Exonic (i.e. coding) variants in genes associated with disease can exert pathogenic effects both at the protein and mRNA level, either by altering the amino acid sequence or by affecting pre-mRNA splicing. The latter is often neglected due to the lack of RNA analyses in genetic diagnostic testing. In this study we considered both pathomechanisms and performed a comprehensive analysis of nine exonic nucleotide changes in OPA1, which is the major gene underlying autosomal dominant optic atrophy (DOA) and is characterized by pronounced allelic heterogeneity. We focused on the GTPase-encoding domain of OPA1, which harbors most of the missense variants associated with DOA. Given that the consensus splice sites extend into the exons, we chose a split codon, namely codon 438, for our analyses. Variants at this codon are the second most common cause of disease in our large cohort of DOA patients harboring disease-causing variants in OPA1. In silico splice predictions, heterologous splice assays, analysis of patient’s RNA when available, and protein modeling revealed different molecular outcomes for variants at codon 438. The wildtype aspartate residue at amino acid position 438 is directly involved in the dimerization of OPA1 monomers. We found that six amino acid substitutions at codon 438 (i.e. all substitutions of the first and second nucleotide of the codon) destabilized dimerization while only substitutions of the first nucleotide of the codon caused exon skipping. Our study highlights the value of combining RNA analysis and protein modeling approaches to accurately assign patients to future precision therapies.

中文翻译：

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OPA1 的 GTPase 编码域中一个分裂密码子的突变通过不同的分子机制导致显性视神经萎缩

与疾病相关的基因中的外显子（即编码）变体可以通过改变氨基酸序列或通过影响前mRNA剪接在蛋白质和mRNA水平上发挥致病作用。由于在基因诊断测试中缺乏 RNA 分析，后者经常被忽视。在这项研究中，我们考虑了两种病理机制并对 OPA1 中的九个外显子核苷酸变化进行了综合分析，OPA1 是常染色体显性遗传性视神经萎缩 (DOA) 的主要基因，其特征是明显的等位基因异质性。我们专注于 OPA1 的 GTPase 编码域，它包含与 DOA 相关的大多数错义变体。鉴于共有剪接位点延伸到外显子，我们选择了一个分裂密码子，即密码子 438，用于我们的分析。这个密码子的变异是我们的一大群 DOA 患者中第二个最常见的疾病原因，这些 DOA 患者在 OPA1 中含有致病变异。在计算机剪接预测、异源剪接分析、可用时患者的 RNA 分析和蛋白质建模中，揭示了密码子 438 处变体的不同分子结果。氨基酸位置 438 处的野生型天冬氨酸残基直接参与 OPA1 单体的二聚化。我们发现密码子 438 的六个氨基酸取代（即密码子的第一个和第二个核苷酸的所有取代）使二聚化不稳定，而只有密码子的第一个核苷酸的取代导致外显子跳跃。我们的研究强调了将 RNA 分析和蛋白质建模方法相结合以准确分配患者接受未来精准治疗的价值。