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Identification of putative regulatory single-nucleotide variants in NTN1 gene associated with NSCL/P

Journal of Human Genetics volume 68, pages 491–497 (2023)Cite this article

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Abstract

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common polygenetic disease. Although genome-wide association studies (GWAS) identified NTN1 gene as a high-priority candidate of NSCL/P, the comprehensive genetic architecture of NTN1 weren’t yet known. Thus, this study aimed to determine full-scale genetic variants of NTN1 for NSCL/P in Chinese Han people. Initially, targeted sequencing of NTN1 gene was performed on 159 NSCL/P patients to identify susceptible single nucleotide polymorphisms (SNPs) associated with NSCL/P. Then, association analysis and burden analysis were separately used to validate the common variants and rare variants identified among large size of samples (1608 NSCL/P cases and 2255 controls). Additionally, NSCL/P subtype association analysis was applied to elucidate the etiology discrepancy of non-syndromic cleft lip with palate (NSCLP) and non-syndromic cleft lip only (NSCLO). Lastly, bioinformatics analysis was performed to annotate and prioritize candidate variants. We found 15 NSCL/P-associated SNPs including rs4791774 (P = 1.10E-08, OR = 1.467, 95% CI: 1.286~1.673) and rs9788972 (P = 1.28E-07, OR = 1.398, 95% CI : 1.235~1.584) originally detected by previous GWASs in Chinese Han ancestry. Four NSCLO risk-associated SNPs and eight specific NSCLP associated SNPs were found. Three SNPs (rs4791331, rs4791774 and rs9900753) were predicted to locate at regulatory region of NTN1. Our study validated the association between NTN1 gene and pathogenesis of NSCL/P and reinforced the hypothesis that NSCLP have a different etiology from NSCLO. We also identified three putative regulatory SNPs in NTN1 gene.

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References

  1. Tanaka SA, Mahabir RC, Jupiter DC, Menezes JM. Updating the epidemiology of cleft lip with or without cleft palate. Plast Reconstr Surg. 2012;129:511e–8e.

    Article  PubMed  Google Scholar 

  2. Fan D, Wu S, Liu L, Xia Q, Tian G, Wang W, et al. Prevalence of non-syndromic orofacial clefts: based on 15,094,978 Chinese perinatal infants. Oncotarget. 2018;9:13981–90.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Sivertsen A, Wilcox A, Skjaerven R, Vindenes H, Abyholm F, Harville E, et al. Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. BMJ. 2008;336:432–4.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Marazita ML. The evolution of human genetic studies of cleft lip and cleft palate. Annu Rev Genomics Hum Genet. 2012;13:263–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mossey P, Little J, Munger R, Dixon M, Shaw W. Cleft lip and palate. Lancet 2009;374:1773–85.

    Article  PubMed  Google Scholar 

  6. Harville E, Wilcox A, Lie R, Vindenes H, Abyholm F. Cleft lip and palate versus cleft lip only: are they distinct defects? Am J Epidemiol. 2005;162:448–53.

    Article  PubMed  Google Scholar 

  7. Grosen D, Chevrier C, Skytthe A, Bille C, Mølsted K, Sivertsen A, et al. A cohort study of recurrence patterns among more than 54,000 relatives of oral cleft cases in Denmark: support for the multifactorial threshold model of inheritance. J Med Genet. 2010;47:162–8.

    Article  PubMed  Google Scholar 

  8. 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 

  9. 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 

  10. 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 

  11. 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 

  12. 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  PubMed  Google Scholar 

  13. Mukhopadhyay N, Feingold E, Moreno-Uribe L, Wehby G, Valencia-Ramirez LC, Muneton CPR, et al. Genome-wide association study of non-syndromic orofacial clefts in a multiethnic sample of families and controls identifies novel regions. Front Cell Dev Biol. 2021;9:621482.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Leslie EJ, Taub MA, Liu H, Steinberg KM, Koboldt DC, Zhang Q, et al. Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci. Am J Hum Genet. 2015;96:397–411.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Li D, Zhu G, Lou S, Ma L, Zhang C, Pan Y, et al. The functional variant of NTN1 contributes to the risk of nonsyndromic cleft lip with or without cleft palate. Eur J Hum Genet. 2020;28:453–60.

    Article  CAS  PubMed  Google Scholar 

  16. Tam V, Patel N, Turcotte M, Bosse Y, Pare G, Meyre D. Benefits and limitations of genome-wide association studies. Nat Rev Genet. 2019;20:467–84.

    Article  CAS  PubMed  Google Scholar 

  17. Bonnefond A, Froguel P. Rare and common genetic events in type 2 diabetes: what should biologists know? Cell Metab. 2015;21:357–68.

    Article  CAS  PubMed  Google Scholar 

  18. Sazonovs A, Barrett JC. Rare-variant studies to complement genome-wide association studies. Annu Rev Genomics Hum Genet. 2018;19:97–112.

