Synonymous single nucleotide variants (sSNVs) have been implicated in various genetic disorders through alterations of pre-mRNA splicing, mRNA structure and miRNA regulation. However, their impact on synonymous codon usage and protein translation remains to be elucidated in clinical context. Here, we explore the functional impact of sSNVs in the Sonic Hedgehog (SHH) gene, identified in patients affected by holoprosencephaly, a congenital brain defect resulting from incomplete forebrain cleavage. We identified eight sSNVs in SHH, selectively enriched in holoprosencephaly patients as compared to healthy individuals, and systematically assessed their effect at both transcriptional and translational levels using a series of in silico and in vitro approaches. Although no evidence of impact of these sSNVs on splicing, mRNA structure or miRNA regulation was found, five sSNVs introduced significant changes in codon usage and were predicted to impact protein translation. Cell assays demonstrated that these five sSNVs are associated with a significantly reduced amount of the resulting protein, ranging from 5% to 23%. Inhibition of the proteasome rescued the protein levels for four out of five sSNVs, confirming their impact on protein stability and folding. Remarkably, we found a significant correlation between experimental values of protein reduction and computational measures of codon usage, indicating the relevance of in silico models in predicting the impact of sSNVs on translation. Considering the critical role of SHH in brain development, our findings highlight the clinical relevance of sSNVs in holoprosencephaly and underline the importance of investigating their impact on translation in human pathologies.

中文翻译：

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全前脑同义变体会改变密码子使用并影响Sonic Hedgehog蛋白。

通过改变前mRNA剪接，mRNA结构和miRNA调节，同义单核苷酸变体（sSNV）已被牵涉到各种遗传疾病中。但是，它们对同义密码子使用和蛋白质翻译的影响在临床背景下仍有待阐明。在这里，我们探讨了sSNVs在Sonic Hedgehog（SHH）基因中的功能影响，该基因在受前脑缺损（由于前脑分裂不完全导致的先天性脑缺损）影响的患者中确定。我们在SHH中鉴定出8个sSNV，与健康个体相比，在全前脑患者中选择性富集，并使用一系列计算机模拟和体外系统评估其在转录和翻译水平上的作用方法。尽管没有发现这些sSNV对剪接，mRNA结构或miRNA调控的影响的证据，但五种sSNV引入了密码子使用的重大变化，并预计会影响蛋白质翻译。细胞分析表明，这五个sSNV与产生的蛋白质量显着降低有关，范围为5％至23％。蛋白酶体的抑制挽救了五分之四的sSNV的蛋白质水平，证实了它们对蛋白质稳定性和折叠的影响。值得注意的是，我们发现蛋白质还原的实验值与密码子使用的计算指标之间存在显着相关性，表明计算机模型在预测sSNV对翻译的影响方面具有相关性。考虑SHH的关键作用 在大脑发育中，我们的发现突出了sSNV在全脑前脑的临床相关性，并强调了研究其对人类病理学翻译的影响的重要性。