Association of TIMP-1 and COL4A4 Gene Polymorphisms with Keratoconus in an Iranian Population


Purpose: Keratoconus (KC) is a bilateral and noninflammatory disease, characterized by progressive thinning and anterior protrusion of the cornea and may result in severe visual impairment due to irregular astigmatism. Matrix metalloproteinases (MMP) are the main group of enzymes that degrade extracellular matrix proteins including collagens; Type IV collagen is found in the corneal stroma. MMP enzymatic activity is inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1). A decrease in TIMP-1 level is associated with the development of KC. In the present study, we investigated the impact of COL4A4 rs2228557 C/T and TIMP-1 rs4898 C/T (X-chromosome) variants on the odds of KC development in a sample of Iranian population.

Methods: This case–control study was conducted on 140 patients with KC and 150 healthy control subjects. We used modified methods of Nested-PCR and ARMS-PCR in combination (Nested- ARMS-PCR) and confirmed their validity with RFLP–PCR.

Results: Significant differences were noticed between KC patients and healthy individuals regarding the genotype TY or T allele frequencies of rs4898 in the male subjects (OR = 0.43, 95%CI: 0.20–0.92, P = 0.03), whereas no significant differences were identified in the female subjects (OR = 1.07, 95%CI: 0.52–2.20, P = 0.85). The rs2228557, T allele was associated with KC (OR = 0.69, 95% CI: 0.50–0.97, P = 0.035).

Conclusion: In the rs2228557 variant, T allele acts as a protective factor from the disease and decreases the risk of KC compared with the C allele. Also, in our investigation about rs4898, we found that TY genotype or T allele decreased the risk of KC compared with the C allele in males and was a protective factor for KC in our population.


Collagen, COL4A4, Keratoconus, Polymorphism, TIMP-1

1. Burdon KP, Vincent AL. Insights into keratoconus from a genetic perspective. Clin Exp Optom 2013;96:146–154.

2. Gajecka M, Radhakrishna U, Winters D, Nath SK, Rydzanicz M, Ratnamala U, et al. Localization of a gene for keratoconus to a 5.6-Mb interval on 13q32. Invest Ophthalmol Vis Sci 2009;50:1531–1539.

3. Lee LR, Hirst LW, Readshaw G. Clinical detection of unilateral keratoconus. Aust N Z J Ophthalmol 1995;23:129–133.

4. Li X, Bykhovskaya Y, Canedo AL, Haritunians T, Siscovick D, Aldave AJ, et al. Genetic association of COL5A1 variants in keratoconus patients suggests a complex connection between corneal thinning and keratoconus. Invest Ophthalmol Vis Sci 2013;54:2696–2704.

5. Grewal DS, Brar GS, Grewal SP. Assessment of central corneal thickness in normal, keratoconus, and postlaser in situ keratomileusis eyes using Scheimpflug imaging, spectral domain optical coherence tomography, and ultrasound pachymetry. J Cataract Refract Surg 2010;36:954–964.

6. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998;42:297–319.

7. Pearson AR, Soneji B, Sarvananthan N, Sandford-Smith JH. Does ethnic origin influence the incidence or severity of keratoconus? Eye 2000;14:625–628.

8. Sahebjada S, Schache M, Richardson AJ, Snibson G, MacGregor S, Daniell M, et al. Evaluating the association between keratoconus and the corneal thickness genes in an independent Australian population. Invest Ophthalmol Vis Sci 2013;54:8224–8228.

9. Bae HA, Mills RA, Lindsay RG, Phillips T, Coster DJ, Mitchell P, et al. Replication and meta-analysis of candidate loci identified variation at RAB3GAP1 associated with keratoconus. Invest Ophthalmol Vis Sci 2013;54:5132– 5135.

10. Hasanian-Langroudi F, Saravani R, Validad MH, Bahari G, Yari D. Association of Lysyl oxidase (LOX) polymorphisms with the risk of Keratoconus in an Iranian population. Ophthalmic Genet 2014;0:1–6.

11. Heon E, Greenberg A, Kopp KK, Rootman D, Vincent AL, Billingsley G, et al. VSX1: a gene for posterior polymorphous dystrophy and keratoconus. Hum Mol Genet 2002;11:1029–1036.

