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Офтальмохирургия. 2020; : 6-13

Изучение ассоциации с кератоконусом однонуклеотидных вариантов в гене COL5A1 и прилегающем межгенном пространстве в когорте пациентов из России

Скородумова Л. О., Белодедова А. В., Захарова Е. С., Шарова Е. И., Бикбов М. М., Усубов Э. Л., Антонова О. П., Малюгин Б. Э.

https://doi.org/10.25276/0235-4160-2020-1-6-13

Аннотация

Цель. Выявить ассоциацию с кератоконусом маркеров вблизи гена COL5A1 и в его интроне. Изучить взаимосвязь наличия маркерной аллели с уровнем экспрессии гена COL5A1 в роговице пациентов с кератоконусом.

Материал и методы. В исследование было включено 70 пациентов с диагнозом «кератоконус», а также 100 участников контрольной группы. Все участники были генотипированы по двум маркерам, ассоциированным с кератоконусом: rs1536482 и rs7044529. Проведен расчет диагностических показателей для оценки возможности использования маркеров в качестве классификаторов кератоконуса. У пациентов с кератоконусом, которым была выполнена кератопластика, были собраны образцы роговицы (n=9) для выделения РНК. Проведено сравнение уровня экспрессии гена COL5A1 в группе образцов роговицы пациентов с хотя бы одной маркерной аллелью (rs1536482+) и группе образцов без маркерных аллелей (rs1536482-) с помощью ПЦР в реальном времени, сопряженной с обратной транскрипцией.
Результаты. Было выявлено значимое различие в частоте встречаемости маркерной аллели rs1536482 между группой пациентов с кератоконусом и контрольной группой (p=0,0004, критерий Фишера). Хотя бы одна маркерная аллель rs1536482 была обнаружена у 67,1% пациентов (чувствительность маркера) с кератоконусом и 46% участников контрольной группы. Таким образом, специфичность маркера является низкой (0,54). Отношение шансов составило 2,4, а отношение рисков – 1,69. Анализ экспрессии генов в роговице пациентов не выявил ассоциации уровня экспрессии гена COL5A1 с генотипом (p=0,9, критерий Манна-Уитни).

Выводы. В данном исследовании была выявлена ассоциация rs1536482 с кератоконусом в выборке пациентов из России. Однако в связи с низкой специфичностью данного маркера он не может быть использован в качестве классификатора кератоконуса. Наличие маркерной аллели не ассоциировано с уровнем экспрессии гена COL5A1.

Список литературы

1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42(4): 297–319. doi:10.1016/s00396257(97)00119-7.

2. Tanabe U, Fujiki K, Ogawa A, Ueda S, Kanai A. [Prevalence of keratoconus patients in Japan]. Nippon Ganka Gakkai Zasshi. 1985;89(3): 407–11.

3. Waked N, Fayad AM, Fadlallah A, El Rami H. Keratoconus screening in a Lebanese students’ population. J Fr Ophtalmol. 2012;35(1): 23–9. doi:10.1016/j.jfo.2011.03.016.

4. Said A, Hamade IH, Tabbara KF. Late onset corneal ectasia after LASIK surgery. Saudi J Ophthalmol. 2011;25(3): 225–30. doi:10.1016/J.SJOPT.2011.05.003.

5. Randleman JB, Woodward M, Lynn MJ, Stulting RD. Risk assessment for ectasia after corneal refractive surgery. Ophthalmology. 2008;115(1): 37– 50. doi:10.1016/j.ophtha.2007.03.073.

6. Giri P, Azar DT. Risk profiles of ectasia after keratorefractive surgery. Curr Opin Ophthalmol. 2017;28(4): 337–42. doi:10.1097/ ICU.0000000000000383.

7. Owens H, Walters GA. Keratoconus in monozygotic twins in New Zealand. 1995; Clin Exper Optometry. 78(4): 125– 9. doi:10.1111/j.1444-0938.1995.tb00805.x.

8. Bechara SJ, Insler MS. Keratoconus in two pairs of identical twins. Cornea. 1996 Jan;15(1): 90–3.

9. Tuft SJ, Hassan H, George S, Frazer DG, Willoughby CE, Liskova P. Keratoconus in 18 pairs of twins. Acta Ophthalmol. 2012;90(6): e482-6. doi:10.1111/j.1755-3768.2012.02448.x.

