Genome-wide association studies have identified over 80 loci associated with nonsyndromic orofacial cleft (NSOC), yet substantial heritability remains unexplained. Insights from syndromic orofacial cleft (SOC) implicated genes could help bridge this gap.
Design
A case-control association study in a Han Chinese cohort was conducted to evaluate the association between SOC-implicated genes and NSOC subtypes using association, linkage disequilibrium (LD), and haplotype analyses.
Setting
Tertiary medical center.
Patients, Participants
The study included 1626 cases of non-syndromic cleft lip with or without cleft palate, 930 cases of non-syndromic cleft palate only, and 2255 controls.
Interventions
Peripheral blood (cases) and umbilical cord blood (controls) were collected for DNA extraction.
Main Outcome Measures
Allelic (Pearson’ s χ2, 1 df) and genotypic (Pearson’ s χ2, 2 df) associations between SNPs and NSOC subtypes were evaluated, with odds ratios (ORs) and 95% confidence intervals (CIs). LD and sliding-window haplotype association analyses were performed in Haploview. SNPs with minor allele frequency (MAF) >0.05 and in Hardy-Weinberg equilibrium in controls were analyzed. The significance threshold was P < 1.27 × 10−5 after Bonferroni correction.
Results
Allelic analysis identified 23 SNPs that were significantly associated with NSOC subtypes (lowest P = 2.02 × 10−22). Genotypic analysis identified 39 significant SNPs (lowest P = 1.09 × 10−36). Signals mapped to 7 genes. Haplotype analyses revealed a shared causal variant block at MYMK and TWIST2, and allelic heterogeneity at NEDD4L.
Conclusions
We identified MYMK, TWIST2, and NEDD4L as NSOC-associated genes. Using SOC genes as prior knowledge reveals loci missed by standard GWAS, offering key insights into NSOC pathogenesis.
NasreddineGEl HajjJGhassibe-SabbaghM. Orofacial clefts embryology, classification, epidemiology, and genetics. Mutat Res Rev Mutat Res. 2021;787(1):108373. doi:10.1016/j.mrrev.2021.108373
2.
DixonMJMarazitaMLBeatyTHMurrayJC. Cleft lip and palate: Understanding genetic and environmental influences. Nat Rev Genet. 2011;12(3):167‐178. doi:10.1038/nrg2933
3.
AladeAAwotoyeWButaliA. Genetic and epigenetic studies in non-syndromic oral clefts. Oral Dis. 2022;28(5):1339‐1350. doi:10.1111/odi.14146
4.
Robinson KR, Curtis SW, Paschall JE, Adeyemo WL, Beaty TH, Butali A, Buxó CJ, Cutler DJ, Epstein MP, Gowans LJJ, et al. Distinguishing syndromic and nonsyndromic cleft palate through analysis of protein-altering de novo variants in 818 trios. Am J Hum Genet. 2025;112(11):2679‐2692. doi:10.1016/j.ajhg.2025.09.009
5.
Rosero SalazarDHCarvajal MonroyPLWagenerFVon den HoffJW. Orofacial muscles: Embryonic development and regeneration after injury. J Dent Res. 2020;99(2):125‐132. doi:10.1177/0022034519883673
SharifHZiaeiHRezaeiN. Stem cell-based regenerative approaches for the treatment of cleft lip and palate: A comprehensive review. Stem Cell Rev Rep. 2024;20(3):637‐655. doi:10.1007/s12015-024-10676-9
8.
Leslie EJ, Carlson JC, Shaffer JR, Butali A, Buxó CJ, Castilla EE, Christensen K, Deleyiannis FW, Leigh Field L, Hecht JT, et al. Genome-wide meta-analyses of nonsyndromic orofacial clefts identify novel associations between FOXE1 and all orofacial clefts, and TP63 and cleft lip with or without cleft palate. Hum Genet2017;136(3):275‐286. doi:10.1007/s00439-016-1754-7
9.
Ludwig KU, Böhmer AC, Bowes J, Nikolic M, Ishorst N, Wyatt N, Hammond NL, Gölz L, Thieme F, Barth S, et al. Imputation of orofacial clefting data identifies novel risk loci and sheds light on the genetic background of cleft lip ± cleft palate and cleft palate only. Hum Mol Genet. 2017;26(4):829‐842. doi:10.1093/hmg/ddx012
10.
