Univ Brest, Inserm, EFS, UMR1078, GGB, Brest, France.
Laboratory of Excellence GR-Ex, Paris, France.
Transfusion. 2021 Aug;61(8):2468-2476. doi: 10.1111/trf.16538. Epub 2021 Jun 10.
Although D variant phenotype is known to be due to genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed the molecular and cellular mechanisms driving this altered expression. We and others showed previously that splicing is commonly disrupted by SNVs in constitutive splice sites and their vicinity. We thus sought to investigate whether rare missense SNVs located in "deep" exonic regions could also impair this mechanism.
Forty-six missense SNVs reported within exons 6 and 7 were first selected from the Human RhesusBase. Their respective effect on splicing was assessed by using an in vitro assay. An RhD-negative cell model was further generated by using the CRISPR-Cas9 approach. RhD-mutated proteins were overexpressed in the newly created model, and cell membrane expression of the D antigen was measured by flow cytometry.
Minigene splicing assay showed that 14 of 46 (30.4%) missense SNVs alter splicing. Very interestingly, further investigation of two missense SNVs, which both affect codon 338 and confer a weak D phenotype, showed various mechanisms: c.1012C>G (p.Leu338Val) disrupts splicing only, while c.1013T>C (p.Leu338Pro) alters only the protein structure, in agreement with in silico prediction tools and 3D protein structure visualization.
Our functional data set suggests that missense SNVs damage quantitatively D antigen expression by, at least, two different mechanisms (splicing alteration and protein destabilization) that may act independently. These data thereby contribute to extend the current knowledge of the molecular mechanisms governing weakened D expression.
虽然 D 变异表型已知是由于遗传缺陷引起的，包括罕见的错义单核苷酸变异（SNV），位于 RHD 基因内，但很少有研究探讨导致这种改变表达的分子和细胞机制。我们和其他人之前已经表明，剪接通常会被组成性剪接位点及其附近的 SNV 破坏。因此，我们试图研究位于“深”外显子区域的罕见错义 SNV 是否也会损害这种机制。
首先从人类 RhesusBase 中选择报告位于外显子 6 和 7 中的 46 个错义 SNV。通过体外测定评估它们各自对剪接的影响。进一步使用 CRISPR-Cas9 方法生成 RhD-阴性细胞模型。在新创建的模型中过表达 RhD 突变蛋白，并通过流式细胞术测量 D 抗原的细胞膜表达。
小基因剪接测定表明，46 个错义 SNV 中的 14 个（30.4%）改变剪接。非常有趣的是，对两个错义 SNV 的进一步研究，这两个 SNV 都影响密码子 338 并赋予弱 D 表型，显示出不同的机制：c.1012C>G（p.Leu338Val）仅破坏剪接，而 c.1013T>C（p.Leu338Pro）仅改变蛋白质结构，与计算机预测工具和 3D 蛋白质结构可视化一致。
我们的功能数据集表明，错义 SNV 通过至少两种不同的机制（剪接改变和蛋白质不稳定）定量损害 D 抗原表达，这两种机制可能独立作用。这些数据有助于扩展目前对调节减弱的 D 表达的分子机制的认识。
