- Email: [email protected]
C392T polymorphism of the Wnt10a gene in non-syndromic oral cleft in a northeastern Chinese population
Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 52 (2014) 751–755
C392T polymorphism of the Wnt10a gene in non-syndromic oral cleft in a northeastern Chinese population Cuijuan Feng a,∗∗ , Weiyi Duan b , Dan Zhang a , Enjiao Zhang b , Zhongfei Xu b , Li Lu b,∗ a b
Department of Orthodontics, School of Stomatology, China Medical University, Shenyang 110002, PR China Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University, Shenyang 110002, PR China
Accepted 2 June 2014 Available online 20 June 2014
Abstract Non-syndromic oral cleft is one of the most common congenital malformations, and more than 40 genes may be involved in its aetiology. Recent studies have shown that the Wnt10a gene may also contribute. We recruited 198 patients with non-syndromic oral clefts, comprising 96 elementary families (restricted to the patients and their parents) and 187 controls, to investigate their associations with the risk of such clefts and their subgroups in a Chinese Han population. The variant evaluated in this study was a single nucleotide polymorphism – specifically, a missense mutation C392T of Wnt10a. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) was used to genotype the marker, and case–control and family-based associations were analysed. Although in the case-control study there were no significant differences in frequency distributions of genotypes or alleles between cases and controls in the groups with cleft palate and cleft lip and palate, the genotypic and allelic frequencies of C392T in the total groups and the group with cleft lip alone differed significantly from those in the controls (p = 0.04, and 0.01, respectively). A transmission disequilibrium test showed a transmitted disequilibrium in C392T. In conclusion, we found an association between the C392T variant and non-syndromic oral clefts. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: non-syndromic oral cleft; Wnt10a gene polymorphism; polymerase chain reaction; restriction fragment length polymorphism; case-control study; transmission disequilibrium test.
Introduction Non-syndromic oral cleft is one of the most common congenital malformations, and it affects 135,000 babies worldwide each year.1 Chinese newborns have a high prevalence at birth of 1.42/1000,2 and they are a considerable burden on public ∗
Corresponding author. Tel.: +86-13940163503; fax: +86-24-22892645. Address correspondence to: Department of Orthodontics, School of Stomatology, China Medical University, No.117 North Nanjing Street, Shenyang 110002, People’s Republic of China. Phone: +86-18698848565; Fax: +86-24-22892645. E-mail addresses: [email protected] (C. Feng), [email protected] (W. Duan), cmu [email protected] (D. Zhang), [email protected] (E. Zhang), [email protected] (Z. Xu), [email protected] (L. Lu). ∗∗
health services because of the immediate and long-term medical costs and the social impact on patients and their families.3 The identification of genetic and environmental risk factors for non-syndromic oral clefts has been the subject of intensive research for several decades, but the exact mechanism is still not clear.4,5 It has been suggested that more than 40 genes, including IRF6, MSX1, TGFB3, FOXE1, FGFR1, FGFR2, FGF8, PDGFC, CRISPLD2, PVRL1, GABRB3, MSX2, SATB2, TBX10, TBX22, GLI2, JAG2, MTHFR, RARA, LHX8, SKI, SPRY2, Wnt3a, and Wnt9b may be involved in the aetiology.6 However, it is still a challenge to isolate the aetiological roles of the candidate genes. Many studies have indicated that the Wnt family of genes plays a critical part in the development of the lip and palate.7–9 In more recent years, the role of Wnt10a in ectoderm has
http://dx.doi.org/10.1016/j.bjoms.2014.06.001 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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attracted widespread attention, as this interaction is closely related to the development of teeth and skin.10,11 A study by Warner et al. found that the expression of Wnt10a is significantly increased at E13.5 and E14.5 in palatal tissue in fetal rats.12 Beaty et al. also found that the Wnt6-Wnt10a gene cluster is moderately but consistently associated with cleft lip and palate (CLP) in patients from 3 different countries and regions.13 In a recent study we found that downregulation of Wnt10a by interference from RNA inhibited the proliferation of cells, and induced arrest of the cell cycle and apoptosis, in murine embryonic palatal mesenchymal cells.14 Previous studies have shown that the Wnt10a gene may contribute to the aetiology of non-syndromic oral clefts, but there have been no previous reports of the direct association between them. The purpose of the present study, therefore, was to find out if the Wnt10a gene was a risk factor for non-syndromic oral clefts within a specific sample of northeastern Chinese.