Sun Y, Huang Y, Yin A, Pan Y, Wang Y, Wang C, Du Y, Wang M, Lan F, Hu Z, et al. Genome-wide association study identifies a new susceptibility locus for cleft lip with or without a cleft palate. Nat Commun2015;6:6414. doi:10.1038/ncomms7414
11.
Yu Y, Zuo X, He M, Gao J, Fu Y, Qin C, Meng L, Wang W, Song Y, Cheng Y, et al. Genome-wide analyses of non-syndromic cleft lip with palate identify 14 novel loci and genetic heterogeneity. Nat Commun2017;8:14364. doi:10.1038/ncomms14364
12.
Carlson JC, Anand D, Butali A, Buxo CJ, Christensen K, Deleyiannis F, Hecht JT, Moreno LM, Orioli IM, Padilla C, et al. A systematic genetic analysis and visualization of phenotypic heterogeneity among orofacial cleft GWAS signals. Genet Epidemiol2019;43(6):704‐716. doi:10.1002/gepi.22214
13.
Gusev A, Ko A, Shi H, Bhatia G, Chung W, Penninx BW, Jansen R, de Geus EJ, Boomsma DI, Wright FA, et al. Integrative approaches for large-scale transcriptome-wide association studies. Nat Genet2016;48(3):245‐252. doi:10.1038/ng.3506
14.
Gusev A, Lee SH, Trynka G, Finucane H, Vilhjálmsson BJ, Xu H, Zang C, Ripke S, Bulik-Sullivan B, Stahl E, et al. Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases. Am J Hum Genet2014;95(5):535‐552. doi:10.1016/j.ajhg.2014.10.004
15.
Lou S, Zhu G, Xing C, Hao S, Lin J, Xu J, Li D, Du Y, Mi C, Sun L, et al. Transcriptome-wide association identifies KLC1 as a regulator of mitophagy in non-syndromic cleft lip with or without palate. iMeta. 2024;3(6):e262. doi:10.1002/imt2.262
16.
Kichaev G, Yang WY, Lindstrom S, Hormozdiari F, Eskin E, Price AL, Kraft P, Pasaniuc B. Integrating functional data to prioritize causal variants in statistical fine-mapping studies. PLoS Genet2014;10(10):e1004722. doi:10.1371/journal.pgen.1004722
17.
Kondo S, Schutte BC, Richardson RJ, Bjork BC, Knight AS, Watanabe Y, Howard E, de Lima RL, Daack-Hirsch S, Sander A, et al. Mutations in IRF6 cause van der woude and popliteal pterygium syndromes. Nat Genet. 2002;32(2):285‐289. doi:10.1038/ng985
18.
Rahimov F, Marazita ML, Visel A, Cooper ME, Hitchler MJ, Rubini M, Domann FE, Govil M, Christensen K, Bille C, et al. Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet2008;40(11):1341‐1347. doi:10.1038/ng.242
19.
Peyrard-Janvid M, Leslie EJ, Kousa YA, Smith TL, Dunnwald M, Magnusson M, Lentz BA, Unneberg P, Fransson I, Koillinen HK, et al. Dominant mutations in GRHL3 cause van der woude syndrome and disrupt oral periderm development. Am J Hum Genet2014;94(1):23‐32. doi:10.1016/j.ajhg.2013.11.009
20.
Eshete MA, Liu H, Li M, Adeyemo WL, Gowans LJJ, Mossey PA, Busch T, Deressa W, Donkor P, Olaitan PB, et al. Loss-of-Function GRHL3 variants detected in African patients with isolated cleft palate. J Dent Res. 2018;97(1):41‐48. doi:10.1177/0022034517729819
21.
ZhenLYangYDXuLLCaoQLiDZ. Fetal micrognathia in the first trimester: An ominous finding even after a normal array. Eur J Obstet Gynecol Reprod Biol2021;263:176‐180. doi:10.1016/j.ejogrb.2021.06.031
22.
Padula MA, Naing K, Wenger TL, Ahmad I, Coghill CH, 3rd, Wild KT, Rottgers SA, Resnick CM, Goldstein J, Ehsan Z, et al. Spectrum of disease in hospitalized newborns with congenital micrognathia: A cohort of 3,236 infants at north American tertiary-care intensive care units. J Pediatr2024;265:113799. doi:10.1016/j.jpeds.2023.113799
23.
