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

Öz

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.

Anahtar Kelimeler

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

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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

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