Cai Xiaohong, Qian Chengrui, Wu Wenman, Lei Hang, Ding Qiulan, Zou Wei, Xiang Dong, Wang Xuefeng
Blood Transfusion Department, Ruijin Hospital, Medical School of Shanghai Jiao Tong University, Shanghai, China.
Blood Group Reference Laboratory, Shanghai Institute of Blood Transfusion, Shanghai Blood Center, Shanghai, China.
Transfusion. 2017 Sep;57(9):2140-2149. doi: 10.1111/trf.14209. Epub 2017 Jun 26.
The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change.
Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing.
While plasma total GTB transfer capacity was undetectable in this B -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B cDNA and B cDNA. The mRNA expression level of B in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells.
ABO c.28G>A mutation may cause B -like subgroup by affecting RNA splicing of the ABO gene.
ABO基因突变引起的氨基酸替换通常被预测会影响糖基转移酶的功能或其生物合成。在此，我们报告一种ABO外显子错义突变，该突变通过降低ABO基因的mRNA水平而非氨基酸变化来影响B抗原表达。
进行了包括血浆总GTB转移能力在内的血清学研究。通过桑格测序分析ABO基因的外显子序列。将具有c.28G>A（p.G10R）突变的B cDNA在HeLa细胞中表达，并测量细胞上清液中的总GTB转移能力。转染后对这些HeLa细胞进行流式细胞术检测，同时也检测HeLa-B细胞的凝集情况。通过直接测序和实时逆转录聚合酶链反应分析ABO基因的mRNA。制备了一个小基因构建体以评估剪接潜力。
在这个类B个体中，血浆总GTB转移能力检测不到，而表达抗原细胞的相对百分比和B红细胞（RBC）的平均荧光指数分别为正常B RBC的19%和14%。用B cDNA转染的HeLa细胞与B cDNA转染的HeLa细胞相比，细胞上清液中的总GTB转移能力和细胞表面的B抗原表达没有显著差异。外周全血中B的mRNA表达水平显著降低。在K562细胞中转染后，与野生型构建体相比，c.28G>A构建体中的剪接量显著降低。
ABO c.28G>A突变可能通过影响ABO基因的RNA剪接导致类B亚群。
