The effects of IRF6 rs2235373 polymorphism on mRNA expression changes in non-syndromic cleft lip and palate with various phenotypes

Saskia Lenggogeni Nasroen; Ani Melani Maskoen; Hardisiswo Soedjana; Eky Setiawan Soeria Soemantri; Dany Hilmanto

doi:10.24198/pjd.vol30no3.17949

The effects of IRF6 rs2235373 polymorphism on mRNA expression changes in non-syndromic cleft lip and palate with various phenotypes

Saskia Lenggogeni Nasroen, Ani Melani Maskoen, Hardisiswo Soedjana, Eky Setiawan Soeria Soemantri, Dany Hilmanto

Abstract

Introduction: The IRF6 rs2235373 (C/T) is a polymorphism in the intron region that has been associated with non-syndromic cleft lip and palate (NS CLP) among some populations. Polymorphism in introns can also affect the transcription that should be detected through some changes in IRF6 mRNA expression. This study was aimed to evaluate the effects of IRF6 rs2235373 polymorphism on mRNA expression changes in NS CLP with its phenotypes which include complete unilateral (CU) CLP, complete bilateral (CB) CLP, cleft lip only (CLO), and cleft palate only (CPO). Methods: This study was a laboratory analytical study, using samples from 198 individuals in Indonesia (136 individuals with NS CL/P consisted of CU CLP (n=42), CB CLP (n=34), CLO (n=33), CPO (n=27), and 62 individuals as controls). DNA was extracted from venous blood, and the segment of rs2235373 was evaluated by PCR and Sanger sequencing. mRNA expressions analysis preceded by RNA extraction from oral epithelium, then real-time (RT) PCR was performed. mRNA expression changes were analysed by Livak method and Kruskal Wallis test followed by the Mann Whitney test through CC, GT, and TT genotype. Result: The mRNA expression of TT genotype from NS CU CLP and control was 3.36 higher value than CC genotype (p=0.008), and the mRNA expression of CT genotype from NS CB CLP and control was 0.16 lower value than CC genotype (p=0.000). Conclusion: The IRF6 rs2235373 polymorphism of TT genotype tend to cause mRNA overexpression in NS CU CLP, and CT genotype tend to cause mRNA underexpression in NS CB CLP.

Keywords: Non-syndromic cleft lip and palate, NS CLP phenotype, IRF6, rs2235373, mRNA expression.

Keywords

Non-syndromic cleft lip and palate, NS CLP phenotype, IRF6, rs2235373, mRNA expression.

Full Text:

PDF

References

Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet. 2009; 374(9703): 1773-85. DOI: 10.1016/S0140-6736(09)60695-4

Nasroen SL, Tajrin A, Fauziah PN, Maskoen AM, Soemantri ES, Soedjana H, et al. TGFß3 / SfaN1 gene variant and the risk factor of nonsyndromic cleft palate only among Indonesian patients. Cell Mol Biol (Noisy-le-grand). 2017; 63(2): 88-91. DOI: 10.14715/cmb/2017.63.2.13

Leslie EJ, Marazita ML. Genetics of Cleft Lip and Cleft Palate. Am J Med Genet C Semin Med Genet. 2013; 163(4): 246–258. DOI: 10.1002/ajmg.c.31381

Christensen K, Mitchell LE. Familial recurrence-pattern analysis of nonsyndromic isolated cleft palate--a Danish Registry study. Am J Hum Genet. 1996; 58(1): 182-90.

Murray JC. Gene/environment causes of cleft lip and/or palate. Clin Genet. 2002; 61(4): 248-56. DOI: 10.1034/j.1399-0004.2002.610402.x

Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet. 2011; 12(3): 167-78. DOI: 10.1038/nrg2933

Gorlin LJ, Cohen MM, Hennekam RCM. Syndromes of the head and neck. 4th ed. Oxford: Oxford University Press; 2001.

Blanton SH, Cortez A, Stal S, Mulliken JB, Finnell RH, Hecht JT. Variation in IRF6 contributes to nonsyndromic cleft lip and palate. Am J Genet A. 2005; 137A(3): 259-62.

