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Genetic association and functional validation of ZFP36L2 in non-syndromic orofacial cleft subtypes

Journal of Human Genetics volume 69, pages 139–144 (2024)Cite this article

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A Correction to this article was published on 05 March 2024

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Abstract

Background

Non-syndromic orofacial cleft (NSOC) is one of the most common craniofacial malformations with complex etiology. This study aimed to explore the role of specific SNPs in ZFP36L2 and its functional relevance in zebrafish models.

Methods

We analyzed genetic data of the Chinese Han population from two previous GWAS, comprising of 2512 cases and 2255 controls. Based on the Hardy-Weinberg Equilibrium (HWE) and minor allele frequency (MAF), SNPs in the ZFP36L2 were selected for association analysis. In addition, zebrafish models were used to clarify the in-situ expression pattern of zfp36l2 and the impact of its Morpholino-induced knockdown.

Results

Via association analysis, rs7933 in ZFP36L2 was significantly associated with various non-syndromic cleft lip-only subtypes, potentially conferring a protective effect. Zebrafish embryos showed elevated expression of zfp36l2 in the craniofacial region during critical stages of oral cavity formation. Furthermore, Morpholino-induced knockdown of zfp36l2 led to craniofacial abnormalities, including cleft lip, which was partially rescued by the addition of zfp36l2 mRNA.

Conclusion

Our findings highlight the significance of ZFP36L2 in the etiology of NSOC, supported by both human genetic association data and functional studies in zebrafish. These results pave the way for further exploration of targeted interventions for craniofacial malformations.

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References

  1. Smarius B, Loozen C, Manten W, Bekker M, Pistorius L, Breugem C. Accurate diagnosis of prenatal cleft lip/palate by understanding the embryology. World J Methodol. 2017;7:93–100.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Rahimov F, Jugessur A, Murray JC. Genetics of nonsyndromic orofacial clefts. Cleft Palate Craniofac J. 2012;49:73–91.

    Article  PubMed  Google Scholar 

  3. Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet. 2011;12:167–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beaty TH, Hetmanski JB, Zeiger JS, Fan YT, Liang KY, VanderKolk CA, et al. Testing candidate genes for non-syndromic oral clefts using a case-parent trio design. Genet Epidemiol. 2002;22:1–11.

    Article  PubMed  Google Scholar 

  5. Birnbaum S, Ludwig KU, Reutter H, Herms S, Steffens M, Rubini M, et al. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet. 2009;41:473–7.

    Article  CAS  PubMed  Google Scholar 

  6. Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet. 2010;42:24–6.

    Article  CAS  PubMed  Google Scholar 

  7. Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet. 2010;42:525–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, Paul A, et al. Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet. 2012;44:968–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sun Y, Huang Y, Yin A, Pan Y, Wang Y, Wang C, et al. Genome-wide association study identifies a new susceptibility locus for cleft lip with or without a cleft palate. Nat Commun. 2015;6:6414.

    Article  ADS  PubMed  Google Scholar 

  10. Leslie EJ, Carlson JC, Shaffer JR, Feingold E, Wehby G, Laurie CA, et al. A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13. Hum Mol Genet 2016;25:2862–72.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu Y, Zuo X, He M, Gao J, Fu Y, Qin C, et al. Genome-wide analyses of non-syndromic cleft lip with palate identify 14 novel loci and genetic heterogeneity. Nat Commun. 2017;8:14364.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang L, Jia Z, Shi Y, Du Q, Shi J, Wang Z, et al. Genetic factors define CPO and CLO subtypes of nonsyndromicorofacial cleft. PLoS Genet. 2019;15:e1008357.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Li B, Yong L, Yu Y, Yu Y, Zhen Q, Ge H, et al. Genome-wide analyses of nonsyndromic cleft lip with or without palate identify 20 new risk loci in the Chinese Han population. J Genet Genomics. 2022;49:903–5.

    Article  CAS  PubMed  Google Scholar 

  14. Leslie EJ, Liu H, Carlson JC, Shaffer JR, Feingold E, Wehby G, et al. A genome-wide association study of nonsyndromic cleft palate identifies an etiologic missense variant in GRHL3. Am J Hum Genet. 2016;98:744–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Butali A, Mossey PA, Adeyemo WL, Eshete MA, Gowans LJJ, Busch TD, et al. Genomic analyses in African populations identify novel risk loci for cleft palate. Hum Mol Genet. 2019;28:1038–51.

