Screening for Novel LOX and SOD1 Variants in Keratoconus Patients from Brazil


Purpose: To investigate the presence of the variants of lysyl oxygenase (LOX) and superoxide dismutase 1 (SOD1) genes in Brazilian patients with advanced keratoconus.

Methods: Donor genomic DNA extracted from blood samples was screened for 5’UTR, exonic LOX, and SOD1 variants in a subset of 26 patients presenting with advanced keratoconus (KISA > 1000% and I–S > 2.0) by Sanger sequencing. The impact of non-synonymous amino acid changes was evaluated by SIFT, PMUT, and PolyPhen algorithms. The Mutation Taster tool was used to evaluate the potential impact of formation of new donor and acceptor splice sites in the promoter region of affected volunteers carrying sequence variants. A 7-base SOD1 deletion (IVS2 + 50del7bp) previously associated with keratoconus was screened in 140 patients presenting classical keratoconus by gel fragment analysis, and positive samples were sequenced for confirmation.

Results: We found an unreported missense variant in LOX exon 6 in one heterozygous patient, leading to substitution of proline with threonine at residue 392 (p. Thr392Pro) of LOX protein sequence. This mutation was predicted to be potentially damaging to LOX protein. Another LOX variant, Arg158Gln, was also detected in another patient but predicted to be non-pathogenic. Two additional new polymorphisms in LOX 5’UTR region (–116C > T and –58C > T) were found in two patients presenting with advanced keratoconus and were predicted to modulate or create donor/acceptor splice sites in LOX transcripts. Additionally, SOD1 deletion was detected in one patient presenting with severe keratoconus, not in control samples.

Conclusion: We described three novel LOX polymorphisms identified for the first time in Brazilian patients with advanced keratoconus, as well as a previously described SOD1 deletion strongly associated with keratoconus. A possible role of these variants in modulating transcript levels in the cornea of affected individual requires further investigation.


Keratoconus, Mutation, LOX, SOD1

1. ABTO - Brazilian Association of Organ and Tissue Transplantation. Report on performed transplants - Jan/Dec 2018 [Internet]; 2019. Available from:

2. Lechner J, Bae HA, Guduric-Fuchs J, Rice A, Govindarajan G, Siddiqui S, et al. Mutational analysis of MIR184 in sporadic keratoconus and myopia. Invest Ophthalmol Vis Sci 2013;54:5266–5272.

3. Moussa S, Grabner G, Ruckhofer J, Dietrich M, Reitsamer H. Genetics in keratoconus – What is new? Open Ophthalmol J 2017;11:201–210.

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

5. de Araujo AL. Corneal stem cells and tissue engineering: current advances and future perspectives. World J Stem Cells 2015;7:806.

6. Gordon-Shaag A, Millodot M, Shneor E, Liu Y. The genetic and environmental factors for keratoconus. Biomed Res Int 2015;2015:24–32.

7. Gordon-Shaag A, Millodot M, Kaiserman I, Sela T, Itzhaki GB, Zerbib Y, et al. Risk factors for keratoconus in Israel: a case–control study. Ophtalmic Physiol Opt 2015;35:673– 681.

8. Vazirani J, Basu S. Keratoconus: current perspectives. Clin Ophthalmol 2013;7:2019–2030.

9. Sugar J, Macsay MS. What causes keratoconus? Cornea 2012;31:716–719.

10. Davidson AE, Hayes S, Hardcastle AJ, Tuft SJ. The pathogenesis of keratoconus. Eye 2014;28:189–195.

11. Galvis V, Sherwin T, Tello A, Merayo J, Barrera R, Acera A. Keratoconus: an inflammatory disorder? Eye 2015;29:843–859.

12. Wang Y, Rabinowitz YS, Rotter JI, Yang H. Genetic epidemiological study of keratoconus: evidence for major gene determination. Am J Med Genet 2000;93:403–409.

13. Tuft SJ, Hassan H, George S, Frazer DG, Willoughby CE, Liskova P. Keratoconus in 18 pairs of twins. Acta Ophthalmol 2012;90:482–486.

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

15. Zhang J, Zhang L, Hong J, Wu D, Xu J. Association of common variants in LOX with keratoconus: a metaanalysis. PLOS ONE 2015;10:e0145815.

16. Hasanian-Langroudi F, Saravani R, Validad M-H, Bahari G, Yari D. Association of lysyl oxidase (LOX) polymorphisms with the risk of keratoconus in an Iranian population. Ophthalmic Genet 2015;36:309–314.

17. Saravani R, Farzaneh Hasanian-Langroudi M-HVDYGB, Mahmood Faramarzi M, Khateri M, Bahadoram S. Evaluation of possible relationship between COL4A4 gene polymorphisms and risk of keratoconus. Cornea 2015;34:318–322.

18. Wojcik KA, Kaminska A, Blasiak J, Szaflik J, Szaflik JP. Oxidative stress in the pathogenesis of keratoconus and Fuchs endothelial corneal dystrophy. Int J Mol Sci 2013;14:19294–19308.