Zucchero T, Cooper ME, Maher BS, Daack-Hirsch S, Nepomuceno B, Ribeiro L, et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med. 2004; 351(8): 769-80. DOI: 10.1056/NEJMoa032909

Jugessur A, Farlie PG, Kilpatrick N. The genetics of isolated orofacial clefts: from genotypes to subphenotypes. Oral Dis. 2009; 15(7): 437-53. DOI: 10.1111/j.1601-0825.2009.01577.x

Kondo S, Schutte BC, Richardson RJ, Bjork BC, Knight AS, Watanabe Y, et al. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat Genet. 2002; 32(2): 285-9. DOI: 10.1038/ng985

Nasroen SL, Maskoen AM, Soemantri ESS, Soedjana H, Hilmanto D. The IRF6 mRNA expression changes in oral epithelium of non syndromic cleft lip and palate with various phenotypes. Ind J Oral Maxillofac Surg. 2017; 4: 3-11.

Ingraham CR, Kinoshita A, Kondo S, Yang B, Sajan S, Trout KJ, et al. Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (IRF6). Nat Genet. 2006; 38(11): 1335-40. DOI: 10.1038/ng1903

Richardson RJ, Dixon J, Malhotra S, Hardman MJ, Knowles L, Boot-Hanford RP, et al. IRF6 is a key determinant keratinocyte proliferation-differentiation switch. Nat Genet. 2006; 38(11): 1329-34. DOI: 10.1038/ng1894

Biggs LC, Rhea L, Schutte B, Dunnwald M. Interferon Regulatory Factor 6 Is Necessary, but Not Sufficient, for Keratinocyte Differentiation. J Invest Dermatol. 2012; 132(1): 50-8. DOI: 10.1038/jid.2011.272

Leslie EJ. Advances in understanding the genetic architecture of cleft lip and palate disorders [dissertation]. Iowa: University of Iowa; 2012.

Kousa YA, Schutte BC. Toward an Orofacial Gene Regulatory Network. Dev Dyn. 2016; 245(3): 220–32. DOI: 10.1002/dvdy.24341

Salahshourifar I, Wan Sulaiman WA, Halim AS, Zilfalil BA. Mutation screening of IRF6 among families with non-syndromic oral clefts and identification of two novel variants: Review of literature. Eur J Med Genet. 2012; 55(6-7): 389-93. DOI: 10.1016/j.ejmg.2012.02.006

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(11): 1341-7. DOI: 10.1038/ng.242

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(9): 968-71. DOI: 10.1038/ng.2360

Fogh-Andersen P. Inheritance of harelip and cleft palate. Copenhagen: NYT Nordisk Forlag; 1942.

Pegelow M, Peyrard-Janvid M, Zucchelli M, Fransson I, Larson O, Kere J, et al. Familial non-syndromic cleft lip and palate - analysis of the IRF6 gene and clinical phenotypes. Eur J Orthod. 2008; 30(2): 169-75. DOI: 10.1093/ejo/cjm097

Harville EW, Wilcox AJ, Lie RT, Vindenes H, Abyholm F. Cleft Lip and Palate versus Cleft Lip Only: Are They Distinct Defects?. Am J Epidemiol. 2005; 162(5): 448–53. DOI: 10.1093/aje/kwi214

Kreiborg S, Hermann NV. Craniofacial morphology and growth in infants and young children with cleft lip and palate. In: Wyszynski DF. Cleft lip and palate: From origin to treatment. New York: Oxford University Press; 2002. p. 87-97.

Pan Y, Ma J, Zhang W, Du Y, Niu Y, Wang M, et al. IRF6 polymorphisms are associated with nonsyndromic orofacial clefts in a Chinese Han population. Am J Med Genet A. 2010; 152A(10): 2505-11. DOI: 10.1002/ajmg.a.33624

Ensembl Asia. [homepage on internet]. Cambridge: retrieved 2015 October; [about 2 screens]. Available from https://asia.ensembl.org/index.html

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(4): 402-8. DOI: 10.1006/meth.2001.1262

Fakhouri WD, Rhea L, Du T, Sweezer E, Morrison H, Fitzpatrick D, et.al. MCS9.7 Enhancer activity is Highly, but not Completely, Associated with Expression of Irf6 and p63. Dev Dyn. 2012; 241(2): 340-9. DOI: 10.1002/dvdy.22786

