Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Targeted next-generation sequencing analysis in Italian patients with keratoconus

Eye (2024)Cite this article

Subjects

Abstract

Objective

To report variants in 26 candidate genes and describe the clinical features of Italian patients with keratoconus (KC).

Subjects/methods

Sixty-four patients with a confirmed diagnosis of KC were enrolled in this genetic association study. Patients were classified into two study groups according to whether they had a confirmed diagnosis of progressive or stable KC. A purpose-developed Next Generation Sequencing (NGS) panel was used to identify and analyse the coding exons and flanking exon/intron boundaries of 26 genes known to be associated with KC and corneal dystrophies. Interpretation of the pathogenic significance of variants was performed using in silico predictive algorithms.

Result

The targeted NGS research identified a total of 167 allelic variants of 22 genes in the study population; twenty-four patients had stable keratoconus (n. 54 variants) and forty patients had progressive disease (n. 113 variants). We identified genetic variants of certain pathogenic significance in five patients with progressive KC; in addition, eight novel genetic variants were found in eight patients with progressive KC. Mutations of FLG, LOXHD1, ZNF469, and DOCK9 genes were twice more frequently identified in patients with progressive than stable disease. Filaggrin gene variants were found in 49 patients (76% of total), of whom 32 patients (80% of progressive KC group) had progressive disease.

Conclusions

Targeted NGS research provided new insights into the causative effect of candidate genes in the clinical phenotype of keratoconus. Filaggrin mutations were found to represent a genetic risk factor for development of progressive disease in Italy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Study methodology and results.

Similar content being viewed by others

Data availability

Data sharing is applicable to this article upon reasonable request.

References

  1. Bykhovskaya Y, Rabinowitz YS. Update on the genetics of keratoconus. Exp Eye Res. 2021;202:108398.

    Article  CAS  PubMed  Google Scholar 

  2. Ahuja P, Dadachanji Z, Shetty R, Nagarajan SA, Khamar P, Sethu S, et al. Relevance of IgE, allergy and eye rubbing in the pathogenesis and management of Keratoconus. Indian J Ophthalmol. 2020;68:2067–74.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Nishtala K, Pahuja N, Shetty R, Nuijts RMMA, Ghosh A. Tear biomarkers for keratoconus. Eye Vis. 2016;3:19.

    Article  Google Scholar 

  4. McMonnies CW. Inflammation and Keratoconus. Optom Vis Sci. 2015;92:e35–41.

    Article  PubMed  Google Scholar 

  5. Jun AS, Cope L, Speck C, Feng X, Lee S, Meng H, et al. Subnormal cytokine profile in the tear fluid of keratoconus patients. PLoS One. 2011;6:e16437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wheeler J, Hauser MA, Afshari NA, Allingham RR, Liu Y. The genetics of keratoconus: a review. Reprod Syst Sex Disord: Curr Res. 2012;(Suppl 6):001.

  7. Woodward MA, Blachley TS, Stein JD. The association between sociodemographic factors, common systemic diseases, and keratoconus: an analysis of a nationwide heath care claims database. Ophthalmology. 2016;123:457–65.e2.

    Article  PubMed  Google Scholar 

  8. Pearson AR, Soneji B, Sarvananthan N, Sandford-Smith JH. Does ethnic origin influence the incidence or severity of keratoconus? Eye. 2000;14:625–8.

    Article  PubMed  Google Scholar 

  9. Hashemi H, Heydarian S, Yekta A, Ostadimoghaddam H, Aghamirsalim M, Derakhshan A, et al. High prevalence and familial aggregation of keratoconus in an Iranian rural population: a population-based study. Ophthalmic Physiol Opt. 2018;38:447–55.

    Article  PubMed  Google Scholar 

  10. Owens H, Gamble G. A profile of keratoconus in New Zealand. Cornea 2003;22:122–5.

    Article  PubMed  Google Scholar 

  11. Wang Y, Rabinowitz YS, Rotter JI, Yang H. Genetic epidemiological study of keratoconus: evidence for major gene determination. Am J Med Genet. 2000;93:403–9.

    Article  CAS  PubMed  Google Scholar 

  12. Tuft SJ, Hassan H, George S, Frazer DG, Willoughby CE, Liskova P. Keratoconus in 18 pairs of twins. Acta Ophthalmol. 2012;90:e482–6.

    Article  PubMed  Google Scholar 

  13. Weed KH, MacEwen CJ, McGhee CN. The variable expression of keratoconus within monozygotic twins: dundee University Scottish Keratoconus Study (DUSKS). Cont Lens Anterior Eye. 2006;29:123–6.

