In Silico Functional Assessment of COL4A3, COL4A4, and COL4A5 SNPs in Alport Syndrome

Abstract

Aim: Alport syndrome is a rare genetic disorder characterized by hematuria, proteinuria, progressive renal failure, and, in some cases, hearing and visual impairment. This study aims to identify and prioritize deleterious missense single-nucleotide polymorphisms (SNPs) in the COL4A3, COL4A4, and COL4A5 genes associated with Alport syndrome and to evaluate their effects on protein stability and three-dimensional structure using an integrated multi-tool in silico approach.

Method: SNP data and protein amino acid sequences were obtained from the NCBI dbSNP and UniProt databases; these data were used as input in in silico analyses performed using various bioinformatics tools, including SIFT, PolyPhen-2, SNPs&GO, PANTHER, PROVEAN, SNAP2, Mutation Assessor, I-Mutant 2.0, MUpro, and Project HOPE, to evaluate the potential structural and functional effects on COL4A3, COL4A4, and COL4A5 proteins. Only variants predicted as deleterious by all applied tools were selected for further analysis.

Results: A large number of SNPs were initially identified for COL4A3, COL4A4, and COL4A5 genes; however, multi-tool consensus analysis identified 12, 14, and 146 deleterious missense variants in COL4A3, COL4A4, and COL4A5, respectively.  Protein stability analyses produced divergent results: I-Mutant 2.0 predicted increased stability for 160 of 172 variants, whereas MUpro predicted decreased stability for 147 variants. In addition, a substantial number of deleterious variants involved glycine substitutions, which are likely to affect protein flexibility, folding, and intermolecular interactions, potentially impairing the structural integrity of the glomerular basement membrane.

Conclusion: This study represents a comprehensive consensus-based in silico evaluation of deleterious missense SNPs in COL4A3, COL4A4, and COL4A5 genes. The findings highlight the potential role of glycine substitutions in disease pathogenesis and provide an important basis for future experimental studies.

Keywords

• Alport syndrome
• single nucleotide polymorphism
• bioinformatics
• COL4A3
• COL4A4
• COL4A5

Alport Sendromunda COL4A3, COL4A4 ve COL4A5 SNP’lerinin İn Silico Fonksiyonel Değerlendirilmesi

Abstract

Amaç: Alport sendromu, hematüri, proteinüri, ilerleyici böbrek yetmezliği ve bazı olgularda işitme ve görme bozuklukları ile karakterize nadir görülen bir genetik hastalıktır. Bu çalışma, Alport sendromuyla ilişkili COL4A3, COL4A4 ve COL4A5 genlerindeki olası patojenik yanlış anlamlı tek nükleotid polimorfizmlerini (SNP’leri) belirlemek ve önceliklendirmek ve bu SNP’lerin protein stabilitesi ve üç boyutlu yapısı üzerindeki etkilerini çoklu in silico yaklaşım kullanarak değerlendirmektir.

Yöntem: SNP verileri ve protein amino asit dizileri NCBI dbSNP ve UniProt veri tabanlarından elde edildi; bu veriler, COL4A3, COL4A4 ve COL4A5 proteinleri üzerindeki potansiyel yapısal ve fonksiyonel etkileri değerlendirmek amacıyla SIFT, PolyPhen-2, SNPs&GO, PANTHER, PROVEAN, SNAP2, Mutation Assessor, I-Mutant 2.0, MUpro ve Project HOPE gibi çeşitli biyoenformatik araçları kullanılarak in silico analizler yapıldı. Kullanılan tüm araçlar tarafından zararlı olarak tahmin edilen varyantlar ileri analizler için seçildi.

Bulgular: Başlangıçta COL4A3, COL4A4 ve COL4A5 genlerinde çok sayıda SNP tespit edildi, ancak çoklu araçlara dayalı konsensüs analizi sonucunda sırasıyla 12, 14 ve 146 zararlı missense varyant tespit edildi. Protein stabilitesi analizleri farklı sonuçlar ortaya koymuştur:    I-Mutant 2.0, 172 varyantın 160’ında stabilite artışı ön görürken MUpro, 147 varyantta stabilite azalması öngörmüştür. Ayrıca, zararlı varyantların önemli bir kısmının glisin substitüsyonlarını içerdiği ve bu değişimlerin protein esnekliği, katlanması ve moleküller arası etkileşimleri etkileyerek glomerüler bazal membranın yapısal bütünlüğünü bozabileceği belirlenmiştir.

Sonuç: Bu çalışma, COL4A3, COL4A4 ve COL4A5 genlerindeki zararlı missense SNP’lerin konsensüs temelli kapsamlı bir in silico değerlendirmesini sunmaktadır. Elde edilen bulgular, özellikle glisin substitüsyonlarının hastalık patogenezindeki olası rolünü ortaya koymakta ve gelecekte yapılacak deneysel çalışmalar için önemli bir temel sağlamaktadır.