12. Yari D, Saravani R, Saravani S, Ebrahimian K, Galavi HR. Genetic polymorphisms of catalase and glutathione peroxidase-1 in keratoconus. Iran J Public Health 2018;47:1567–1574.

13. Guan T, Liu C, Ma Z, Ding S. The point mutation and polymorphism in keratoconus candidate gene TGFBI in Chinese population. Gene 2012;503:137–139.

14. Stabuc-Silih M, Ravnik-Glavac M, Glavac D, Hawlina M, Strazisar M. Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus. Mol Vis 2009;15:2848–2860.

15. Lee JE, Oum BS, Choi HY, Lee SU, Lee JS. Evaluation of differentially expressed genes identified in keratoconus. Mol Vis 2009;15:2480–2487.

16. Fullerton J, Paprocki P, Foote S, Mackey DA, Williamson R, Forrest S. Identity-by-descent approach to gene localisation in eight individuals affected by keratoconus from north-west Tasmania, Australia. Hum Genet 2002;110:462–470.

17. Patel D, McGhee C. Understanding keratoconus: what have we learned from the New Zealand perspective? Clin Exp Optom 2013;96:183–187.

18. Boye E, Mollet G, Forestier L, Cohen-Solal L, Heidet L, Cochat P, et al. Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome. Am J Hum Genet 1998;63:1329–1340.

19. Mariyama M, Zheng K, Yang-Feng TL, Reeders ST. Colocalization of the genes for the alpha 3(IV) and alpha 4(IV) chains of type IV collagen to chromosome 2 bands q35-q37. Genomics 1992;13:809–813.

20. Stachs O, Bochert A, Gerber T, Koczan D, Thiessen HJ, Guthoff RF. [The extracellular matrix structure in keratoconus]. Ophthalmologe 2004;101:384–389.

21. Bochert A, Berlau J, Koczan D, Seitz B, Thiessen HJ, Guthoff R. [Gene expression in keratoconus. Initial results using DNA microarrays]. Ophthalmologe 2003;100:545– 549.

22. Nielsen K, Hjortdal J, Pihlmann M, Corydon TJ. Update on the keratoconus genetics. Acta Ophthalmol 2013;91:106– 113.

23. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477:267–283.

24. Brew K, Nagase H. The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta 2010;1803:55–71.

25. Guedez L, Stetler-Stevenson WG, Wolff L, Wang J, Fukushima P, Mansoor A, et al. In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1. J Clin Invest 1998;102:2002–2010.

26. Usher PA, Sieuwerts AM, Bartels A, Lademann U, Nielsen HJ, Holten-Andersen L, et al. Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue. Mol Oncol 2007;1:205–215.

27. Akahane T, Akahane M, Shah A, Connor CM, Thorgeirsson UP. TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms. Exp Cell Res 2004;301:158–167.

28. Johnson MD, Kim HR, Chesler L, Tsao-Wu G, Bouck N, Polverini PJ. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase. J Cell Physiol 1994;160:194–202.

29. Brown D, Chwa MM, Opbroek A, Kenney MC. Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors. Curr Eye Res 1993;12:571–581.

30. Saravani R, Hasanian-Langroudi F, Validad MH, Yari D, Bahari G, Faramarzi M, et al. Evaluation of possible relationship between COL4A4 gene polymorphisms and risk of keratoconus. Cornea 2015;34:318–322.

31. Kokolakis NS, Gazouli M, Chatziralli IP, Koutsandrea C, Gatzioufas Z, Peponis VG, et al. Polymorphism analysis of COL4A3 and COL4A4 genes in Greek patients with keratoconus. Ophthalmic Genet 2014;35:226–228.

32. Wang HX, Yang QD, Liu BQ, Zhang L, Ma MM, Hu ZY, et al. TIMP-1 polymorphisms in a Chinese Han population with intracerebral hemorrhage. Int J Neurosci 2014;124:61–67.

33. Skarmoutsou E, D’Amico F, Marchini M, Malaponte G, Scorza R, Mazzarino MC. Association of TIMP-1 +372 SNP with digital ulcer manifestation in female systemic sclerosis patients. Hum Immunol 2012;73:950–953.