    Article  CAS  PubMed  Google Scholar 

  19. International HapMap C. A haplotype map of the human genome. Nature. 2005;437:1299–320.

    Article  Google Scholar 

  20. Altshuler D, Daly MJ, Lander ES. Genetic mapping in human disease. Science. 2008;322:881–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 2012;337:1190–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Thieme F, Ludwig KU. The role of noncoding genetic variation in isolated orofacial clefts. J Dent Res. 2017;96:1238–47.

    Article  CAS  PubMed  Google Scholar 

  23. 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 

  24. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics.2009;25:2078–9.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Yang J, Bakshi A, Zhu Z, Hemani G, Vinkhuyzen A, Lee S, et al. Genetic variance estimation with imputed variants finds negligible missing heritability for human height and body mass index. Nat Genet. 2015;47:1114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. 2013;Chapter 7:Unit7 20. https://doi.org/10.1002/0471142905.hg0720s76.

  28. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003;31:3812–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 2010;7:575–6.

    Article  CAS  PubMed  Google Scholar 

  30. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019;47:D886–D94.

    Article  CAS  PubMed  Google Scholar 

  31. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007;81:559–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA, Kasowski M, et al. Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 2012;22:1790–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Quan C, Ping J, Lu H, Zhou G, Lu Y. 3DSNP 2.0: update and expansion of the noncoding genomic variant annotation database. Nucleic Acids Res. 2022;50:D950–D5.

    Article  CAS  PubMed  Google Scholar 

  34. Ward L, Kellis M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 2012;40:D930–4.

    Article  CAS  PubMed  Google Scholar 

  35. Kou I, Otomo N, Takeda K, Momozawa Y, Lu HF, Kubo M, et al. Genome-wide association study identifies 14 previously unreported susceptibility loci for adolescent idiopathic scoliosis in Japanese. Nat Commun. 2019;10:3685.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Barbeira AN, Bonazzola R, Gamazon ER, Liang Y, Park Y, Kim-Hellmuth S, et al. Exploiting the GTEx resources to decipher the mechanisms at GWAS loci. Genome Biol. 2021;22:49.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Chen S, Jia Z, Cai M, Ye M, Wu D, Wan T, et al. viaSP1-mediated upregulation of long noncoding RNA ZFAS1 involved in non-syndromic cleft lip and palate inactivating WNT/β-catenin signaling pathway. Front Cell Dev Biol. 2021;9:662780.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Carlson J, Standley J, Petrin A, Shaffer J, Butali A, Buxó C, et al. Identification of 16q21 as a modifier of nonsyndromic orofacial cleft phenotypes. Genet Epidemiol. 2017;41:887–97.

    Article  PubMed  PubMed Central  Google Scholar 

  39. 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 

  40. Çalışkan M, Manduchi E, Rao H, Segert J, Beltrame M, Trizzino M, et al. Genetic and epigenetic fine mapping of complex trait associated loci in the human. Liver 2019;105:89–107.

    Google Scholar 

  41. Blanco-Gómez A, Castillo-Lluva S, Del Mar Sáez-Freire M, Hontecillas-Prieto L, Mao J, Castellanos-Martín A, et al. Missing heritability of complex diseases: enlightenment by genetic variants from intermediate phenotypes. Bioessays. 2016;38:664–73.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Field A, Adelman K. Evaluating enhancer function and transcription. Annu Rev Biochem. 2020;89:213–34.

    Article  CAS  PubMed  Google Scholar 

  43. Cramer P. Organization and regulation of gene transcription. Nature 2019;573:45–54.

    Article  CAS  PubMed  Google Scholar 

  44. Edwards S, Beesley J, French J, Dunning AM. Beyond GWASs: illuminating the dark road from association to function. Am J Hum Genet. 2013;93:779–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Guo Q, Li D, Meng X, Liu T, Shi J, Hao Y, et al. Association between PAX7 and NTN1 gene polymorphisms and nonsyndromic orofacial clefts in a northern Chinese population. Med (Baltim). 2017;96:e6724.

    Article  CAS  Google Scholar 

  46. Xu Y, Xie B, Shi J, Li J, Zhou C, Lu W, et al. Distinct expression of miR-378 in nonsyndromic cleft lip and/or cleft palate: a cogitation of skewed sex ratio in prevalence. Cleft Palate Craniofac J. 2021;58:61–71.

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank all the participants provided samples and friends donated helpful comments in this study.

Funding

This project was supported by the National Science Funds of China (No.82170919 and No. 81600849) and the Major frontier issues of the application foundation project of Sichuan Science and Technology Department (2020YJ0211).

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Authors and Affiliations

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

    Hong-Xu Tao, Yi-Xin Yang, Bing Shi & Zhong-Lin Jia

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Contributions

HXT Conceptualization, Methodology, Investigation, Writing-Original Draft. YXYang validation, Investigation, Writing-original draft, BS Validation, Software, Writing-Review/Editing, ZLJ Conceptualization, Methodology, Software, Supervision, Project administration, Funding acquisition, Writing-Review/Editing.

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Correspondence to Zhong-Lin Jia.

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Tao, HX., Yang, YX., Shi, B. et al. Identification of putative regulatory single-nucleotide variants in NTN1 gene associated with NSCL/P. J Hum Genet 68, 491–497 (2023). https://doi.org/10.1038/s10038-023-01137-1

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