Keywords

• Alport sendromu
• tek nükleotid polimorfizmi
• biyoenformatik
• COL4A3
• COL4A4
• COL4A5

References

1. 1. Williamson DA. Alport's syndrome of hereditary nephritis with deafness. Lancet. 1961;2(7216):1321-1323. doi: 10.1016/S0140-6736(61)90899-6.
2. 2. Gubler MC, Levy M, Broyer M, et al. Alport's syndrome: A report of 58 cases and a review of the literature. Am J Med. 1981;71(4):493-505. doi: 10.1016/0002-9343(81)90571-4.
3. 3. Temme J, Peters F, Lange K, et al. Incidence of renal failure and nephroprotection by RAAS inhibition in heterozygous carriers of X-linked and autosomal recessive Alport mutations. Kidney Int. 2012;81(8):779-783. doi: 10.1038/ki.2011.452.
4. 4. Gross O, Weber M. From the molecular genetics of Alport's syndrome to principles of organo-protection in chronic renal diseases. Med Klin (Munich). 2005;100(12):826-831.
5. 5. Migeon BR. X inactivation, female mosaicism, and sex differences in renal diseases. J Am Soc Nephrol. 2008;19(11):2052-2059. doi: 10.1681/ASN.2008020198.
6. 6. Kashtan CE. Alport syndrome: Achieving early diagnosis and treatment. Am J Kidney Dis. 2021;77(2):272-279. doi: 10.1053/j.ajkd.2020.03.026.
7. 7. Izzedine H, Tankere F, Launay-Vacher V, Deray G. Ear and kidney syndromes: Molecular versus clinical approach. Kidney Int. 2004;65(4):1290-1302.
8. 8. Shaw EA, Colville D, Wang YY, et al. Characterization of the peripheral retinopathy in X-linked and autosomal recessive Alport syndrome. Nephrol Dial Transplant. 2007;22(1):104-108. doi: 10.1093/ndt/gfl607.

