通过迷你基因实验证实4个与X连锁Alport 综合征相关的同义变异改变了IV型 胶原α5链基因的剪接过程

Minigene Assays Validate That Four Synonymous Variants Associated with X-Linked Alport Syndrome Alter the Splicing of the Collagen IV α5 Gene

摘要: 背景：X连锁Alport综合征(X-linked Alport syndrome, XLAS)是一种由COL4A5基因突变所致的遗传性肾小球疾病。剪接变异是XLAS的核心致病机制之一。同义变异虽不直接改变基因编码序列，但可能通过干扰剪接调控信号、破坏前体mRNA的正常剪接过程，从而影响基因的正常表达。然而，这类变异的致病性判定常存在较大挑战，需要通过转录本分析来确定剪接模式并评估致病性，但此类分析并非总能实现。方法：我们从ClinVar数据库中收集了COL4A5基因中所有的同义变异。运用SpliceAI、BDGP、HSF等生物信息学工具预测这些变异对经典剪接位点及剪接调控辅助元件的影响，进而筛选出候选剪接变异。通过迷你基因实验进一步验证这些变异对前体mRNA剪接过程的影响。最后，根据美国医学遗传学和基因组学学会(ACMG)标准和指南对识别出来的剪接变异重新分类。结果：迷你基因实验结果证实：变异c.465G>A，c.3942G>A导致相应外显子完全跳跃；变异c.4953C>T显著增加了外显子25完全跳跃的比例，但仍保留少量正常转录本；变异c.1683A>T导致24号外显子部分缺失。最后，根据ACMG标准和指南，我们将识别出来的这4个影响剪接的同义变异重新判定为致病性变异。结论：本研究结合生物信息学分析工具与体外迷你基因实验，对已知同义变异的致病性重新评估，为XLAS的变异解读及临床诊断提供理论依据。

Abstract: Background: X-linked Alport syndrome (XLAS) is a hereditary glomerular disease caused by pathogenic variants in the COL4A5 gene. Splicing variants represent one of the key pathogenic mechanisms underlying XLAS. Although synonymous variants do not directly alter the coding sequence, they may disrupt splicing regulatory signals and interfere with normal pre-mRNA splicing, thereby impairing gene expression. Nevertheless, determining the pathogenicity of such variants remains challenging. Transcript analysis is required to characterize splicing patterns and evaluate clinical significance, yet functional verification is not always feasible in routine practice. Methods: All synonymous variants in the COL4A5 gene were retrieved from the ClinVar database. Bioinformatic tools, including SpliceAI, BDGP, and HSF, were employed to predict the effects of these variants on canonical splice sites and auxiliary splicing regulatory elements, followed by screening candidate splice-altering variants. The impact of these variants on pre-mRNA splicing was further validated using minigene assays. Finally, all identified splicingrelated synonymous variants were reclassified according to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines. Results: Minigene assays confirmed that variants c.465G>A and c.3942G>A led to complete skipping of their corresponding exons; variant c.4953C>T significantly increased the proportion of exon 25 skipping, while retaining a small amount of normal transcript; and variant c.1683A>T resulted in partial deletion of exon 24. Based on the ACMG standards and guidelines, these four synonymous variants were reclassified as pathogenic. Conclusion: This study combined bioinformatics analysis tools with in vitro minigene assays to re-evaluate the pathogenicity of known synonymous variants, providing a theoretical basis for variant interpretation and clinical diagnosis of XLAS.