin MD, et al. (2010) Evidence of gene-environment interaction for the IRF6 gene and maternal multivitamin supplementation in controlling the risk of cleft lip with/without cleft palate. Hum Genet 128: 401–410.
- 26. Li MJ, Zhu WL, Wang Y, Guo JZ, Li SQ, et al. (2012) [Association between polymorphism of IRF6 rs2235371 locus and nonsyndromic cleft lip with or without cleft palate]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 29: 149–154.
- 27. Stein J, Mulliken JB, Stal S, Gasser DL, Malcolm S, et al. (1995) Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families. Am J Hum Genet 57: 257–272.
- 28. Blanco R, Suazo J, Santos JL, Paredes M, Sung H, et al. (2004) Association between 10 microsatellite markers and nonsyndromic cleft lip palate in the Chilean population. Cleft Palate Craniofac J 41: 163–167.
- 29. Ichikawa E, Watanabe A, Nakano Y, Akita S, Hirano A, et al. (2006) PAX9 and TGFB3 are linked to susceptibility to nonsyndromic cleft lip with or without cleft palate in the Japanese: population-based and family-based candidate gene analyses. J Hum Genet 51: 38–46.
- 30. Vieira AR, Orioli IM, Castilla EE, Cooper ME, Marazita ML, et al. (2003) MSX1 and TGFB3 contribute to clefting in South America. J Dent Res 82: 289–292.
- 31. Jugessur A, Wilcox AJ, Lie RT, Murray JC, Taylor JA, et al. (2003) Exploring the effects of methylenetetrahydrofolate reductase gene variants C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads. Am J Epidemiol 157: 1083–1091.
- 32. Avila JR, Jezewski PA, Vieira AR, Orioli IM, Castilla EE, et al. (2006) PVRL1 variants contribute to non-syndromic cleft lip and palate in multiple populations. Am J Med Genet A 140: 2562–2570.
- 33. Alkuraya FS, Saadi I, Lund JJ, Turbe-Doan A, Morton CC, et al. (2006) SUMO1 haploinsufficiency leads to cleft lip and palate. Science 313: 1751.
- 34. Song T, Li G, Jing G, Jiao X, Shi J, et al. (2008) SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate. Biochem Biophys Res Commun 377: 1265–1268.
- 35. Erdogan F, Kirchner R, Mann W, Ropers HH, Nuber UA (2001) Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays. Nucleic Acids Res 29: E36.
- 36. Jagomagi T, Nikopensius T, Krjutskov K, Tammekivi V, Viltrop T, et al. (2010) MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci 118: 213–220.
- 37. Vieira AR (2006) Association between the transforming growth factor alpha gene and nonsyndromic oral clefts: a HuGE review. Am J Epidemiol 163: 790–810.
- 38. Shiang R, Lidral AC, Ardinger HH, Buetow KH, Romitti PA, et al. (1993) Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO). Am J Hum Genet 53: 836–843.
- 39. Letra A, Fakhouri W, Fonseca RF, Menezes R, Kempa I, et al. (2012) Interaction between IRF6 and TGFA genes contribute to the risk of nonsyndromic cleft lip/palate. PLoS One 7: e45441.
- 40. Birnbaum S, Ludwig KU, Reutter H, Herms S, de Assis NA, et al. (2009) IRF6 gene variants in Central European patients with non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci 117: 766–769.
- 41. Brito LA, Bassi CF, Masotti C, Malcher C, Rocha KM, et al. (2012) IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population. Am J Med Genet A 158A: 2170–2175.
- 42. Yeetong P, Mahatumarat C, Siriwan P, Rojvachiranonda N, Suphapeetiporn K, et al. (2009) Three novel mutations of the IRF6 gene with one associated with an unusual feature in Van der Woude syndrome. Am J Med Genet A 149A: 2489–2492.
- 43. Richardson RJ, Dixon J, Malhotra S, Hardman MJ, Knowles L, et al. (2006) Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch. Nat Genet 38: 1329–1334.
- 44. Ingraham CR, Kinoshita A, Kondo S, Yang B, Sajan S, et al. (2006) Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat Genet 38: 1335–1340.
- 45. Blanton SH, Cortez A, Stal S, Mulliken JB, Finnell RH, et al. (2005) Variation in IRF6 contributes to nonsyndromic cleft lip and palate. Am J Med Genet A 137A: 259–262.
- 46. Ghassibe M, Bayet B, Revencu N, Verellen-Dumoulin C, Gillerot Y, et al. (2005) Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population. Eur J Hum Genet 13: 1239–1242.
- 47. Huang Y, Wu J, Ma J, Beaty TH, Sull JW, et al. (2009) Association between IRF6 SNPs and oral clefts in West China. J Dent Res 88: 715–718.
- 48. Pauws E, Stanier P (2007) FGF signalling and SUMO modification: new players in the aetiology of cleft lip and/or palate. Trends Genet 23: 631–640.
- 49. Jia ZL, Li Y, Meng T, Shi B (2010) Association between polymorphisms at small ubiquitin-like modifier 1 and nonsyndromic orofacial clefts in Western China. DNA Cell Biol 29: 675–680.