Materials and Methods Subjects We studied 198 patients with non-syndromic oral clefts, including 96 elementary families (patients and their parents), and 187 controls. All subjects were recruited between 2008 and 2011 from the Department of Oral and Maxillofacial Surgery of the Affiliated Stomatological Hospital of China Medical University (Shenyang, Liaoning, the People’s Republic of China). After clinical evaluation and taking detailed family histories, syndromic patients were excluded and only those with an isolated cleft were studied. All subjects were of Han Chinese origin. The controls who had congenital malformations of the body, malignant tumours, or a family history of genetic disease were also excluded. The controls were selected to be, as far as possible, born in the same region and to have the same sex distribution as the cases (Table 1). Subphenotypes of clefts
only (CP), cleft lip with cleft palate (CLP), and the total group of patients with clefts. Selection of the single nucleotide polymorphism The single nucleotide polymorphism C392T in Wnt10a that was selected in this study was based on the reports of Sadia et al,15 and is proposed to be a pathogenic variant of odonto-onychodermal dysplasia (MIM 257980). It has not yet, however, been confirmed to be associated with nonsyndromic oral clefts. Amplification and genotyping by polymerase chain reaction (PCR) Genomic DNA were extracted from peripheral blood using the DNA isolation kit for mammalian blood (Tiangen Biotech, Beijing, China). The reference sequence (wildtype sequence) of the Wnt10a gene was obtained from the NCBI GenBank (NM 025216.2). The PCR–restriction fragment length polymorphism assay was applied to genotype Wnt10a C392T. Specific primer pairs were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto CA, USA). The PCR primers were F-5 GAACAGGAG AAGGGCGTACAA-3 , and R-5 TGTAAGCGGTGCAGCTTCCTAC-3 . The restriction enzyme was Eco47 III and the fragment length was 403 bp. A representative gel of the digestion patterns is shown in Fig. 1. For quality control, we retyped 10% of the samples by DNA sequencing (Sangon Biotech (Shanghai) Co., Ltd. China) (Fig. 2). The genotypes obtained from DNA sequencing were consistent with the original results. Statistical methods Statistical analysis was aided by SPSS for Windows version 16.0 (SPSS Inc., Chicago, IL). The chi square test was used for comparisons of the genotypic and allelic frequencies among the cases (CL, CP, and CLP) and the controls. The Hardy–Weinberg equilibrium was assessed for frequencies
Individuals with oral clefts were divided by subphenotype into the following 4 categories: cleft lip only (CL), cleft palate Table 1 The distribution of sex, type of cleft, and severity among the cases studied. Type of cleft
Male
Cleft lip: Unilateral 40 Bilateral 6 Cleft palate: Incomplete 20 Complete 0 Cleft lip and palate: Unilateral 42 Bilateral 18 Total 126
Female
Total
Male:female ratio
19 2
59 8
2.1:1 3:1
28 0
48 0
0.7:1 -
15 18 72
57 26 198
2.8:1 2.3:1 1.8:1
Figure 1. Patterns of restriction fragment length polymorphisms for Wnt10a C392T. Lane M is the DNA marker (D2000). Lanes 1, 2, 4, 5, 7, 8 indicate subjects who are wild homozygous genotype CC, having two bands (260 bp and 143 bp); lane 3 indicates the mutated homozygous genotype TT, which had only one band (403 bp); and lane 6 indicates the heterozygous genotype CT, which had 3 bands (260 bp, 143 bp, and 403 bp).