    Article  CAS  PubMed  Google Scholar 

  14. McMonnies CW. Epigenetic mechanisms might help explain environmental contributions to the pathogenesis of keratoconus. Eye Contact Lens. 2014;40:371–5.

    Article  PubMed  Google Scholar 

  15. Bykhovskaya Y, Li X, Taylor KD, Haritunians T, Rotter JI, Rabinowitz YS. Linkage analysis of high-density SNPs confirms keratoconus locus at 5q chromosomal region. Ophthalmic Genet. 2016;37:109–10.

    PubMed  Google Scholar 

  16. Tang YG, Rabinowitz YS, Taylor KD, Li X, Hu M, Picornell Y, et al. Genomewide linkage scan in a multigeneration Caucasian pedigree identifies a novel locus for keratoconus on chromosome 5q14.3-q21.1. Genet Med. 2005;7:397–405.

    Article  CAS  PubMed  Google Scholar 

  17. Choquet H, Melles RB, Yin J, Hoffmann TJ, Thai KK, Kvale MN, et al. A multiethnic genome-wide analysis of 44,039 individuals identifies 41 new loci associated with central corneal thickness. Commun Biol. 2020;3:301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lu Y, Vitart V, Burdon KP, Khor CC, Bykhovskaya Y, Mirshahi A, et al. Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nat Genet. 2013;45:155–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hughes AE, Bradley DT, Campbell M, Lechner J, Dash DP, Simpson DA, et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract. Am J Hum Genet. 2011;89:628–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hardcastle AJ, Liskova P, Bykhovskaya Y, McComish BJ, Davidson AE, Inglehearn CF, et al. A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus. Commun Biol. 2021;4:266.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang J, Zhang L, Hong J, Wu D, Xu J. Association of common variants in LOX with Keratoconus: a meta-analysis. PLoS One. 2015;10:e0145815.

    Article  PubMed  PubMed Central  Google Scholar 

  22. 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 

  23. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. DePristo M, Banks E, Poplin R, Garimela KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hung JH, Weng Z. Mapping short sequence reads to a reference genome. Cold Spring Harb Protoc. 2017;2017:2.

    Google Scholar 

  26. Koboldt DC, Chen K, Wylie T, Larson DE, McLellan MD, Mardis ER, et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics. 2009;25:2283–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Hashemi H, Heydarian S, Hooshmand E, Saatchi M, Yekta A, Aghamirsalim M, et al. The Prevalence and risk factors for keratoconus: a systematic review and meta-analysis. Cornea. 2020;39:263–70.

    Article  PubMed  Google Scholar 

  29. Claessens JLJ, Godefrooij DA, Vink G, Frank L, Wisse RPL. Nationwide epidemiological approach to identify associations between keratoconus and immune-mediated diseases. Br J Ophthalmol. 2022;106:1350–4.

    Article  PubMed  Google Scholar 

  30. Osawa R, Akiyama M, Shimizu H. Filaggrin gene defects and the risk of developing allergic disorders. Allergol Int. 2011;60:1–9.

    Article  PubMed  Google Scholar 

  31. Lapp T, Auw-Haedrich C, Reinhard T, Evans R, Rodríguez E, Weidinger S, et al. Analysis of filaggrin mutations and expression in corneal specimens from patients with or without atopic dermatitis. Int Arch Allergy Immunol. 2014;163:20–4.

    Article  CAS  PubMed  Google Scholar 

  32. Tong L, Corrales RM, Chen Z, Villarreal AL, De Paiva CS, Beuerman R, et al. Expression and regulation of cornified envelope proteins in human corneal epithelium. Investig Ophthalmol Vis Sci. 2006;47:1938–46.

    Article  Google Scholar 

  33. Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet. 2006;38:441–6.9.

    Article  CAS  PubMed  Google Scholar 

  34. Smith FJ, Irvine AD, Terron-Kwiatkowski A, Sandilands A, Campbell LE, Zhao Y, et al. Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat Genet. 2006;38:337–42.

    Article  CAS  PubMed  Google Scholar 

  35. Droitcourt C, Touboul D, Ged C, Ezzedine K, Cario-Andre M, de Verneuil H, et al. A prospective study of filaggrin null mutations in keratoconus patients with or without atopic disorders. Dermatology. 2011;222:336–41.