10. Bykhovskaya Y, Margines B, Rabinowitz YS. Genetics in Keratoconus: where are we? Eye Vis (Lond.). 2016;3(1): 16. doi:10.1186/s40662-016-0047-5.

11. Burdon KP, Vincent AL. Insights into keratoconus from a genetic perspective. Clin Exp Optom. 2013;96(2): 146–54.

12. Nowak DM, Gajecka M. Nonrandom distribution of miRNAs genes and single nucleotide variants in keratoconus loci. PloS one. 2015;10(7): e0132143. doi:10.1371/journal.pone.0132143.

13. Титоян К.Х., Хасанова Р.Р., Лобов С.Л., Джемилева Л.У., Усубов Э.Л., Бикбов М.М., Хуснутдинова Э.К. Анализ мутаций в гене SOD1 у пациентов с кератоконусом. Молекулярная диагностика 2017. Сборник Трудов IХ Всероссийской научно-практической конференции с международным участием. М.; 2017: 435–6. [Titojan KH, Hasanova RR, Lobov SL, Dzhemileva LU, Usubov EL, Bikbov MM, Khusnutdinova EK. Analiz mutacij v gene SOD1 u pacientov s keratokonusom. Molekuljarnaja Diagnostika 2017. Sbornik Trudov IX Vserossijskoj Nauchno-Prakticheskoj Konferencii S Mezhdunarodnym Uchastiem. M.; 2017: 435–6 (In Russ.).]

14. Скородумова Л.О., Белодедова А.В., Шарова Е.И., Малюгин Б.Э. Поиск генетических маркеров для уточняющей диагностики кератоконуса. Биомедицинская химия. 2019;65(1): 9–20. [Skorodumova LO, Belodedova AV, Sharova, EI, Malyugin BE. Search for genetic markers for precise diagnostics of keratoconus. Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry. 2019: 65(1): 9–20 (In Russ.).] doi:10.1134/S1990750819030090.

15. Vitart V, Benčić G, Hayward C, Škunca Herman J, Huffman J, Campbell S, Bućan K, Navarro P, Gunjaca G, Marin J, Zgaga L. New loci associated with central cornea thickness include COL5A1, AKAP13 and AVGR8. Hum Mol Genet. 2010;19(21): 4304–11. doi:10.1093/ hmg/ddq349..

16. Vithana EN, Aung T, Khor CC, Cornes BK, Tay WT, Sim X, Lavanya R, Wu R, Zheng Y, Hibberd ML, Chia KS. Collagen-related genes influence the glaucoma risk factor, central corneal thickness. Hum Mol Genet. 2011; 15;20(4): 649–58. doi:10.1093/hmg/ddq511.

17. Lu Y, Vitart V, Burdon KP, Khor CC, Bykhovskaya Y, Mirshahi A, Hewitt AW, Koehn D, Hysi PG, Ramdas WD, Zeller T. Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nat Genet. 2013;45(2): 155–63. doi:10.1038/ng.2506.

18. Iglesias AI, Mishra A, Vitart V, Bykhovskaya Y, Höhn R, Springelkamp H, Cuellar-Partida G, Gharahkhani P, Bailey JN, Willoughby CE, Li X. Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nature communications. 2018;14;9(1): 1864. doi:10.1038/s41467-018-03646-6.

19. Li X, Bykhovskaya Y, Canedo ALC, 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(4): 2696. doi:10.1167/iovs.13-11601.

20. Sahebjada S, Sahebjada S, Schache M, Richardson AJ, Snibson G, MacGregor S, Daniell M, Baird PN. Evaluating the Association Between Keratoconus and the Corneal Thickness Genes in an Independent Australian Population. Invest Ophthalmol Vis Sci. 2013;54(13): 8224. doi:10.1167/iovs.13-12982.

21. Liskova P, Dudakova L, Krepelova A, Klema J, Hysi PG. Replication of SNP associations with keratoconus in a Czech cohort. PLoS One. 2017;12(2): e0172365. doi:10.1371/journal.pone.0172365.