34. Lorente L, Martin M, Plasencia F, Sole-Violan J, Blanquer J, Labarta L, et al. The 372 T/C genetic polymorphism of TIMP-1 is associated with serum levels of TIMP-1 and survival in patients with severe sepsis. Crit Care 2013;17:R94.

35. Mikami T, Meguro A, Teshigawara T, Takeuchi M, Uemoto R, Kawagoe T, et al. Interleukin 1 beta promoter polymorphism is associated with keratoconus in a Japanese population. Mol Vis 2013;19:845–851.

36. Hashemi M, Amininia S, Ebrahimi M, Hashemi SM, Yousefi J, Eskandari-Nasab E, et al. Association between LAPTM4B gene polymorphism and breast cancer susceptibility in an Iranian population. Med Oncol 2014;31:111.

37. Haff LA. Improved quantitative PCR using nested primers. PCR Methods Appl 1994;3:332–337.

38. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989;17:2503–2516.

39. Dai S, Long Y. Genotyping Analysis Using an RFLP Assay. Methods Mol Biol 2015;1245:91–99.

40. Naychov ZD, Hiyama E, Uchida N, Arihiro K, Nagao M, Takahashi S, et al. TIMP-1 c.T372C Genetic Polymorphism as a Possible Predictor for Acute Aortic Dissection. Hiroshima J Med Sci 2013;62:31–37.

41. Ormonde S. Refractive surgery for keratoconus. Clin Exp Optom 2013;96:173–182.

42. Crawford A, Fassett RG, Geraghty DP, Kunde DA, Ball MJ, Robertson IK, et al. Relationships between single nucleotide polymorphisms of antioxidant enzymes and disease. Gene 2012;501:89–103.

43. Collier SA. Is the corneal degradation in keratoconus caused by matrix-metalloproteinases? Clin Experiment Ophthalmol 2001;29:340–34.

44. Kenney MC, Chwa M, Atilano SR, Tran A, Carballo M, Saghizadeh M, et al. Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci 2005;46:823–832.

45. Krex D, Rohl H, Konig IR, Ziegler A, Schackert HK, Schackert G. Tissue inhibitor of metalloproteinases-1, - 2, and -3 polymorphisms in a white population with intracranial aneurysms. Stroke 2003;34:2817–2821.

46. Hinterseher I, Krex D, Kuhlisch E, Schmidt KG, Pilarsky C, Schneiders W, et al. Tissue inhibitor of metalloproteinase- 1 (TIMP-1) polymorphisms in a Caucasian population with abdominal aortic aneurysm. World J Surg 2007;31:2248– 2254.

47. Meijer MJ, Mieremet-Ooms MA, van Hogezand RA, Lamers CB, Hommes DW, Verspaget HW. Role of matrix metalloproteinase, tissue inhibitor of metalloproteinase and tumor necrosis factor-alpha single nucleotide gene polymorphisms in inflammatory bowel disease. World J Gastroenterol 2007;13:2960–2966.

48. Indelicato M, Chiarenza V, Libra M, Malaponte G, Bevelacqua V, Marchini M, et al. Analysis of TIMP-1 gene polymorphisms in Italian sclerodermic patients. J Clin Lab Anal 2006;20:173–176.

49. Wei JC, Lee HS, Chen WC, Shiu LJ, Yang SF, Wong RH. Genetic polymorphisms of the matrix metalloproteinase-3 (MMP-3) and tissue inhibitors of matrix metalloproteinases- 1 (TIMP-1) modulate the development of ankylosing spondylitis. Ann Rheum Dis 2009;68:1781–1786.

50. Ikebuchi Y, Ishida C, Okamoto K, Murawaki Y. Association of TIMP-1 and TIMP-2 gene polymorphisms with progression of liver fibrosis in patients with type C chronic liver disease. Biochem Genet 2013;51:564–574.

51. Saravani R, Yari D, Saravani S, Hasanian-Langroudi F. Correlation between the COL4A3, MMP-9, and TIMP-1 polymorphisms and risk of keratoconus. Jpn J Ophthalmol 2017;61:218–222.