Analysis Of ABO Subgroup And The Regulation Mechanism Of A Novel B_el Subgroup In Hunan

PurposeThere are many subgroups in human erythrocyte ABO blood types besides normal ABO blood group.They are called ABO subgroups because of weaken antigen intensity.During the process of blood transfusion,it is dangerous when identified the wrong blood type,which will lead to a medical accident.The appearance of ABO subgroups is infrequent,and it is easy to be misjudged because of diseases.Therefore,understanding the occurrence frequency and mechanism of common ABO subgroups in different regions is important to ABO blood group identification.ABO blood groups are dependent on the expression of ABO gene,which are affected by many factors,such as enhancers,transcription factors,etc.In addition,mutations in ABO gene exons also take an important role in ABO gene expression.The purpose of this study was to investigate the types and frequency of ABO subgroups in Hunan province,in addition,we explored the regulation mechanism of a novel Bel subgroup through gene sequencing and dual luciferase reporter assay.MethodsIn this study,blood samples of 175,000 patients selected from Hunan from 2016-2020 in Xiangya Third Hospital were tested by blood group serological methods,including positive and negative stereotyping test,ABH blood group substance determination in saliva,affinity test and absorption and dispersion test,including 3815 healthy individuals.The 34 ABO subgroups were identified by blood group serological methods,including positive and negative stereotype assay,anti-H assay,determination of ABH substances in saliva,and absorption and dispersion assay.The activity of glycosyltransferases in the serum of these 34 samples were measured by the glycosyltransferase assay kit.And the gene type of these 34 samples were performed by PCR-SSP genotyping test kit.To Further confirm the gene types of this 34 samples,exons 1-7 of ABO gene in the 34 samples were sequencing and compared to NCBI gene bank.In addition,we identified the Bel subtype in one blood donor（out-patient）in 34 ABO subtypes and conducted a genetic family investigation.It’s blood type was analyzed by serological method,and the expression of B gene was confirmed by RT-PCR and western blotting.The glycosyltransferase activity was detected by glycosyltransferase test kit.Furthermore,we analyzed the ABO gene in this sample via genotyping and exons 1-7 sequencing.Finally,The effect of mutation in ABO promoter was analysis by dual luciferase reporter assay.We clarify the regulation mechanism of ABO promoter mutation in a novel Bel subgroup.ResultsPart I:Analysis of ABO subgroup in Human province.71 abnormal ABO antigen samples and 34 ABO subgroups were screened in nearly 175,000 blood samples from 2016-2020 at the Third Xiangya Hospital.Among the 34 ABO subgroups,there are 10 B（A）subgroups,6 AB3 subgroups,5 A3 subgroups,4 Bel subgroups,3 B3 subgroup,2 Ael and 2 Bend subgroups,and 1Bw and 1 A2B subgroup.And then,we analysed the ABO gene of 34 samples via genotyping and exons 1-7 sequencing,and found some new mutations in ABO gene.Finally,through statistical analysis,we found that ABO subgroup B（A）had the highest occurrence frequency in Hunan,accounting for 5.7/100,000（10 cases）,Bw subgroup and A2B subgroup had the lowest occurrence frequency,accounting for 0.57/100,000（1 case each）,and AB3 subgroups were accounting for 3.43/100,000（6 cases）.A3 subgroups were 2.86/100,000（5 cases）;Bel subgroups were about 2.29/100,000（4 cases）,B3 subgroups were about 1.71/100,000（3 cases）,Ael subgroup and Bend subgroup were about 1.15/100,000（2 cases each）.Part II:the mechanism of ABO gene regulation in a Bel subgroupIn this study,we found that the blood type of the sample（Out-patient）from loudi central blood station is Bel via blood group serology assay,glycosyltransferase activity assay,RT-PCR and western blotting.In addition,we found that there is insertion mutation in the ABO gene of this sample via genotyping and ABO exon 1-7 sequecing.The insertion mutation TGTTG located at the position of 15-16 in ABO exon 1（c.15₁6insTGTTG）.It is inherited from proband’s mother.At last,we confirmed that this mutation can attenuate ABO promoter activity induced by GAT A-1 and RUNX1 via dual luciferase reporter assay,which maybe the reason on downregulation of B antibody activity.Conclusion1.The occurrence frequency of ABO subgroups in Hunan was relatively low,with an overall of 19.43/100,000,among which B（A）subgroup have the highest occurrence frequency,and the Bw and A2B were the lowest.2.The c.15₁6insTGTTG mutation in ABO gene exon 1 attenuate the promoter activity induced by GATA-1 and RUNX1,resulting in reduced expression of B antigen,which maybe an important molecular mechanism of Bel subgroup formation.