Washbourne BJ, Cox TC. Expression profiles of cIRF6, cLHX6 and cLHX7 in the facial primordia suggest specific roles during parimary palatogenesis. BMC Dev Biol. 2006; 6: 18. DOI: 10.1186/1471-213X-6-18

Riley BM, Mansilla MA, Ma J, Daack-Hirsch S, Maher BS, Raffensperger LM, et al. FGF signaling contributes to cleft lip and palate. Proc Natl Acad Sci U S A. 2007; 104(11): 4512-7. DOI: 10.1073/pnas.0607956104

Riley BM, Murray JC. Sequence evaluation of FGF and FGFR gene conserved non-coding elements in non-syndromic cleft lip and palate cases. Am J Med Genet A. 2007; 143A(24): 3228-34. DOI: 10.1002/ajmg.a.31965

Tian M, Shi JY, Wu M, Wang Y, Li L, Liu Y, et.al. Overexpression of mouse TTF-2 gene causes cleft palate. J Cell Mol Med. 2012; 16(10): 2362-8. DOI: 10.1111/j.1582-4934.2012.01546.x

Cox TC. Taking it to the max: the genetic and developmental mechanisms coordinating midfacial morphogenesis and dysmorphology. Clin Genet. 2004; 65(3): 163-76. DOI: 10.1111/j.0009-9163.2004.00225.x

Smane L, Pilmane M. IRF6, RYK, and PAX9 Expression in Facial Tissue of Children with Cleft Palate. Int J Morphol. 2015; 33(2): 647-52. DOI: 10.4067/S0717-95022015000200037

Knight AS, Schutte BC, Jiang R, Dixon MJ. Developmental expression analysis of the mouse and chick orthologues of IRF6: the gene mutated in Van der Woude syndrome. Dev Dyn. 2006; 235(5): 1441-7. DOI: 10.1002/dvdy.20598

Park JW, McIntosh I, Hetmanski JB, Jabs EW, Vander Kolk CA, Wu-Chou YH, et al. Association between IRF6 and nonsyndromic cleft lip with or without cleft palate in four populations. Genet Med. 2007; 9(4): 219-27. DOI: 10.1097GIM.0b013e3180423cca

Chorley BN, Wang X, Campbell MR, Pittman GS, Noureddine MA, Bell DA. Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: current and developing technologies. Mutat Res. 2008; 659(1-2): 147-57. DOI: 10.1016/j.mrrev.2008.05.001

Sadee W, Wang D, Papp AC, Pinsonneault JK, Smith RM, Moyer RA, et al. Pharmacogenomics of the RNA world: structural RNA polymorphisms in drug therapy. Clin Pharmacol Ther. 2011; 89(3): 355-65. DOI: 10.1038/clpt.2010.314

Liao PY, Lee KH. From SNPs to functional polymorphism: the insight into biotechnology applications. Biochem Eng J. 2010; 49(2): 149-58. DOI: 10.1016/j.bej.2009.12.021

Albert PR. What is a functional genetic polymorphism? Defining classes of functionality. J Psychiatry Neurosci. 2011; 36(6): 363-5. DOI: 10.1503/jpn.110137

Ke CY, Xiao WL, Chen CM, Lo LJ, Wong FH. IRF6 is the mediator of TGFß3 during regulation of the epithelial mesenchymal transition and palatal fusion. Sci Rep. 2015; 5: 12791. DOI: 10.1038/srep12791

Moretti F, Marinari B, Lo Iacono N, Botti E, Giunta A, Spallone G, et al. A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias. J Clin Invest. 2010; 120(5): 1570-7. DOI: 10.1172/JCI40267

Iwata J, Suzuki A, Pelikan RC, Ho TV, Sanchez-Lara PA, Urata M, et al. Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice. Development. 2013; 140: 1220-30. DOI: 10.1242/dev.089615

Gritli-Linde A. p63 and IRF6: brothers in arms against cleft palate. J Clin Invest. 2010; 120(5): 1386-9. DOI: 10.1172/JCI42821

DOI: https://doi.org/10.24198/pjd.vol30no3.17949