C. Feng et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 751–755
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Table 3 Genotypic and allelic distributions of Wnt10a C392T in patients and their parents. Patient
Father
Genotype (n = 96): CC 79 (82) CT 15 (16) TT 2 (2) Allele (n = 192): C 173 (90) T 19 (10)
Mother
85 (89) 11 (12) 0
80 (83) 15 (16) 1 (1)
181 (94) 11 (6)
175 (91) 17 (9)
Table 4 Results of the transmission disequilibrium test.
C T Chi square p value OR 95% CI
Figure 2. DNA sequencing results. (A) wild homozygous genotype CC. (B) mutated homozygous genotype TT. (C) heterozygous genotype CT.
in the variant in the cases, parents and control groups. We did a transmission disequilibrium test with the help of the Statistical Analysis for Genetic Epidemiology (S.A.G.E) software, version 6.3, to test for excess transmission of the target allele in these case–parents trios. Probabilities of less than 0.05 were accepted as significant, and an odds ratio (OR) > 1 designated an increased risk of disease.
Results For the parents, all cases, and the controls, genotypic and allelic distributions were within the Hardy-Weinberg
Transmitted
Not transmitted
15 19 6.07 0.01 3.52 1.27 to 9.75
25 9 -
equilibrium (data not shown). In the case-control analysis, we found an association between the Wnt10a gene and the phenotypes for the total clefts group and the cleft lip only group (Table 2). Table 3 shows the genotypic and allelic distributions of patients and their parents. For the heterozygote informative families, a transmission disequilibrium test was used to test whether the target alleles were over-transmitted or under-transmitted. There was a transmitted disequilibrium in C392T (Table 4).
Discussion Non-syndromic oral clefts are commonly divided into 3 groups: CL only, CLP, and CP only.16 Traditionally, the former two (thought to be aetiologically distinct from CP), are amalgamated into cleft lip with or without cleft palate (CL/P). Nevertheless, recent research has suggested that the latter 2 have distinct genetic origins and should be
Table 2 Genotypic and allelic distributions of Wnt10a C392T in patients and controls. Data are number (%) unless otherwise stated. Total Genotypes: CC CT TT Chi square p value Alleles: C T Chi square p value OR * 95% CI ∗
167 (84) 29 (15) 2 (1) 6.22 0.05 363 (92) 33 (8) 6.15 0.01 0.46 0.245 to 0.861
Cleft lip 55 (82) 11 (16) 1 (2) 6.73 0.04 121 (90) 13 (10) 6.14 0.01 0.389 0.180 to 0.841
Cleft palate 40 (83) 8 (17) 0 3.23 0.07 88 (92) 8 (8) 3.07 0.08 0.46 0.189 to 1.118
Cleft lip and palate 72 (87) 10 (12.) 1 (1) 3.43 0.18 154 (93) 12 (7) 2.51 0.11 0.536 0.245 to 1.172
CC (wild homozygous genotype) compared with CT/TT (heterozygous genotype)/mutated homozygous genotype).
Controls 172 (92) 15 (8) 0
359 (96) 15 (4)
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analysed separately.17,18 We have therefore also divided nonsyndromic oral clefts into 3 groups. The Wnt genes encode a large family of secreted glycoproteins that specify various cell lineages during embryogenesis.19,20 It has been suggested that the Wnt signalling pathway has an important role in formation of the lip and palate.7,13,21 Variations in Wnt genes (for example Wnt3, Wnt3a, Wnt5a, Wnt7a, Wnt8a, Wnt9b, and Wnt11), are associated with non-syndromic cleft lip with or without cleft palate.22,23 In this study we found an association between Wnt10a C392T and non-syndromic oral clefts. Although there were no significant differences in the case control study between the genotypic or allelic frequency distributions among cases and controls in the CP and CLP groups, the genotypic and allelic frequencies of C392T in the total group and the CL group did differ significantly from the controls. This suggested to us that because the OR (CC (wild hymozygous genotype) compared with CT (heterozygous genotype)/TT (mutated homozygous genotype)) was <1, the allele C might prove to be a protective factor. At the same time, the transmission disequilibrium test showed a transmitted disequilibrium in C392T and an OR of 3.519, which showed that allele T might be a risk factor for non-syndromic oral cleft. The C392T mutation is located in exon 3 and