22. Segev F, Héon E, Cole WG, Wenstrup RJ, Young F, Slomovic AR, Rootman DS, Whitaker-Menezes D, Chervoneva I, Birk DE. Structural abnormalities of the cornea and lid resulting from collagen V mutations. Invest Ophthalmol Vis Sci. 2006;47(2): 565–73.

23. Smith SM, Birk DE. Focus on molecules: collagens V and XI. Exp Eye Res. 2012;98: 105–6. doi:10.1016/j.exer.2010.08.003.

24. Schwarze U, Atkinson M, Hoffman GG, Greenspan DS, Byers PH. Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II). Am J Hum Genet. 2000;66(6): 1757–65. doi:10.1086/302933.

25. De Paepe A, Nuytinck L, Hausser I, AntonLamprecht I, Naeyaert JM. Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II. Am J Hum Genet. 1997;60(3): 547–54.

26. Alkanaan A, Barsotti R, Kirat O, Khan A, Almubrad T, Akhtar S. Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea-Ultrastructure and 3D transmission electron tomography. Scientific Reports. 2019;9(1): 19963. doi:10.1038/s41598-019-56529-1.

27. Mathew JH, Goosey JD, Bergmanson JP. Quantified histopathology of the keratoconic cornea. Optom Vis Sci. 2011;88(8): 988–97. doi:10.1097/ OPX.0b013e31821ffbd4.

28. Takahashi A, Nakayasu K, Okisaka S, et al. Quantitative analysis of collagen fiber in keratoconus. Nihon Ganka Gakkai Zasshi. 1990;94: 1068–73.

29. Khaled ML, Helwa I, Drewry M, Seremwe M, Estes A, Liu Y. Molecular and histopathological changes associated with keratoconus. BioMed research international. 2017;7803029. doi:10.1155/2017/7803029.

30. Chaerkady R, Shao R, Scott SG, Pandey A, Jun AS, Chakravarti S. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J Proteomics. 2013;87: 122–31. doi:10.1016/j. jprot.2013.05.023.

31. Bykhovskaya Y, Gromova A, Makarenkova HP, Rabinowitz YS. Abnormal regulation of extracellular matrix and adhesion molecules in corneas of patients with keratoconus. Int J Keratoconus Ectatic Corneal Dis. 2016;5(2): 63–70. doi:10.5005/jp-journals-10025-1123.

Fyodorov Journal of Ophthalmic Surgery. 2020; : 6-13

Association of single nucleotide variants in the COL5A1 gene and adjacent intergenic space with keratoconus in a cohort of patients from Russia

Skorodumova L. O., Belodedova A. V., Zakharova E. S., Sharova Е. I., Bikbov M. M., Usubov E. L., Antonova O. P., Malyugin B. E.

https://doi.org/10.25276/0235-4160-2020-1-6-13

Abstract

Purpose. To assess an association of markers near the COL5A1 gene and in its intron with keratoconus. To study the relationship between the presence of marker alleles and the level of expression of the COL5A1 gene in the cornea of patients with keratoconus.

Material and methods.The study included 70 patients diagnosed with keratoconus, and 100 participants of the control group. All participants were genotyped for two markers associated with keratoconus: rs1536482 and rs7044529. The diagnostic indicators were calculated to assess a possibility of application of these markers as a keratoconus classifiers. In patients with keratoconus who underwent keratoplasty, corneal samples (n=9) were collected to isolate the RNA. The COL5A1 gene expression level was compared between the group of patient corneal samples with at least one marker allele (rs1536482 +) and the group of samples without marker alleles (rs1536482-) using real-time PCR coupled with reverse transcription.

Results. A significant difference of the rs1536482 marker allele occurrence was found between the group of keratoconus patients and the control group (p=0.0004, Fisher test). At least one marker allele rs1536482 was found in 67.1% of patients (marker sensitivity) with keratoconus and in 46% of control group participants. Thus, the specificity of the marker is low (0.54). The odds ratio was 2.4, and the risk ratio was 1.69. Gene expression analysis in the keratoconus patients’ cornea did not reveal an association of the COL5A1 gene expression level with the genotype (p=0.9, Mann-Whitney test).

Conclusion. In this study, an association of rs1536482 with keratoconus was identified in a cohort of patients from Russia. However, due to the low specificity of this marker, it cannot be used as a keratoconus classifier. The presence of a marker allele is not associated with the COL5A1 gene expression level.

