RHD外显子9的5' ss与剪接体U1 snRNP结合力降低在“亚洲型”DEL血型mRNA异常剪接中的作用及机理研究

“亚洲型”DEL为常见变异血型，一直被误检测为D阴性稀有血型，但研究发现其不会发生抗-D免疫，完全可作为D+患者对待，但由于尚缺乏深入研究，此策略暂未得到应用。以往发现其基因变异可导致转录异常，表达大量缺乏外显子9转录本和可能少量全长转录本，具体机制不明。我们前期在“亚洲型”DEL红细胞中首次检测到低丰度全长转录本，随后对突变所在5’ ss信息学分析发现1227A合并IVS9+6a变异可导致5’ ss剪接效能显著下降。据此我们推测在剪接起始步骤，此突变型5' ss与剪接体U1 snRNP结合能力下降但仍有少量结合可能是异常剪接的机制。本项目拟通过体外表达、增强5' ss 与U1 snRNA结合纠正外显子9缺失，分析突变5’ ss区域与U1 snRNP结合变化等，阐明其在“亚洲型”DEL异常剪接和表型形成中的作用，这将为建立“亚洲型”DEL血型患者D+血液输注和正确孕期监测策略提供重要依据。

“Asia type” DEL is one of common variant blood group of red blood cell (RBCs), which is always typed as D negative rare blood group as lacking of the suitable routine testing. In recent years, many studies showed that no alloanti-D occurrence was identified in “Asia type” DEL individuals immunized with D+ red blood cells (RBCs), especially during the pregnancy. So, based on it, it is indicated that“Asia type” DEL patients could be treated as common D+ patients to receive the common D+ blood rather the rare D negative blood. But lacking of comprehensive mechanism studies about the formation of “Asia type” DEL phenotype, the strategy is still be treated with caution and not applied in clinic. In previous studies, aberrant splicing of RHD was observed in “Asia type” DEL RBCs, in which aberrant isoforms lacking of exon 9 were detected predominately and low copy of complete isoform was deduced. In our previous study, low copy of complete RHD isoform was firstly identified in cultured RBCs from “Asia type” DEL individuals. Then, the bioinformatics analysis of the mutant 5’ splice site (5’ ss) of RHD*1227A allele showed that the value of Maximum Entropy of 5’ ss was decreased significantly when two variants exists on the conserved sites (1227A, IVS9+6a). So, it is predicted that the decreasing binding but not completely absent binding between the mutant 5’ ss with the complementary U1 snRNP maybe account for it. Minigene splicing analysis in vitro, adapted U1 snRNA co-expression with minigene carried RHD exon 9, the binding changes analysis between the mutant 5’ ss with U1 snRNP will be conducted to clarify this issue. The aim of this project is to clarify the mechanism of the aberrant mRNA splicing and the “Asia type” DEL phenotype. The study will help us to establish the correct strategy to transfusion of D+ to “Asia type” DEL patients and avoid the anti-D prophylaxis in “Asia type” DEL pregnant women in clinic.