PriceKEHaddadYFakhouriWD. Analysis of the relationship between micrognathia and cleft palate: A systematic review. Cleft Palate Craniofac J. 2016;53(2):e34‐e44. doi:10.1597/14-238
24.
Motch PerrineSMWuMHolmesGBjorkBCJabsEWRichtsmeierJT. Phenotypes, Developmental basis, and genetics of Pierre Robin complex. J Dev Biol. 2020;8(4):30. doi:10.3390/jdb8040030
25.
Huang L, Jia Z, Shi Y, Du Q, Shi J, Wang Z, Mou Y, Wang Q, Zhang B, Wang Q, et al. Genetic factors define CPO and CLO subtypes of nonsyndromicorofacial cleft. PLoS Genet2019;15(10):e1008357. doi:10.1371/journal.pgen.1008357
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, et al. PLINK: A tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet2007;81(3):559‐575. doi:10.1086/519795
28.
LinckEBatteyCJ. Minor allele frequency thresholds strongly affect population structure inference with genomic data sets. Mol Ecol Resour2019;19(3):639‐647. doi:10.1111/1755-0998.12995
29.
Wittke-ThompsonJKPluzhnikovACoxNJ. Rational inferences about departures from hardy-weinberg equilibrium. Am J Hum Genet. 2005;76(6):967‐986. doi:10.1086/430507
30.
BarrettJCFryBMallerJDalyMJ. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21(2):263‐265. doi:10.1093/bioinformatics/bth457
31.
Yu Y, Zhen Q, Chen W, Yu Y, Li Z, Wang Y, Fan W, Luo S, Wang D, Bai Y, et al. Genome-wide meta-analyses identify five new risk loci for nonsyndromic orofacial clefts in the Chinese han population. Mol Genet Genomic Med. 2023;11(10):e2226. doi:10.1002/mgg3.2226
32.
Millay DP, O'Rourke JR, Sutherland LB, Bezprozvannaya S, Shelton JM, Bassel-Duby R, Olson EN Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature. 2013;499(7458):301‐305. doi:10.1038/nature12343
33.
Di Gioia SA, Connors S, Matsunami N, Cannavino J, Rose MF, Gilette NM, Artoni P, de Macena Sobreira NL, Chan WM, Webb BD, et al. A defect in myoblast fusion underlies carey-fineman-ziter syndrome. Nat Commun. 2017;8:16077. doi:10.1038/ncomms16077
34.
Huang W, Qian Z, Zhang J, Ding Y, Wang B, Lin J, Zhang X, Zhao H, Chen F. Single-cell atlas of developing mouse palates reveals cellular and molecular transitions in periderm cell fate. Genomics Proteomics Bioinformatics. 2025;23(1):qzaf013. doi:10.1093/gpbjnl/qzaf013
35.
NaranSFordMLoseeJE. What’s new in cleft palate and velopharyngeal dysfunction management?Plast Reconstr Surg2017;139(6):1343e‐1355e. doi:10.1097/prs.0000000000003335
36.
Marchegiani S, Davis T, Tessadori F, van Haaften G, Brancati F, Hoischen A, Huang H, Valkanas E, Pusey B, Schanze D, et al. Recurrent mutations in the basic domain of TWIST2 cause ablepharon macrostomia and barber-say syndromes. Am J Hum Genet. 2015;97(1):99‐110. doi:10.1016/j.ajhg.2015.05.017
37.
ZengWGuSYuYFengYXiaoMFengXH. ZNF451 Stabilizes TWIST2 through SUMOylation and promotes epithelial-mesenchymal transition. Am J Cancer Res2021;11(3):898‐915.
38.
HuangZZhiYCaoHBianZHeM. Exosomes derived from human palatal mesenchymal cells mediate intercellular communication during palatal fusion by promoting oral epithelial cell migration. Int J Nanomed2024;19:3109‐3121. doi:10.2147/ijn.S451491
39.
Liu H, Wang N, Yang R, Luan J, Cao M, Zhai C, Wang S, Wei M, Wang D, Qiao J, et al. E3 ubiquitin ligase NEDD4L negatively regulates skin tumorigenesis by inhibiting IL-6/GP130 signaling pathway. J Invest Dermatol. 2024;144(11):2453‐2464.e11. doi:10.1016/j.jid.2024.03.030
40.