    Article  CAS  PubMed  Google Scholar 

  36. Karolak JA, Polakowski P, Szaflik J, Szaflik JP, Gajecka M. Molecular screening of keratoconus susceptibility sequence variants in VSX1, TGFBI, DOCK9, STK24, and IPO5 genes in Polish patients and novel TGFBI variant identification. Ophthalmic Genet. 2016;37:37–43.

    CAS  PubMed  Google Scholar 

  37. Chen B, Yu X, Zhang X, Yang H, Cui Y, Shentu X. Novel mutations identified in the chinese han population with Keratoconus by Next-Generation Sequencing. J Ophthalmol. 2022;2022:9991910.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Cheng WY, Yang SY, Huang XY, Zi FY, Li HP, Sheng XL. Identification of genetic variants in five Chinese families with keratoconus: pathogenicity analysis and characteristics of parental corneal topography. Front Genet. 2022;13:978684.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yohe S, Thyagarajan B. Review of clinical next-generation sequencing. Arch Pathol Lab Med. 2017;141:1544–57.

    Article  CAS  PubMed  Google Scholar 

  40. Nakagawa H, Fujita M. Whole genome sequencing analysis for cancer genomics and precision medicine. Cancer Sci. 2018;109:513–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Akiyama M. FLG mutations in ichthyosis vulgaris and atopic eczema: spectrum of mutations and population genetics. Br J Dermatol. 2010;162:472–7.

    Article  CAS  PubMed  Google Scholar 

  42. Lincoln SE, Truty R, Lin C-F, Zook JM, Paul J, Ramey VH, et al. A rigorous interlaboratory examination of the need to confirm next-generation sequencing-detected variants with an orthogonal method in clinical genetic testing. J Mol Diagn. 2019;21:318–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rodenburg RJ. The functional genomics laboratory: functional validation of genetic variants. J Inherit Metab Dis. 2018;41:297–307.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Studio Italiano di Oftalmologia, Via Livenza 3, 00198, Rome, Italy

    Marco Lombardo & Sebastiano Serrao

  2. Vision Engineering Italy srl, Via Livenza 3, 00198, Rome, Italy

    Marco Lombardo, Sebastiano Serrao & Giuseppe Lombardo

  3. Department of Biomedical Sciences, Ophthalmology Clinic, University of Messina, Via Consolare Valeria 1, 98124, Messina, Italy

    Umberto Camellin & Anna Maria Roszkowska

  4. Department of Ophthalmology, University Magna Graecia of Catanzaro, Viale Europa, 88100, Catanzaro, Italy

    Raffaella Gioia & Vincenzo Scorcia

  5. Ophthalmology Department, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski University, Krakow, Poland

    Anna Maria Roszkowska

  6. CNR-IPCF, Istituto per i Processi Chimico-Fisici, Viale F. Stagno D’Alcontres 37, 98158, Messina, Italy

    Giuseppe Lombardo

  7. MAGI’s Lab srl, Via Maioliche 57, 38068, Rovereto, Italy

    Matteo Bertelli & Maria Chiara Medori

  8. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via A. Moro 2, Siena, Italy

    Maria Chiara Medori

  9. Department of Public Health and infectious diseases, University of Rome “La Sapienza”, Piazzale Aldo Moro 5, 00185, Rome, Italy

    Danilo Alunni Fegatelli & Annarita Vestri

  10. Ophthalmology Clinic, AOU Careggi, University of Florence, Largo Brambilla 3, 50134, Firenze, Italy

    Rita Mencucci

  11. Fondazione G.B. Bietti IRCCS, Via S. Stefano Rotondo 6, 00184, Rome, Italy

    Domenico Schiano Lomoriello

Authors
  1. Marco Lombardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Umberto Camellin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Raffaella Gioia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sebastiano Serrao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vincenzo Scorcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anna Maria Roszkowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Giuseppe Lombardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matteo Bertelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Maria Chiara Medori
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Danilo Alunni Fegatelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Annarita Vestri
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rita Mencucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Domenico Schiano Lomoriello
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Each author has contributed sufficiently to the intellectual content of the submission and qualifies as a contributing author. The corresponding author confirms that he has full access to the data and final responsibility for the decision to submit for publication.

Corresponding author

Correspondence to Marco Lombardo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Informed consent

Informed consent was obtained from each participant. Subject-identifying data were coded and kept anonymous and confidential.

Ethical approval

The study protocol was granted by the IFO ethics committee in Rome (604/14).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lombardo, M., Camellin, U., Gioia, R. et al. Targeted next-generation sequencing analysis in Italian patients with keratoconus. Eye (2024). https://doi.org/10.1038/s41433-024-03090-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41433-024-03090-5

Search

Advanced search

Quick links