“亚洲型”DEL表达完整D抗原的分子机制研究

D-elute (DEL) is a very weak form of D antigen expressed on the surface of red blood cell. Clinically DEL individuals have been commonly typed and treated as D negative for a long time. DEL individuals should prevent hemolytic transfusion reactions and hemolytic disease of the fetus and newborn caused by anti-D alloimmunization. Approximately 25% of the individuals shown to be RhD-negative by conventional serological techniques in China were identified to be DEL. The RHD*1227A, which was termed “Asia type” DEL, accounted for up to 90% of the DEL phenotype in China. Previously several studies reported that a series of aberrant RHD transcripts rather than normal full-length RHD transcript were identified in the “Asia type” DEL. All of these aberrant RHD transcripts skipped exon 9 and could not express a complete repertoire of D epitopes. However, our own study with more than 600 “Asia type” DEL pregnant women cases and other studies with 493 “Asia type” DEL pregnant women cases showed that none of these “Asia type” DEL pregnant women formed alloanti-D. All of these “Asia type” DEL pregnant women had delivered RhD-positive infant. These indicate the existence of compatibility and same repertoire of D antigen between the pregnant women and the RhD positive infant, which is contradictory with the aberrant mRNA transcript result. In our previous study, very low abundance transcripts with exon 9 were identified from the cultured erythroblast from the “Asia type” DEL individuals. Based on this data, we speculated that very low number of complete repertoire of D antigens of “Asia type” DEL were expressed by low abundant of normal full-length RHD mRNA transcripts, D antigens on the surface of red blood cell were too few to be detected by the routine methods used in previous studies. In this study, third generation sequencing technology, which is more sensitive, will be used to confirm whether normal full-length RHD transcript exists in “Asia type” DEL. In addition, expression studies in vitro will be conducted to clarify the molecular basis for expression of complete repertoire of D epitopes in “Asia type” DEL. This study will provide the basic theoretical evidence for the establishment of specific strategy about transfusion and pregnancy for “Asia type” DEL patients in the future.

D放散型（DEL）的D抗原极弱，长期被检测为D阴性，临床需预防抗-D免疫所致输血反应和溶血病。DEL在我国初筛D阴性人群中占25%，其中>90%为携带RHD*1227A等位基因的亚洲型DEL。以往报道该等位基因可导致异常剪接而无法产生正常转录本，仅形成多种外显子9缺失的异常转录本，这些异常转录本无法表达完整D抗原。但在我们超600例和其他地区493例有D阳性胎儿孕产史的亚洲型DEL孕妇中无一例产生抗-D，表明母婴D血型相合，即其表达完整D抗原，这与其未发现正常转录本相矛盾。我们从亚洲型DEL个体分离培养的前体红细胞中检出微量含外显子9的转录本。据此推测亚洲型DEL中存在传统方法无法检测的低丰度正常转录本。本研究拟通过灵敏的三代测序获得其存在低丰度正常转录本的证据，并通过体外表达阐明其表达完整D抗原的分子基础，为未来指导亚洲型DEL患者输注D阳性血和孕妇免除抗-D免疫球蛋白注射提供理论依据。

D放散型（DEL）个体的红细胞上D抗原极弱，DEL个体长期被检测为D阴性并按D阴性对待。目前DEL病人仍需要输注长期供应短缺的D阴性血，DEL孕妇需要注射抗-D免疫球蛋白来预防新生儿溶血病。DEL在我国初筛D阴性人群中约占25%，其中>90%为携带RHD*1227A等位基因的亚洲型DEL。亚洲型DEL个体表达完整D抗原，在胎母免疫中从未发生抗-D同种免疫。虽然有学者建议亚洲型DEL孕妇应作为普通D阳性对待，但目前亚洲型DEL表达完整D抗原的分子机制尚未阐明，目前临床上仍按D阴性对待。对于亚洲型DEL表达完整D抗原的分子机制，有学者使用一代Sanger测序发现亚洲型DEL中不存在RHD mRNA全长转录本，仅存在多种异常剪接的RHD mRNA转录本（均不含外显子9），并推测部分异常转录本可能表达出完整表位的D抗原。我们前期使用定量PCR从亚洲型DEL个体分离培养的前体红细胞中检出微量含外显子9的转录本。据此推测亚洲型DEL中存在传统方法无法检测的低丰度全长转录本。本项目首先筛选出7例亚洲型DEL，从其外周血中分离培养前体红细胞后提取总RNA并反转录成cDNA，随后使用特异性引物对整个RHD基因编码区进行PCR扩增，并首次通过灵敏且高通量的三代Nanopore测序技术发现在亚洲型DEL个体中存在着低丰度的（占转录本总数的0.18%）携带1227G>A突变的正常RHD mRNA全长转录本。随后通过体外表达试验证实亚洲型DEL中存在的6种主要异常RHD mRNA转录本均不能表达出D抗原，而全长转录本可表达出完整表位的D抗原。本项目结果表明亚洲型DEL中存在的低丰度的RHD mRNA全长转录本翻译出少量完整表位的D抗原，从而阐明了亚洲型DEL表达完整D抗原的分子机制。亚洲型DEL个体表达完整表位的D抗原，可通过免疫耐受从而避免发生抗-D同种免疫。在完成本项目计划基础上，本项目还通过多中心临床试验发现亚洲型DEL患者（n=46例）输注D阳性血不会产生抗-D同种免疫；亚洲型DEL孕妇（n=1032例）生产D阳性胎儿也不会产生抗-D同种免疫。本研究有临床应用前景：本研究为建立亚洲型DEL患者常规输注普通D阳性血液的临床输血规范提供了理论和临床依据，如果在全国推广可缓解目前D阴性血液供应紧缺的问题。此外，亚洲型DEL孕妇也可以不必注射抗D免疫球蛋白和进行频繁的孕期抗-D同种免疫监测。