results in a p.A131 V substitution. Although this homozygous C392T transition was excluded on 400 control chromosomes of Pakistani origin and 400 control chromosomes of Swedish origin in a previous study by Sadia et al.,15 we found a Wnt10a C392T polymorphism and confirmed an association with non-syndromic oral clefts. Differences in the results may be the result of differences in race or ethnicity among the subject studied. The SNP C392T was found in 17 different, apparently unrelated, families who shared a relatively rare T allele inherited from their parents. These cases were all sporadic, and categorised as 3 patients with CP, 8 with CL, and 6 with CLP. Although it seemed that the T allele variant was inherited from both the mothers and fathers in these families, it was possibly more often inherited from their mothers, as there were 6 fathers who did not carry the T allele and only one mother who did. In one family, the mother’s genotype was TT and the father’s was CT, yet the patient’s genotype was CT. Our study has several potential limitations. First, we detected only one single nucleotide polymorphism, so the detection of more than one might strengthen our results. Secondly, the number of cases and elementary families was relatively limited. Increasing the size of the sample would increase the robustness of our investigations. Thirdly, the gene-genetic and gene-environmental interactions were not evaluated. Consequently, other well-designed studies of different ethnic groups are warranted to verify our findings. Collectively, our results show for the first time to our knowledge that Wnt10a is particularly attractive as a candidate gene for non-syndromic oral cleft in Chinese people of Han ethnicity. This may be helpful in extending our understanding of the aetiology of human oral clefts.
Conflict of interest statement We have no conflict of interest.
Statement of ethics and confirmation of patients’ permission to be studied The protocol was approved by the Ethics Committee of the University. Written informed consent was obtained from all the subjects or their guardians.
Ethics Statement/confirmation of patient permission The study protocol was approved by the Ethics Committee of the Affiliated Stomatological Hospital of China Medical University. Written informed consent was obtained from all the subjects or their guardians.
Acknowledgments We are grateful to all of the individuals who made this study possible, especially the patients, their parents, and the controls who voluntarily cooperated with this study. This research was supported by the Public Welfare Fund Project for Science of Liaoning Province (No: 2012002015), and a grant from the Science and Technology Project of Shenyang (grant no.: F13-220-9-73).
References 1. Yuan Q, Blanton SH, Hecht JT. Association of ABCA4 and MAFB with non-syndromic cleft lip with or without cleft palate. Am J Med Genet A 2011;155A:1469–71. 2. Dai L, Zhu J, Mao M, et al. Time trends in oral clefts in Chinese newborns: data from the Chinese National Birth Defects Monitoring Network. Birth Defects Res A Clin Mol Teratol 2010;88:41–7. 3. Berk NW, Cooper ME, Liu YE, Marazita ML. Social anxiety in Chinese adults with oral-facial clefts. Cleft Palate Craniofac J 2001;38:126–33. 4. Jugessur A, Murray JC. Orofacial clefting: recent insights into a complex trait. Curr Opin Genet Dev 2005;15:270–8. 5. Lidral AC, Moreno LM. Progress toward discerning the genetics of cleft lip. Curr Opin Pediatr 2005;17:731–9. 6. Jugessur A, Farlie PG, Kilpatrick N. The genetics of isolated orofacial clefts: from genotypes to subphenotypes. Oral Dis 2009;15:437–53. 7. Menezes R, Letra A, Kim AH, et al. Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol 2010;88:995–1000. 8. Andrade Filho PA, Letra A, Cramer A, et al. Insights from studies with oral cleft genes suggest associations between WNT-pathway genes and risk of oral cancer. J Dent Res 2011;90:740–6. 9. Chiquet BT, Blanton SH, Burt A, et al. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum Mol Genet 2008;17:2212–8. 10. Reddy S, Andl T, Bagasra A, et al. Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a
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11.
