Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto City, 862-0973, Japan.
Global Center for Natural Resources Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Clin Exp Nephrol. 2024 Sep;28(9):874-881. doi: 10.1007/s10157-024-02503-9. Epub 2024 Apr 24.
Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping.
We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed.
Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart.
Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.
Alport 综合征（AS）是一种遗传性肾脏疾病，由 IV 型胶原 α3、α4 和 α5 的突变引起，这些突变通常作为三聚体 α345(IV)分泌。这些基因中的无义突变导致严重的 AS 表型。我们之前的研究表明，通过外显子跳跃去除 α5(IV)中的无义突变可改善 AS 病理。然而，去除外显子对三聚体形成的影响尚不清楚。在这里，我们评估了外显子缺失对三聚体形成的影响，以评估其潜在的治疗适用性，并预测与外显子跳跃相关的突变的严重程度。
我们通过诱变产生外显子缺失构建体（ΔExon）、无义和错义突变体，并使用我们之前开发的基于纳米荧光素酶的测定法评估它们的三聚体形成和分泌活性。
外显子跳跃对 α345(IV)的三聚体分泌有不同的影响。一些 ΔExon 可以形成和分泌 α345(IV)三聚体，并且比无义突变体具有更高的活性。其他 ΔExon 的分泌活性较低，特别是对于靠近 C 末端的外显子缺失的 ΔExon，尽管细胞内三聚体化正常。错义突变体及其 ΔExon 对应物的分泌没有差异。
外显子跳跃对具有严重表型和早期肾功能衰竭的 AS 中的无义突变体有利，但应用可能仅限于能够正常三聚体化和分泌的 ΔExon。本研究为 Alport 综合征中 α5(IV)外显子跳跃的可能治疗应用和与外显子跳跃相关的三聚体行为的预测提供了信息。