    Article  CAS  PubMed  Google Scholar 

  16. He M, Zuo X, Liu H, Wang W, Zhang Y, Fu Y, et al. Genome-wide analyses identify a novel risk locus for nonsyndromic cleft palate. J Dent Res. 2020;99:1461–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li MJ, Kumari P, Lin YS, Yao ML, Zhang BH, Yin B, et al. A variant in the IRF6 promoter associated with the risk for orofacial clefting. J Dent Res. 2023;102:806–13.

    Article  CAS  PubMed  Google Scholar 

  18. Sun JL, Shi JY, Yin B, Lin YS, Shi B, Jia ZL. Association analysis of SNPs in GRHL3, FAF1, and KCNJ2 with NSCPO sub-phenotypes in Han Chinese. Oral Dis. 2022;28:2204–14.

    Article  PubMed  Google Scholar 

  19. Zhang JX, Arneja JS. Evidence-based medicine: the bilateral cleft lip repair. Plast Reconstr Surg. 2017;140:152E–65E.

    Article  CAS  PubMed  Google Scholar 

  20. Carlson JC, Taub MA, Feingold E, Beaty TH, Murray JC, Marazita ML, et al. Identifying genetic sources of phenotypic heterogeneity in orofacial clefts by targeted sequencing. Birth Defects Res. 2017;109:1030–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Curtis SW, Chang D, Lee MK, Shaffer JR, Indencleef K, Epstein MP, et al. The PAX1 locus at 20p11 is a potential genetic modifier for bilateral cleft lip. HGG Adv. 2021;2:100025.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Yin B, Shi JY, Lin YS, Shi B, Jia ZL. SNPs at TP63 gene was specifically associated with right-side cleft lip in Han Chinese population. Oral Dis. 2021;27:559–66.

    Article  PubMed  Google Scholar 

  23. Zheng W, Sha QQ, Hu H, Meng F, Zhou Q, Chen X, et al. Biallelic variants in ZFP36L2 cause female infertility characterised by recurrent preimplantation embryo arrest. J Med Genet. 2022;59:850–7.

    Article  CAS  PubMed  Google Scholar 

  24. Xing R, Zhou Y, Yu J, Yu Y, Nie Y, Luo W, et al. Whole-genome sequencing reveals novel tandem-duplication hotspots and a prognostic mutational signature in gastric cancer. Nat Commun. 2019;10:2037.

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  25. Vogel KU, Bell LS, Galloway A, Ahlfors H, Turner M. The RNA-binding proteins Zfp36l1 and Zfp36l2 enforce the Thymic β-selection checkpoint by limiting DNA damage response signaling and cell cycle progression. J Immunol. 2016;197:2673–85.

    Article  CAS  PubMed  Google Scholar 

  26. Cook ME, Bradstreet TR, Webber AM, Kim J, Santeford A, Harris KM, et al. The ZFP36 family of RNA binding proteins regulates homeostatic and autoreactive T-cell responses. Sci Immunol. 2022;7:eabo0981.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yonemori K, Seki N, Kurahara H, Osako Y, Idichi T, Arai T, et al. ZFP36L2 promotes cancer cell aggressiveness and is regulated by antitumor microRNA-375 in pancreatic ductal adenocarcinoma. Cancer Sci. 2017;108:124–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Priestley P, Baber J, Lolkema MP, Steeghs N, de Bruijn E, Shale C, et al. Pan-cancer whole-genome analyses of metastatic solid tumours. Nature. 2019;575:210–6.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li MJ, Shi JY, Zhu QS, Shi B, Jia ZL. Targeted re-sequencing of the 2p21 Locus identifies non-syndromic cleft lip only novel susceptibility gene ZFP36L2. Front Genet. 2022;13:802229.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 2008;3:59–69.

    Article  CAS  PubMed  Google Scholar 

  31. Sun H, Li D, Chen S, Liu Y, Liao X, Deng W, et al. Zili inhibits transforming growth factor-beta signaling by interacting with Smad4. J Biol Chem. 2010;285:4243–50.

    Article  CAS  PubMed  Google Scholar 

  32. Beaty TH, Taub MA, Scott AF, Murray JC, Marazita ML, Schwender H, et al. Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study. Hum Genet. 2013;132:771–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Moreno Uribe LM, Fomina T, Munger RG, Romitti PA, Jenkins MM, Gjessing HK, et al. A population-based study of effects of genetic loci on orofacial clefts. J Dent Res. 2017;96:1322–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pan Y, Han Y, Zhang H, Zhou L, Li D, Cai Q, et al. Association and cumulative effects of GWAS-identified genetic variants for nonsyndromic orofacial clefts in a Chinese population. Environ Mol Mutagen. 2013;54:261–7.