References

1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42(4): 297–319. doi:10.1016/s00396257(97)00119-7.

2. Tanabe U, Fujiki K, Ogawa A, Ueda S, Kanai A. [Prevalence of keratoconus patients in Japan]. Nippon Ganka Gakkai Zasshi. 1985;89(3): 407–11.

3. Waked N, Fayad AM, Fadlallah A, El Rami H. Keratoconus screening in a Lebanese students’ population. J Fr Ophtalmol. 2012;35(1): 23–9. doi:10.1016/j.jfo.2011.03.016.

4. Said A, Hamade IH, Tabbara KF. Late onset corneal ectasia after LASIK surgery. Saudi J Ophthalmol. 2011;25(3): 225–30. doi:10.1016/J.SJOPT.2011.05.003.

5. Randleman JB, Woodward M, Lynn MJ, Stulting RD. Risk assessment for ectasia after corneal refractive surgery. Ophthalmology. 2008;115(1): 37– 50. doi:10.1016/j.ophtha.2007.03.073.

6. Giri P, Azar DT. Risk profiles of ectasia after keratorefractive surgery. Curr Opin Ophthalmol. 2017;28(4): 337–42. doi:10.1097/ ICU.0000000000000383.

7. Owens H, Walters GA. Keratoconus in monozygotic twins in New Zealand. 1995; Clin Exper Optometry. 78(4): 125– 9. doi:10.1111/j.1444-0938.1995.tb00805.x.

8. Bechara SJ, Insler MS. Keratoconus in two pairs of identical twins. Cornea. 1996 Jan;15(1): 90–3.

9. Tuft SJ, Hassan H, George S, Frazer DG, Willoughby CE, Liskova P. Keratoconus in 18 pairs of twins. Acta Ophthalmol. 2012;90(6): e482-6. doi:10.1111/j.1755-3768.2012.02448.x.

10. Bykhovskaya Y, Margines B, Rabinowitz YS. Genetics in Keratoconus: where are we? Eye Vis (Lond.). 2016;3(1): 16. doi:10.1186/s40662-016-0047-5.

11. Burdon KP, Vincent AL. Insights into keratoconus from a genetic perspective. Clin Exp Optom. 2013;96(2): 146–54.

12. Nowak DM, Gajecka M. Nonrandom distribution of miRNAs genes and single nucleotide variants in keratoconus loci. PloS one. 2015;10(7): e0132143. doi:10.1371/journal.pone.0132143.

13. Titoyan K.Kh., Khasanova R.R., Lobov S.L., Dzhemileva L.U., Usubov E.L., Bikbov M.M., Khusnutdinova E.K. Analiz mutatsii v gene SOD1 u patsientov s keratokonusom. Molekulyarnaya diagnostika 2017. Sbornik Trudov IKh Vserossiiskoi nauchno-prakticheskoi konferentsii s mezhdunarodnym uchastiem. M.; 2017: 435–6. [Titojan KH, Hasanova RR, Lobov SL, Dzhemileva LU, Usubov EL, Bikbov MM, Khusnutdinova EK. Analiz mutacij v gene SOD1 u pacientov s keratokonusom. Molekuljarnaja Diagnostika 2017. Sbornik Trudov IX Vserossijskoj Nauchno-Prakticheskoj Konferencii S Mezhdunarodnym Uchastiem. M.; 2017: 435–6 (In Russ.).]

14. Skorodumova L.O., Belodedova A.V., Sharova E.I., Malyugin B.E. Poisk geneticheskikh markerov dlya utochnyayushchei diagnostiki keratokonusa. Biomeditsinskaya khimiya. 2019;65(1): 9–20. [Skorodumova LO, Belodedova AV, Sharova, EI, Malyugin BE. Search for genetic markers for precise diagnostics of keratoconus. Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry. 2019: 65(1): 9–20 (In Russ.).] doi:10.1134/S1990750819030090.

15. Vitart V, Benčić G, Hayward C, Škunca Herman J, Huffman J, Campbell S, Bućan K, Navarro P, Gunjaca G, Marin J, Zgaga L. New loci associated with central cornea thickness include COL5A1, AKAP13 and AVGR8. Hum Mol Genet. 2010;19(21): 4304–11. doi:10.1093/ hmg/ddq349..