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as a target of Sonic hedgehog in hair follicle morphogenesis. Mech Devel 2001;107:69–82. Yamashiro T, Zheng L, Shitaku Y, et al. Wnt10a regulates dentin sialophosphoprotein mRNA expression and possibly links odontoblast differentiation and tooth morphogenesis. Differentiation 2007;75:452–62. Warner DR, Smith HS, Webb CL, Greene RM, Pisano MM. Expression of Wnts in the developing murine secondary palate. Int J Dev Biol 2009;53:1105–12. Beaty TH, Hetmanski JB, Fallin MD, et al. Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations. Hum Genet 2006;120:501–18. Feng C, Xu Z, Li Z, Zhang D, Liu Q, Lu L. Down-regulation of Wnt10a by RNA interference inhibits proliferation and promotes apoptosis in mouse embryonic palatal mesenchymal cells through Wnt/beta-catenin signaling pathway. J Physiol Biochem 2013;69:855–63. Nawaz S, Klar J, Wajid M, et al. WNT10A missense mutation associated with a complete odonto-onycho-dermal dysplasia syndrome. Eur J Hum Genet 2009;17:1600–5. Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374:1773–85.
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17. Rahimov F, Marazita ML, Visel A, Cooper ME, Hitchler MJ, Rubini M, et al. Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet 2008;40:1341–7. 18. Pan Y, Ma J, Zhang W, et al. IRF6 polymorphisms are associated with nonsyndromic orofacial clefts in a Chinese Han population. Am J Med Genet A 2010;152A:2505–11. 19. Nusse R. Wnt signaling and stem cell control. Cell Res 2008;18: 523–7. 20. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004;20:781–810. 21. Kokkinos MI, Wafai R, Wong MK, Newgreen DF, Thompson EW, Waltham M. Vimentin and epithelial-mesenchymal transition in human breast cancer–observations in vitro and in vivo. Cells Tissues Organs 2007;185:191–203. 22. Cawthorn WP, Bree AJ, Yao Y, Du B, Hemati N, Martinez-Santibanez G, et al. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a beta-catenin-dependent mechanism. Bone 2012;50:477–89. 23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8.
British Journal of Oral and Maxillofacial Surgery 52 (2014) 751–755
C392T polymorphism of the Wnt10a gene in non-syndromic oral cleft in a northeastern Chinese population Cuijuan Feng a,∗∗ , Weiyi Duan b , Dan Zhang a , Enjiao Zhang b , Zhongfei Xu b , Li Lu b,∗ a b
Department of Orthodontics, School of Stomatology, China Medical University, Shenyang 110002, PR China Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University, Shenyang 110002, PR China
Accepted 2 June 2014 Available online 20 June 2014
Abstract Non-syndromic oral cleft is one of the most common congenital malformations, and more than 40 genes may be involved in its aetiology. Recent studies have shown that the Wnt10a gene may also contribute. We recruited 198 patients with non-syndromic oral clefts, comprising 96 elementary families (restricted to the patients and their parents) and 187 controls, to investigate their associations with the risk of such clefts and their subgroups in a Chinese Han population. The variant evaluated in this study was a single nucleotide polymorphism – specifically, a missense mutation C392T of Wnt10a. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) was used to genotype the marker, and case–control and family-based associations were analysed. Although in the case-control study there were no significant differences in frequency distributions of genotypes or alleles between cases and controls in the groups with cleft palate and cleft lip and palate, the genotypic and allelic frequencies of C392T in the total groups and the group with cleft lip alone differed significantly from those in the controls (p = 0.04, and 0.01, respectively). A transmission disequilibrium test showed a transmitted disequilibrium in C392T. In conclusion, we found an association between the C392T variant and non-syndromic oral clefts. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: non-syndromic oral cleft; Wnt10a gene polymorphism; polymerase chain reaction; restriction fragment length polymorphism; case-control study; transmission disequilibrium test.