19. Vallabh NA, Romano V, Willoughby CE. Mitochondrial dysfunction and oxidative stress in corneal disease. Mitochondrion [Internet] 2017;36:103–113. Available from: http: //

20. Bykhovskaya Y, Li X, Epifantseva I, Haritunians T, Siscovick D, Aldave A, et al. Variation in the lysyl oxidase (LOX) gene is associated with keratoconus in family-based and case-control studies. Investig Ophthalmol Vis Sci 2012;53:4152–4157.

21. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci 2006;63:2304–2316.

22. Dudakova L, Jirsova K. The impairment of lysyl oxidase in keratoconus and in keratoconus-associated disorders. J Neural Transm 2013;120:977–982.

23. Shetty R, Sathyanarayanamoorthy A, Ramachandra RA, Arora V, Ghosh A, Srivatsa PR, et al. Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity. Mol Vis 2015;21:12–25.

24. Behndig A, Karlsson K, Johansson BO, Brännström T, Marklund SL. Superoxide dismutase isoenzymes in the normal and diseased human cornea. Investig Ophthalmol Vis Sci 2001;42:2293–2296.

25. Wheeler J, Hauser M, Afshari N, Allingham RR, Liu Y. The genetics of keratoconus: a review. Reprod Syst Sex Disord Curr Res 2012;6:1–19.

26. Atilano SR, Coskun P, Chwa M, Jordan N, Reddy V, Le K, et al. Accumulation of mitochondrial DNA damage in keratoconus corneas. Investig Ophthalmol Vis Sci 2005;46:1256–1263.

27. Udar N, Atilano SR, Brown DJ, Holguin B, Small K, Nesburn AB, et al. SOD1: a candidate gene for keratoconus. Investig Ophthalmol Vis Sci 2006;47:3345–3351.

28. Udar N, Atilano SR, Small K, Nesburn AB, Kenney MC. SOD1 haplotypes in familial keratoconus. Cornea 2009;28:902–907.

29. Moschos MM, Kokolakis N, Gazouli M, Chatziralli IP, Droutsas D, Anagnou NP, et al. Polymorphism analysis of VSX1 and SOD1 genes in Greek patients with keratoconus. Ophthalmic Genet 2013;36:213–217.

30. De Bonis P, Laborante A, Pizzicoli C, Stallone R, Barbano R, Longo C, et al. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus. Mol Vision 2011;1:2482–2494.

31. Canizales L, Rodriguez L, Rivera C, Martinez A, Mendez F, Castillo A. Low-level expression of SOD1 in peripheral blood samples of patients diagnosed with primary openangle glaucoma. Biomark Med 2016;10:1219–1223.

32. Loukovitis E, Sfakianakis K, Syrmakesi P. Genetic aspects of keratoconus: a literature review exploring potential genetic contributions and possible genetic relationships with comorbidities. Ophthalmol Ther 2018;7:263–292.

33. Panahi Y, Azimi A, Naderi M, Jadidi K, Sahebkar A. An analytical enrichment-based review of structural genetic studies on keratoconus. J Cell Biochem 2018;9:1–9.

34. Valgaeren H, Koppen C, Van Camp G. A new perspective on the genetics of keratoconus: why have we not been more successful? Ophthalmic Genet 2018;39:158–174.

35. Silva D, Gadelha B, Feitosa A, Silva R, Albuquerque T, Santos D, et al. Analysis of VSX1 variations in Brazilian subjects with keratoconus. J Ophthalmic Vis Res 2018;13:266–273.

36. Saee-Rad S, Hashemi H, Miraftab M, Noori-Daloii MR, Chaleshtori MH, Raoofian R, et al. Mutation analysis of VSX1 and SOD1 in Iranian patients with keratoconus. Mol Vis 2011;17:3128–3136.

37. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009;4:1073–1081.

38. Ferrer-Costa C, Gelpí JL, Zamakola L, Parraga I, de la Cruz X, Orozco M. PMUT: a web-based tool for the annotation of pathological mutations on proteins. Bioinformatics 2005;21:3176–3178.

39. Zeschau A, Balestrin IG, Stock RA, Bonamigo EL. Indications of keratoplasty: a retrospective study in a University Hospital. Brazilian J Ophthalmol 2013;72:316–320.

40. Almeida HG, Hida RY, Kara-Junior N. Trends in corneal transplantation from 2001 to 2016 in Brazil. Arq Bras Oftalmol 2018;81:3–6.

41. Ingre C, Wuolikainen A, Marklund SL, Birve A, Press R, Andersen PM. A 50 bp deletion in the SOD1 promoter lowers enzyme expression but is not associated with ALS in Sweden. Amyotroph Lateral Scler Front Degener 2016;17:452–457.

42. Saify K, Saadat M. Influence of a 50bp Ins/Del polymorphism at promoter of the superoxide dismutase-1 on gene expression and risk of heroin dependency. Environ Health Prev Med 2017;22:4–9.

43. Abu-Amero KK, Al-Muammar AM, Kondkar AA. Genetics of keratoconus: where do we stand? J Ophthalmol 2014;1–11.

44. Hao XD, Chen ZL, Qu ML, Zhao XW, Li SX, Chen PC. Decreased integrity, content, and increased transcript level of mitochondrial DNA are associated with keratoconus. PLOS ONE 2016;11:1–14.

45. Nita M, Grzybowski A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev 2016;2016.