16. Vithana EN, Aung T, Khor CC, Cornes BK, Tay WT, Sim X, Lavanya R, Wu R, Zheng Y, Hibberd ML, Chia KS. Collagen-related genes influence the glaucoma risk factor, central corneal thickness. Hum Mol Genet. 2011; 15;20(4): 649–58. doi:10.1093/hmg/ddq511.

17. Lu Y, Vitart V, Burdon KP, Khor CC, Bykhovskaya Y, Mirshahi A, Hewitt AW, Koehn D, Hysi PG, Ramdas WD, Zeller T. Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nat Genet. 2013;45(2): 155–63. doi:10.1038/ng.2506.

18. Iglesias AI, Mishra A, Vitart V, Bykhovskaya Y, Höhn R, Springelkamp H, Cuellar-Partida G, Gharahkhani P, Bailey JN, Willoughby CE, Li X. Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nature communications. 2018;14;9(1): 1864. doi:10.1038/s41467-018-03646-6.

19. Li X, Bykhovskaya Y, Canedo ALC, 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(4): 2696. doi:10.1167/iovs.13-11601.

20. Sahebjada S, Sahebjada S, Schache M, Richardson AJ, Snibson G, MacGregor S, Daniell M, Baird PN. Evaluating the Association Between Keratoconus and the Corneal Thickness Genes in an Independent Australian Population. Invest Ophthalmol Vis Sci. 2013;54(13): 8224. doi:10.1167/iovs.13-12982.

21. Liskova P, Dudakova L, Krepelova A, Klema J, Hysi PG. Replication of SNP associations with keratoconus in a Czech cohort. PLoS One. 2017;12(2): e0172365. doi:10.1371/journal.pone.0172365.

22. Segev F, Héon E, Cole WG, Wenstrup RJ, Young F, Slomovic AR, Rootman DS, Whitaker-Menezes D, Chervoneva I, Birk DE. Structural abnormalities of the cornea and lid resulting from collagen V mutations. Invest Ophthalmol Vis Sci. 2006;47(2): 565–73.

23. Smith SM, Birk DE. Focus on molecules: collagens V and XI. Exp Eye Res. 2012;98: 105–6. doi:10.1016/j.exer.2010.08.003.

24. Schwarze U, Atkinson M, Hoffman GG, Greenspan DS, Byers PH. Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II). Am J Hum Genet. 2000;66(6): 1757–65. doi:10.1086/302933.

25. De Paepe A, Nuytinck L, Hausser I, AntonLamprecht I, Naeyaert JM. Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II. Am J Hum Genet. 1997;60(3): 547–54.

26. Alkanaan A, Barsotti R, Kirat O, Khan A, Almubrad T, Akhtar S. Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea-Ultrastructure and 3D transmission electron tomography. Scientific Reports. 2019;9(1): 19963. doi:10.1038/s41598-019-56529-1.

27. Mathew JH, Goosey JD, Bergmanson JP. Quantified histopathology of the keratoconic cornea. Optom Vis Sci. 2011;88(8): 988–97. doi:10.1097/ OPX.0b013e31821ffbd4.

28. Takahashi A, Nakayasu K, Okisaka S, et al. Quantitative analysis of collagen fiber in keratoconus. Nihon Ganka Gakkai Zasshi. 1990;94: 1068–73.

29. Khaled ML, Helwa I, Drewry M, Seremwe M, Estes A, Liu Y. Molecular and histopathological changes associated with keratoconus. BioMed research international. 2017;7803029. doi:10.1155/2017/7803029.

30. Chaerkady R, Shao R, Scott SG, Pandey A, Jun AS, Chakravarti S. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J Proteomics. 2013;87: 122–31. doi:10.1016/j. jprot.2013.05.023.

31. Bykhovskaya Y, Gromova A, Makarenkova HP, Rabinowitz YS. Abnormal regulation of extracellular matrix and adhesion molecules in corneas of patients with keratoconus. Int J Keratoconus Ectatic Corneal Dis. 2016;5(2): 63–70. doi:10.5005/jp-journals-10025-1123.