Introduction Non-syndromic oral cleft is one of the most common congenital malformations, and it affects 135,000 babies worldwide each year.1 Chinese newborns have a high prevalence at birth of 1.42/1000,2 and they are a considerable burden on public ∗
Corresponding author. Tel.: +86-13940163503; fax: +86-24-22892645. Address correspondence to: Department of Orthodontics, School of Stomatology, China Medical University, No.117 North Nanjing Street, Shenyang 110002, People’s Republic of China. Phone: +86-18698848565; Fax: +86-24-22892645. E-mail addresses: [email protected] (C. Feng), [email protected] (W. Duan), cmu [email protected] (D. Zhang), [email protected] (E. Zhang), [email protected] (Z. Xu), [email protected] (L. Lu). ∗∗
health services because of the immediate and long-term medical costs and the social impact on patients and their families.3 The identification of genetic and environmental risk factors for non-syndromic oral clefts has been the subject of intensive research for several decades, but the exact mechanism is still not clear.4,5 It has been suggested that more than 40 genes, including IRF6, MSX1, TGFB3, FOXE1, FGFR1, FGFR2, FGF8, PDGFC, CRISPLD2, PVRL1, GABRB3, MSX2, SATB2, TBX10, TBX22, GLI2, JAG2, MTHFR, RARA, LHX8, SKI, SPRY2, Wnt3a, and Wnt9b may be involved in the aetiology.6 However, it is still a challenge to isolate the aetiological roles of the candidate genes. Many studies have indicated that the Wnt family of genes plays a critical part in the development of the lip and palate.7–9 In more recent years, the role of Wnt10a in ectoderm has
http://dx.doi.org/10.1016/j.bjoms.2014.06.001 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
752
C. Feng et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 751–755
attracted widespread attention, as this interaction is closely related to the development of teeth and skin.10,11 A study by Warner et al. found that the expression of Wnt10a is significantly increased at E13.5 and E14.5 in palatal tissue in fetal rats.12 Beaty et al. also found that the Wnt6-Wnt10a gene cluster is moderately but consistently associated with cleft lip and palate (CLP) in patients from 3 different countries and regions.13 In a recent study we found that downregulation of Wnt10a by interference from RNA inhibited the proliferation of cells, and induced arrest of the cell cycle and apoptosis, in murine embryonic palatal mesenchymal cells.14 Previous studies have shown that the Wnt10a gene may contribute to the aetiology of non-syndromic oral clefts, but there have been no previous reports of the direct association between them. The purpose of the present study, therefore, was to find out if the Wnt10a gene was a risk factor for non-syndromic oral clefts within a specific sample of northeastern Chinese.
Materials and Methods Subjects We studied 198 patients with non-syndromic oral clefts, including 96 elementary families (patients and their parents), and 187 controls. All subjects were recruited between 2008 and 2011 from the Department of Oral and Maxillofacial Surgery of the Affiliated Stomatological Hospital of China Medical University (Shenyang, Liaoning, the People’s Republic of China). After clinical evaluation and taking detailed family histories, syndromic patients were excluded and only those with an isolated cleft were studied. All subjects were of Han Chinese origin. The controls who had congenital malformations of the body, malignant tumours, or a family history of genetic disease were also excluded. The controls were selected to be, as far as possible, born in the same region and to have the same sex distribution as the cases (Table 1). Subphenotypes of clefts
only (CP), cleft lip with cleft palate (CLP), and the total group of patients with clefts. Selection of the single nucleotide polymorphism The single nucleotide polymorphism C392T in Wnt10a that was selected in this study was based on the reports of Sadia et al,15 and is proposed to be a pathogenic variant of odonto-onychodermal dysplasia (MIM 257980). It has not yet, however, been confirmed to be associated with nonsyndromic oral clefts. Amplification and genotyping by polymerase chain reaction (PCR) Genomic DNA were extracted from peripheral blood using the DNA isolation kit for mammalian blood (Tiangen Biotech, Beijing, China). The reference sequence (wildtype sequence) of the Wnt10a gene was obtained from the NCBI GenBank (NM 025216.2). The PCR–restriction fragment length polymorphism assay was applied to