WiszniakSHarveyNSchwarzQ. Cell autonomous roles of Nedd4 in craniofacial bone formation. Dev Biol2016;410(1):98‐107. doi:10.1016/j.ydbio.2015.12.001
41.
Wiszniak S, Kabbara S, Lumb R, Scherer M, Secker G, Harvey N, Kumar S, Schwarz Q. The ubiquitin ligase Nedd4 regulates craniofacial development by promoting cranial neural crest cell survival and stem-cell like properties. Dev Biol2013;383(2):186‐200. doi:10.1016/j.ydbio.2013.09.024
42.
Broix L, Jagline H, Ivanova E, Schmucker S, Drouot N, Clayton-Smith J, Pagnamenta AT, Metcalfe KA, Isidor B, Louvier UW, et al. Mutations in the HECT domain of NEDD4L lead to AKT-mTOR pathway deregulation and cause periventricular nodular heterotopia. Nat Genet2016;48(11):1349‐1358. doi:10.1038/ng.3676
43.
Kato K, Miya F, Hori I, Ieda D, Ohashi K, Negishi Y, Hattori A, Okamoto N, Kato M, Tsunoda T, et al. A novel missense mutation in the HECT domain of NEDD4L identified in a girl with periventricular nodular heterotopia, polymicrogyria and cleft palate. J Hum Genet2017;62(9):861‐863. doi:10.1038/jhg.2017.53
44.
ChoKYKelleyBPMonierDLeeBSzabo-RogersHNapieralaD. Trps1 regulates developmenT of craniofacial skeleton and is required for the initiation of palatal shelves fusion. Front Physiol2019;10:513. doi:10.3389/fphys.2019.00513
McMillin MJ, Beck AE, Chong JX, Shively KM, Buckingham KJ, Gildersleeve HI, Aracena MI, Aylsworth AS, Bitoun P, Carey JC, et al. Mutations in PIEZO2 cause gordon syndrome, marden-walker syndrome, and distal arthrogryposis type 5. Am J Hum Genet. 2014;94(5):734‐744. doi:10.1016/j.ajhg.2014.03.015
47.
HuangXGoudySLKetovaTLitingtungYChiangC. Gli3-deficient mice exhibit cleft palate associated with abnormal tongue development. Dev Dyn. 2008;237(10):3079‐3087. doi:10.1002/dvdy.21714
48.
HallCFloresMVMurisonGCrosierKCrosierP. An essential role for zebrafish Fgfrl1 during gill cartilage development. Mech Dev2006;123(12):925‐940. doi:10.1016/j.mod.2006.08.006
49.
Wong SY, Beamer LJ, Gadomski T, Honzik T, Mohamed M, Wortmann SB, Brocke Holmefjord KS, Mork M, Bowling F, Sykut-Cegielska J, et al. Defining the phenotype and assessing severity in phosphoglucomutase-1 deficiency. J Pediatr. 2016;175:130‐136.e8. doi:10.1016/j.jpeds.2016.04.021
50.
Kumari P, Friedman RZ, Curtis SW, Pi L, Paraiso K, Visel A, Rhea L, Dunnwald M, Patni AP, Mar D, et al. Identification of functional non-coding variants associated with orofacial cleft. Nat Commun2025;16(1):6545. doi:10.1038/s41467-025-61734-w
51.
Li MJ, Kumari P, Lin YS, Yao ML, Zhang BH, Yin B, Duan SJ, Cornell RA, Marazita ML, Shi B, et al. A variant in the IRF6 promoter associated with the risk for orofacial clefting. J Dent Res2023;102(7):806‐813. doi:10.1177/00220345231165210
52.
BushJOJiangR. Palatogenesis: Morphogenetic and molecular mechanisms of secondary palate development. Development. 2012;139(2):231‐243. doi:10.1242/dev.067082
53.
Sivertsen A, Wilcox AJ, Skjaerven R, Vindenes HA, Abyholm F, Harville E, Lie RT. Familial risk of oral clefts by morphological type and severity: Population based cohort study of first degree relatives. Br Med J2008;336(7641):432‐434. doi:10.1136/bmj.39458.563611.AE
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