    Article  CAS  PubMed  Google Scholar 

  35. Deplancke B, Alpern D, Gardeux V. The genetics of transcription factor DNA binding variation. Cell. 2016;166:538–54.

    Article  CAS  PubMed  Google Scholar 

  36. Shen X, Song S, Li C, Zhang J. Synonymous mutations in representative yeast genes are mostly strongly non-neutral. Nature. 2022;606:725–31.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Einarsson H, Salvatore M, Vaagensø C, Alcaraz N, Bornholdt J, Rennie S, et al. Promoter sequence and architecture determine expression variability and confer robustness to genetic variants. Elife. 2022;11:e80943.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rochard L, Monica SD, Ling IT, Kong Y, Roberson S, Harland R, et al. Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis. Development 2016;143:2541–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Yu Y, Alvarado R, Petty LE, Bohlender RJ, Shaw DM, Below JE, et al. Polygenic risk impacts PDGFRA mutation penetrance in non-syndromic cleft lip and palate. Hum Mol Genet. 2022;31:2348–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Maili L, Tandon B, Yuan Q, Menezes S, Chiu F, Hashmi SS, et al. Disruption of fos causes craniofacial anomalies in developing zebrafish. Front Cell Dev Biol. 2023;11:1141893.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Tréguer K, Faucheux C, Veschambre P, Fédou S, Thézé N, Thiébaud P. Comparative functional analysis of ZFP36 genes during Xenopus development. PLoS One. 2013;8:e54550.

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  42. Stumpo DJ, Broxmeyer HE, Ward T, Cooper S, Hangoc G, Chung YJ, et al. Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis. Blood. 2009;114:2401–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Méndez-Maldonado K, Vega-López GA, Aybar MJ, Velasco I. Neurogenesis from neural crest cells: molecular mechanisms in the formation of cranial nerves and Ganglia. Front Cell Dev Biol. 2020;8:635.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Keyte A, Hutson MR. The neural crest in cardiac congenital anomalies. Differentiation. 2012;84:25–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Klaus A, Müller M, Schulz H, Saga Y, Martin JF, Birchmeier W. Wnt/β-catenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells. Proc Natl Acad Sci USA. 2012;109:10921–6.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. Graf D, Malik Z, Hayano S, Mishina Y. Common mechanisms in development and disease: BMP signaling in craniofacial development. Cytokine Growth Factor Rev. 2016;27:129–39.

    Article  CAS  PubMed  Google Scholar 

  47. Manzari-Tavakoli A, Babajani A, Farjoo MH, Hajinasrollah M, Bahrami S, Niknejad H. The cross-talks among Bone Morphogenetic Protein (BMP) signaling and other prominent pathways involved in neural differentiation. Front Mol Neurosci. 2022;15:827275.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Liu J, Xiao Q, Xiao J, Niu C, Li Y, Zhang X, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022;7:3.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Heijlen M, Houbrechts AM, Bagci E, Van Herck SL, Kersseboom S, Esguerra CV, et al. Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and early larval development in zebrafish. Endocrinology. 2014;155:1547–59.

    Article  PubMed  Google Scholar 

  50. Winata CL, Korzh S, Kondrychyn I, Zheng W, Korzh V, Gong Z. Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers. Dev Biol. 2009;331:222–36.

    Article  CAS  PubMed  Google Scholar 

  51. Yin A, Korzh V, Gong Z. Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants. Biochim Biophys Acta. 2012;1823:236–44.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank everyone who contributed to this research. This project was supported by the National Natural Science Foundation of China (No. 82170919) and the Research and Develop Program, West China Hospital of Stomatology Sichuan University (No. RD-03-202301). The authors declare no potential conflicts of interest with respect to the authorship and/ or publication of this article.

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Author notes

  1. These authors contributed equally: Jialin Sun, Mujia Li.

Authors and Affiliations

  1. State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of cleft lip and palate, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China

    Jialin Sun, Mujia Li, Bing Shi & Zhonglin Jia

  2. Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province, Hangzhou, 310000, China

    Mujia Li

  3. SCU-CUHK Joint Laboratory for Reproductive Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, 610041, China

    Huaqin Sun & Ziyuan Lin

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Sun, J., Li, M., Sun, H. et al. Genetic association and functional validation of ZFP36L2 in non-syndromic orofacial cleft subtypes. J Hum Genet 69, 139–144 (2024). https://doi.org/10.1038/s10038-024-01222-z

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