genotype Wnt10a C392T. Specific primer pairs were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto CA, USA). The PCR primers were F-5 GAACAGGAG AAGGGCGTACAA-3 , and R-5 TGTAAGCGGTGCAGCTTCCTAC-3 . The restriction enzyme was Eco47 III and the fragment length was 403 bp. A representative gel of the digestion patterns is shown in Fig. 1. For quality control, we retyped 10% of the samples by DNA sequencing (Sangon Biotech (Shanghai) Co., Ltd. China) (Fig. 2). The genotypes obtained from DNA sequencing were consistent with the original results. Statistical methods Statistical analysis was aided by SPSS for Windows version 16.0 (SPSS Inc., Chicago, IL). The chi square test was used for comparisons of the genotypic and allelic frequencies among the cases (CL, CP, and CLP) and the controls. The Hardy–Weinberg equilibrium was assessed for frequencies
Individuals with oral clefts were divided by subphenotype into the following 4 categories: cleft lip only (CL), cleft palate Table 1 The distribution of sex, type of cleft, and severity among the cases studied. Type of cleft
Male
Cleft lip: Unilateral 40 Bilateral 6 Cleft palate: Incomplete 20 Complete 0 Cleft lip and palate: Unilateral 42 Bilateral 18 Total 126
Female
Total
Male:female ratio
19 2
59 8
2.1:1 3:1
28 0
48 0
0.7:1 -
15 18 72
57 26 198
2.8:1 2.3:1 1.8:1
Figure 1. Patterns of restriction fragment length polymorphisms for Wnt10a C392T. Lane M is the DNA marker (D2000). Lanes 1, 2, 4, 5, 7, 8 indicate subjects who are wild homozygous genotype CC, having two bands (260 bp and 143 bp); lane 3 indicates the mutated homozygous genotype TT, which had only one band (403 bp); and lane 6 indicates the heterozygous genotype CT, which had 3 bands (260 bp, 143 bp, and 403 bp).
C. Feng et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 751–755
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Table 3 Genotypic and allelic distributions of Wnt10a C392T in patients and their parents. Patient
Father
Genotype (n = 96): CC 79 (82) CT 15 (16) TT 2 (2) Allele (n = 192): C 173 (90) T 19 (10)
Mother
85 (89) 11 (12) 0
80 (83) 15 (16) 1 (1)
181 (94) 11 (6)
175 (91) 17 (9)
Table 4 Results of the transmission disequilibrium test.
C T Chi square p value OR 95% CI
Figure 2. DNA sequencing results. (A) wild homozygous genotype CC. (B) mutated homozygous genotype TT. (C) heterozygous genotype CT.
in the variant in the cases, parents and control groups. We did a transmission disequilibrium test with the help of the Statistical Analysis for Genetic Epidemiology (S.A.G.E) software, version 6.3, to test for excess transmission of the target allele in these case–parents trios. Probabilities of less than 0.05 were accepted as significant, and an odds ratio (OR) > 1 designated an increased risk of disease.
Results For the parents, all cases, and the controls, genotypic and allelic distributions were within the Hardy-Weinberg
Transmitted
Not transmitted
15 19 6.07 0.01 3.52 1.27 to 9.75
25 9 -
equilibrium (data not shown). In the case-control analysis, we found an association between the Wnt10a gene and the phenotypes for the total clefts group and the cleft lip only group (Table 2). Table 3 shows the genotypic and allelic distributions of patients and their parents. For the heterozygote informative families, a transmission disequilibrium test was used to test whether the target alleles were over-transmitted or under-transmitted. There was a transmitted disequilibrium in C392T (Table 4).
Discussion Non-syndromic oral clefts are commonly divided into 3 groups: CL only, CLP, and CP only.16 Traditionally, the former two (thought to be aetiologically distinct from CP), are amalgamated into cleft lip with or without cleft palate (CL/P). Nevertheless, recent research has suggested that the latter 2 have distinct genetic origins and should be
Table 2 Genotypic and allelic distributions of Wnt10a C392T in patients and controls. Data are number (%) unless otherwise stated. Total Genotypes: CC CT TT Chi square p value Alleles: C T Chi square p value OR * 95% CI ∗
167 (84) 29 (15) 2 (1) 6.22 0.05 363 (92) 33 (8) 6.15 0.01 0.46 0.245 to 0.861
Cleft lip 55 (82) 11 (16) 1 (2) 6.73 0.04 121 (90) 13 (10) 6.14 0.01 0.389 0.180 to 0.841
Cleft palate 40 (83) 8 (17) 0 3.23 0.07 88 (92) 8 (8) 3.07 0.08 0.46 0.189 to 1.118
Cleft lip and palate 72 (87) 10 (12.) 1 (1) 3.43 0.18 154 (93) 12 (7) 2.51 0.11 0.536 0.245 to 1.172
CC (wild homozygous genotype) compared with CT/TT (heterozygous genotype)/mutated homozygous genotype).
