Supplementary Material for: A Novel Mutation Eliminates GATA-1 and RUNX1-Mediated Promoter Activity in Galactosyltransferase Gene

<b><i>Introduction:</i></b> Mutations in the promoter region and exons of ABO gene may cause changes in the expression of blood group antigens, often showing a weak ABO phenotype. Here, we identified a novel weak ABO subgroup allele that caused B_el phenotype and explored its mechanisms. <b><i>Methods:</i></b> The ABO phenotype of subjects (Chinese Han nationality) was classified by serological method. The plasma activity of erythrocyte glycosyltransferase was detected by the phosphate coupling method. ABO subtype genotyping was performed by PCR-SSP and exon sequencing. The activity of the promoter was evaluated by a dual-luciferase reporter assay. <b><i>Results:</i></b> We identified a mutation exon 1 c.15_16insTGTTG of the B allele in a B_el subject. Genealogical investigation showed that the mutation was inherited from her mother. The mutation was located in the promoter region of the ABO gene. The dual-luciferase reporter assay showed that the mutation inactivated GATA-1 and RUNX1-mediated activity of the ABO gene promoter, leading to a decrease in the expression and activity of B glycosyltransferase. <b><i>Conclusion:</i></b> A novel B_var ABO subgroup allele was identified. The novel mutation can reduce the promoter activity that activated by GATA-1 and RUNX1, subsequently causing the B_el phenotype.

<b><i>引言：</i></b> ABO基因启动子区及外显子发生突变可导致血型抗原表达异常，常表现为弱ABO表型。本研究鉴定出1个可引发B_el表型（B_el phenotype）的新型弱ABO亚型等位基因，并对其致病机制进行了探究。<b><i>方法：</i></b> 采用血清学方法对中国汉族研究对象的ABO血型表型进行分型；通过磷酸偶联法检测红细胞糖基转移酶血浆活性；利用聚合酶链反应-序列特异性引物（PCR-SSP）技术及外显子测序完成ABO亚型基因分型；采用双荧光素酶报告基因检测（dual-luciferase reporter assay）评估启动子活性。<b><i>结果：</i></b> 本研究在1例B_el表型个体中发现B等位基因外显子1存在c.15_16insTGTTG突变。家系调查显示，该突变由其母亲遗传而来。经确认，该突变定位于ABO基因的启动子区域。双荧光素酶报告基因检测结果表明，该突变可使GATA结合蛋白1（GATA-1）及RUNX转录因子1（RUNX1）介导的ABO基因启动子活性失活，进而导致B糖基转移酶的表达与活性降低。<b><i>结论：</i></b> 本研究鉴定出1个新型B_var型ABO亚型等位基因。该新型突变可削弱GATA-1与RUNX1介导的ABO基因启动子活性，最终引发B_el表型。