自发现RAD51C以来的20多年里,科学家对这种抑癌基因的错义变异如何影响其功能和致病性感到困惑。与乳腺癌和卵巢癌有很强的联系,在诊断和治疗RAD51C变异的患者时,将这些变异分为致病性或良性辅助。特别是,翻译起始位点的变体是破坏性的,因为它们阻止蛋白质表达。这些变异通常被归类为致病性的,除非显示替代的翻译起始产生功能性同种型以挽救蛋白质表达。在这项研究中,我们利用核糖体分析数据库GWIPS-VIZ鉴定了人RAD51C中甲硫氨酸一和甲硫氨酸十的两个活性翻译起始位点。甲硫氨酸10处的第二个翻译起点在97%的哺乳动物中都是保守的,并且在80%的哺乳动物中是唯一的翻译起点。已在47个个体中鉴定出任一蛋氨酸的错义变体,阻止这两个起始位点之一的表达。因此,我们稳定表达了两种野生型亚型,以及RAD51CCRISPR/Cas9敲除U2OS细胞中的RAD51CM1和M10变体,并比较了它们的同源重组功能。令人惊讶的是,我们发现,从任一起始位点表达人RAD51C可以通过姐妹染色单体重组试验等效地挽救RAD51CCRISPR/Cas9敲除U2OS细胞的同源重组。同样,我们的每个RAD51CCRISPR/Cas9KO细胞在M1或M10处与RAD51C错义变体稳定互补,均具有同源重组能力.一起,我们的数据表明,RAD51C有两个翻译起始位点,并且任一甲硫氨酸中的变体均可导致同源重组熟练.有了这个关键的发现,M1变异的个体在对这些变异进行重新分类后,将更准确地了解其癌症风险.
In the 20+ years since the discovery of
RAD51C, scientists have been perplexed as to how missense variants in this tumor suppressor gene impacts its function and pathogenicity. With a strong connection to breast and ovarian cancer, classifying these variants as pathogenic or benign aids in the diagnosis and treatment of patients with RAD51C variants. In particular, variants at translational starts sites are disruptive as they prevent protein expression. These variants are often classified as pathogenic, unless an alternative translational start is shown to produce a functional isoform to rescue protein expression. In this study, we utilized the ribosome profiling database GWIPS-VIZ to identify two active translational start sites in human RAD51C at methionine one and methionine ten. This second translational start at methionine ten is both conserved in 97 % of mammals and is the sole translational start in 80 % of mammals. Missense variants at either methionine have been identified in 47 individuals, preventing expression from one of these two start sites. Therefore, we stably expressed both wildtype isoforms, as well as the
RAD51C M1 and M10 variants in a
RAD51C CRISPR/Cas9 knockout U2OS cell and compared their homologous recombination function. Surprisingly, we find that expression of human
RAD51C from either start site can equivalently rescue homologous recombination of RAD51C CRISPR/Cas9 knockout U2OS cells through a sister chromatid recombination assay. Similarly, each of our
RAD51C CRISPR/Cas9 KO cells stably complemented with
RAD51C missense variants at either M1 or M10 are homologous recombination proficient. Together, our data demonstrate that RAD51C has two translational start sites and that variants in either methionine result in homologous recombination proficiency. With this critical discovery, individuals with variants at M1 will be more accurately informed of their cancer risk upon reclassification of these variants.