Controls 172 (92) 15 (8) 0
359 (96) 15 (4)
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analysed separately.17,18 We have therefore also divided nonsyndromic oral clefts into 3 groups. The Wnt genes encode a large family of secreted glycoproteins that specify various cell lineages during embryogenesis.19,20 It has been suggested that the Wnt signalling pathway has an important role in formation of the lip and palate.7,13,21 Variations in Wnt genes (for example Wnt3, Wnt3a, Wnt5a, Wnt7a, Wnt8a, Wnt9b, and Wnt11), are associated with non-syndromic cleft lip with or without cleft palate.22,23 In this study we found an association between Wnt10a C392T and non-syndromic oral clefts. Although there were no significant differences in the case control study between the genotypic or allelic frequency distributions among cases and controls in the CP and CLP groups, the genotypic and allelic frequencies of C392T in the total group and the CL group did differ significantly from the controls. This suggested to us that because the OR (CC (wild hymozygous genotype) compared with CT (heterozygous genotype)/TT (mutated homozygous genotype)) was <1, the allele C might prove to be a protective factor. At the same time, the transmission disequilibrium test showed a transmitted disequilibrium in C392T and an OR of 3.519, which showed that allele T might be a risk factor for non-syndromic oral cleft. The C392T mutation is located in exon 3 and results in a p.A131 V substitution. Although this homozygous C392T transition was excluded on 400 control chromosomes of Pakistani origin and 400 control chromosomes of Swedish origin in a previous study by Sadia et al.,15 we found a Wnt10a C392T polymorphism and confirmed an association with non-syndromic oral clefts. Differences in the results may be the result of differences in race or ethnicity among the subject studied. The SNP C392T was found in 17 different, apparently unrelated, families who shared a relatively rare T allele inherited from their parents. These cases were all sporadic, and categorised as 3 patients with CP, 8 with CL, and 6 with CLP. Although it seemed that the T allele variant was inherited from both the mothers and fathers in these families, it was possibly more often inherited from their mothers, as there were 6 fathers who did not carry the T allele and only one mother who did. In one family, the mother’s genotype was TT and the father’s was CT, yet the patient’s genotype was CT. Our study has several potential limitations. First, we detected only one single nucleotide polymorphism, so the detection of more than one might strengthen our results. Secondly, the number of cases and elementary families was relatively limited. Increasing the size of the sample would increase the robustness of our investigations. Thirdly, the gene-genetic and gene-environmental interactions were not evaluated. Consequently, other well-designed studies of different ethnic groups are warranted to verify our findings. Collectively, our results show for the first time to our knowledge that Wnt10a is particularly attractive as a candidate gene for non-syndromic oral cleft in Chinese people of Han ethnicity. This may be helpful in extending our understanding of the aetiology of human oral clefts.
Conflict of interest statement We have no conflict of interest.
Statement of ethics and confirmation of patients’ permission to be studied The protocol was approved by the Ethics Committee of the University. Written informed consent was obtained from all the subjects or their guardians.
Ethics Statement/confirmation of patient permission The study protocol was approved by the Ethics Committee of the Affiliated Stomatological Hospital of China Medical University. Written informed consent was obtained from all the subjects or their guardians.
Acknowledgments We are grateful to all of the individuals who made this study possible, especially the patients, their parents, and the controls who voluntarily cooperated with this study. This research was supported by the Public Welfare Fund Project for Science of Liaoning Province (No: 2012002015), and a grant from the Science and Technology Project of Shenyang (grant no.: F13-220-9-73).
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