Abstract：
OBJECTIVE: To study the molecular basis for a proband with A subtype B of the ABO blood group and explore the influence of amino acid variant on the activity of glycosyltransferase (GT).
METHODS: A proband who had presented at the First Affiliated Hospital of Zhengzhou University on July 2, 2020 was selected as the study subject. Serological identification of the ABO blood groups of the proband and her family members were performed by gel card and test tube methods. The ABO gene of the proband was identified by PCR-sequence specific primers (PCR-SSP) and DNA sequencing. A 3D molecular homologous model was constructed to predict the impact of the variant on the stability of α-(1→3)-D-N-acetylgalactosamine transferase (GTA).
RESULTS: The red blood cells of the proband, her mother and two younger brothers showed weak agglutination with anti-A and strong agglutination with anti-B. The sera showed 1~2+ agglutination with Ac and no agglutination with Bc. Based on the serological characteristics, the proband was identified as AwB subtype. Pedigree analysis suggested that the variant was inherited from her mother. The blood group of the proband was identified as A223B type by PCR-SSP. ABO gene sequencing analysis showed that the proband has harbored heterozygous variants of c.297A>G, c.467C>T, c.526C>G, c.657C>T, c.703G>A, c.796C>A, c.803G>C, c.930G>A and c.1055insA. Based on the results of clone sequencing, it was speculated that the genotype was ABO*A223/ABO*B.01. There were c.467C>T and c.1055insA variants compared with ABO*A1.01, and c.1055insA variant compared with ABO*A1.02. Homologous modeling showed that the C-terminal of A223 GT was significantly prolonged, and the local amino acids and hydrogen bond network have changed.
CONCLUSIONS: Above results revealed the molecular genetics mechanism of A223B subtype. The c.1055insA variant carried by the proband may affect the enzymatic activity of GTA and ultimately lead to weakening of A antigen.
METHODS: A proband who had presented at the First Affiliated Hospital of Zhengzhou University on July 2, 2020 was selected as the study subject. Serological identification of the ABO blood groups of the proband and her family members were performed by gel card and test tube methods. The ABO gene of the proband was identified by PCR-sequence specific primers (PCR-SSP) and DNA sequencing. A 3D molecular homologous model was constructed to predict the impact of the variant on the stability of α-(1→3)-D-N-acetylgalactosamine transferase (GTA).
RESULTS: The red blood cells of the proband, her mother and two younger brothers showed weak agglutination with anti-A and strong agglutination with anti-B. The sera showed 1~2+ agglutination with Ac and no agglutination with Bc. Based on the serological characteristics, the proband was identified as AwB subtype. Pedigree analysis suggested that the variant was inherited from her mother. The blood group of the proband was identified as A223B type by PCR-SSP. ABO gene sequencing analysis showed that the proband has harbored heterozygous variants of c.297A>G, c.467C>T, c.526C>G, c.657C>T, c.703G>A, c.796C>A, c.803G>C, c.930G>A and c.1055insA. Based on the results of clone sequencing, it was speculated that the genotype was ABO*A223/ABO*B.01. There were c.467C>T and c.1055insA variants compared with ABO*A1.01, and c.1055insA variant compared with ABO*A1.02. Homologous modeling showed that the C-terminal of A223 GT was significantly prolonged, and the local amino acids and hydrogen bond network have changed.
CONCLUSIONS: Above results revealed the molecular genetics mechanism of A223B subtype. The c.1055insA variant carried by the proband may affect the enzymatic activity of GTA and ultimately lead to weakening of A antigen.
摘要：
目的：研究ABO血型A亚型B先证者的分子基础，探讨氨基酸变异对糖基转移酶（GT）活性的影响。
方法：选择2020年7月2日在郑州大学第一附属医院就诊的先证者作为研究对象。先证者及其家庭成员ABO血型的血清学鉴定通过凝胶卡和试管法进行。通过PCR序列特异性引物（PCR-SSP）和DNA测序鉴定了先证者的ABO基因。构建了3D分子同源模型来预测变体对α-（1→3）-D-N-乙酰半乳糖胺转移酶（GTA）稳定性的影响。
结果：先证者的红细胞，她的母亲和两个弟弟显示出抗A的弱凝集和抗B的强凝集。血清与Ac呈1~2+凝集,与Bc无凝集。根据血清学特征，先证者被鉴定为AwB亚型。谱系分析表明，该变体是从她的母亲那里遗传的。通过PCR-SSP鉴定先证者的血型为A223B型。ABO基因测序分析表明，先证者具有c.297A>G的杂合变体，c.467C>T，c.526C>G，c.657C>T，c.703G>A，c.796C>A，c.806G>C，c.930G>A和c.1055insA.根据克隆测序的结果,推测基因型为ABO*A223/ABO*B.01。与ABO*A1.01相比，有c.467C>T和c.1055insA变体，与ABO*A1.02相比，有c.1055insA变体。同源建模表明，A223GT的C端明显延长，局部氨基酸和氢键网络发生了变化。
结论：以上结果揭示了A223B亚型的分子遗传学机制。先证者携带的c.1055insA变体可能会影响GTA的酶活性，并最终导致A抗原的减弱。
方法：选择2020年7月2日在郑州大学第一附属医院就诊的先证者作为研究对象。先证者及其家庭成员ABO血型的血清学鉴定通过凝胶卡和试管法进行。通过PCR序列特异性引物（PCR-SSP）和DNA测序鉴定了先证者的ABO基因。构建了3D分子同源模型来预测变体对α-（1→3）-D-N-乙酰半乳糖胺转移酶（GTA）稳定性的影响。
结果：先证者的红细胞，她的母亲和两个弟弟显示出抗A的弱凝集和抗B的强凝集。血清与Ac呈1~2+凝集,与Bc无凝集。根据血清学特征，先证者被鉴定为AwB亚型。谱系分析表明，该变体是从她的母亲那里遗传的。通过PCR-SSP鉴定先证者的血型为A223B型。ABO基因测序分析表明，先证者具有c.297A>G的杂合变体，c.467C>T，c.526C>G，c.657C>T，c.703G>A，c.796C>A，c.806G>C，c.930G>A和c.1055insA.根据克隆测序的结果,推测基因型为ABO*A223/ABO*B.01。与ABO*A1.01相比，有c.467C>T和c.1055insA变体，与ABO*A1.02相比，有c.1055insA变体。同源建模表明，A223GT的C端明显延长，局部氨基酸和氢键网络发生了变化。
结论：以上结果揭示了A223B亚型的分子遗传学机制。先证者携带的c.1055insA变体可能会影响GTA的酶活性，并最终导致A抗原的减弱。