9. 9. Byrne MC, Budisavljevic MN, Fan Z, et al. Renal transplant in patients with Alport's syndrome. Am J Kidney Dis. 2002;39(4):769-775. doi: 10.1053/ajkd.2002.31997.
10. 10. Perrin D, Jungers P, Grünfeld JP, et al. Perimacular changes in Alport's syndrome. Am J Ophthalmol. 1980;90(1):1-7. doi: 10.1016/s0002-9394(14)75069-7.
11. 11. Shah SN, Weinberg DV. Giant macular hole in Alport syndrome. Ophthalmic Genet. 2010;31(2):89-91. doi: 10.3109/13816811003767128.
12. 12. Gross O, Licht C, Anders HJ, et al. Early angiotensin-converting enzyme inhibition in Alport syndrome delays renal failure and improves life expectancy. Kidney Int. 2012;81(5):494-501. doi: 10.1038/ki.2011.407.
13. 13. GeneCards. COL4A5 gene. GeneCards. https://www.genecards.org/cgi-bin/carddisp.pl?gene=COL4A5. Accessed April 27, 2026.
14. 14. GeneCards. COL4A4 gene. GeneCards. https://www.genecards.org/cgi-bin/carddisp.pl?gene=COL4A4. Accessed April 27, 2026.
15. 15. GeneCards. COL4A3 gene. GeneCards. https://www.genecards.org/cgi-bin/carddisp.pl?gene=COL4A3. Accessed April 27, 2026.
16. 16. Karki R, Pandya D, Elston RC, Ferlini C. Defining “mutation” and “polymorphism” in the era of personal genomics. BMC Med Genomics. 2015;8:37. doi:10.1186/s12920-015-0115-z.
17. 17. Ismail S, Essawi M. Genetic polymorphism studies in humans. Middle East J Med Genet. 2012;1(2):57-63. doi: 10.1097/01.MXE.0000415225.85003.47.
18. 18. MedlinePlus. What are single nucleotide polymorphisms (SNPs)? MedlinePlus Genetics.https://medlineplus.gov/genetics/understanding/genomicresearch/snp/. Accessed April 27, 2026.
19. 19. Lander ES. The new genomics: Global views of biology. Science. 1996;274(5287):536-539.
20. 20. Dabhi B, Mistry KN. In silico analysis of SNP in human TNF-α gene. Meta Gene. 2014;2:586-595. doi: 10.1016/j.mgene.2014.07.005.
21. 21. Cargill M, Altshuler D, Ireland J, et al. Characterization of SNPs in coding regions of human genes. Nat Genet. 1999;22(3):231-238. doi: 10.1038/10290.
22. 22. Rozario LT, Sharker T, Nila TA. In silico analysis of deleterious SNPs of human MTUS1 gene. PLoS One. 2021;16(6):e0252932. doi: 10.1371/journal.pone.0252932.
23. 23. Mah JT, Low ES, Lee E. In silico SNP analysis and bioinformatics tools. Drug Discov Today. 2011;16(17-18):800-809. doi: 10.1016/j.drudis.2011.07.005.
24. 24. The UniProt Consortium. UniProt: The universal protein knowledgebase in 2021. Nucleic Acids Res. 2021;49(D1):D480-D489. doi:10.1093/nar/gkaa1100.
25. 25. 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(7):1073-1081. doi: 10.1038/nprot.2009.86.
26. 26. Ng PC, Henikoff S. Predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet. 2006;7:61-80. doi: 10.1146/annurev.genom.7.080505.115630.
27. 27. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248-249. doi: 10.1038/nmeth0410-248.
28. 28. Choi Y, Chan AP. PROVEAN web server: A tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics. 2015;31(16):2745-2747. doi: 10.1093/bioinformatics/btv195.
29. 29. Hecht M, Bromberg Y, Rost B. Better prediction of functional effects for sequence variants. BMC Genomics. 2015;16(Suppl 8):S1. doi: 10.1186/1471-2164-16-S8-S1.
30. 30. Capriotti E, Fariselli P, Casadio R. I-Mutant2.0: Predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Res. 2005;33(Web Server issue):W306-W310. doi: 10.1093/nar/gki375.
31. 31. Cheng J, Randall A, Baldi P. Prediction of protein stability changes for single-site mutations using support vector machines. Proteins. 2006;62(4):1125-1132. doi: 10.1002/prot.20810.
32. 32. Venselaar H, Te Beek TA, Kuipers RK, Hekkelman ML, Vriend G. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics. 2010;11:548. doi: 10.1186/1471-2105-11-548.
33. 33. Warde-Farley D, Donaldson SL, Comes O, et al. The GeneMANIA prediction server: Biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 2010;38(Web Server issue):W214-W220. doi: 10.1093/nar/gkq537.
34. 34. Wang Z, Moult J. SNPs, protein structure, and disease. Hum Mutat. 2001;17(4):263-270.
35. 35. Yue P, Li Z, Moult J. Loss of protein structure stability in disease. J Mol Biol. 2005;353(2):459-473. doi: 10.1016/j.jmb.2005.08.020.
36. 36. Zhou J, Hertz JM, Tryggvason K. Mutation in COL4A5 chain in Alport syndrome. Am J Hum Genet. 1992;50(6):1291-1300.
37. 37. Knebelmann B, Deschenes G, Gros F, et al. Substitution of arginine for glycine 325 in COL4A5. Am J Hum Genet. 1992;51(1):135-142.
38. 38. Renieri A, Seri M, Myers JC, et al. De novo mutation in COL4A5 gene. Hum Mol Genet. 1992;1(2):127-129. doi: 10.1093/hmg/1.2.127.
39. 39. Choi M, Anistan YM, Eckardt KU, Gollasch M, Nickel P. Possible digenic disease in COL4A3 and COL4A5. Nephron. 2019;141(3):213-218. doi: 10.1159/000495764.
40. 40. Kashtan CE, Fish AJ, Kleppel M, Yoshioka K, Michael AF. Nephritogenic antigens in Alport syndrome. J Clin Invest. 1986;78(4):1035-1044. doi: 10.1172/JCI112658.
41. 41. Hudson BG, Tryggvason K, Sundaramoorthy M, Neilson EG. Alport's syndrome and type IV collagen. N Engl J Med. 2003;348(25):2543-2556. doi: 10.1056/NEJMra022296.
42. 42. Kuivaniemi H, Tromp G. Type III collagen: Structure and diseases. Gene. 2019;707:151-171. doi: 10.1016/j.gene.2019.05.003.
43. 43. Shboul M, Sassi H, Jilani H, et al. Glycine to serine mutation in COL2A1 gene. AIMS Mol Sci. 2021;8(1):76-85. doi: 10.3934/molsci.2021004.
44. 44. Nuytinck L, Tükel T, Kayserili H, Apak MY, De Paepe A. Glycine to tryptophan substitution in collagen. J Med Genet. 2000;37(5):371-375. doi: 10.1136/jmg.37.5.371.
45. 45. Molnár J, Szakács G, Tusnády GE. Disease-associated mutations in transmembrane proteins. PLoS One. 2016;11(3):e0151760. doi: 10.1371/journal.pone.0151760.
46. 46. Wilcox W, Scott L, Cohn D. An N-terminal glycine to cysteine mutation in the collagen COL1A1 gene produces moderately severe osteogenesis imperfecta. Am J Hum Genet. 1994;55(suppl 3).
47. 47. MacArthur MW, Thornton JM. Influence of proline residues on protein conformation. J Mol Biol. 1991;218(2):397-412. doi: 10.1016/0022-2836(91)90721-H.
48. 48. Creighton TE. Disulphide bonds and protein stability. BioEssays. 1988;8(2-3):57-63. doi: 10.1002/bies.950080204.
49. 49. Yavas C, Ozdemir Ozgenturk N, Dogan M, et al. A deeper insight into COL4A3, COL4A4, and COL4A5 variants and genotype-phenotype correlation of a Turkish cohort with Alport syndrome. Mol Syndromol. 2024;15(1):1-13. doi: 10.1159/000533915.