Molecular Mechanisms Of ABO Subgroup Caused By ABO Gene Mutations

Inherited ABO blood group variants resulted from ABO subgroup and para-bombay blood types are important reasons to cause ABO blood grouping discrepancy.in order to investigate the distribution of ABO subgroup,and molecular basis of these two blood groups in Chinese population,we performed a study in Chinese population on relationship between phenotpyes and genetypes of ABO subgroup and para-bombay blood types.We also studied the effect of ABO mutation c.28G>A（p.G10R）and c.538C>T（p.R180C）on ABO-GT’s expression,structure and function to clarify the molecular mechanism of these mutations that lead to weak ABO antigen expression,and further provide reliable and accurate detection markers for molecular diagnosis of ABO subgroup in the future.351 individuals of ABO subgroup were detected in approximate 1.45 million samples with a detection rate of 0.0242%.B（A）was most common,and then B3 and Bx related subgroups.In 19 novel ABO alleles identified,2 alleles with promoter mutation-72G>A was found associated with“3”type subgroup;one allele with mutation in intron 2 may relate to aberrant splicing;the rest 16 alleles may cause amino acide changes directely（including 13 single point mutations,2 interallelic recombinations,1 single base insertion）.FUT1 frameshift mutation c.764-768delC formed a new genotype for para-Bombay in Chinese.When a study was carried out in a B individual with c.28G>A mutation,we found that while plasma GTB transfer capacity was undetectable in this B_end individual with c.28G>A mutation,the relative AEC%and MFI of the B_end RBCs were 19%and 14%of normal B RBCs,respectively.There was no significant difference of GTB transfer capacity in cell supernatant and B antigen expression on cell surfaces between HeLa cells transfected with B^end cDNA and B cDNA.The mRNA expression level of B^end 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.Therefore,ABO c.28G>A mutation may cause B_end subgroup by affecting RNA splicing of ABO gene.When a study was carried out in a AB_x pedigree with c.538C>T mutation,we found that while plasma GTB transfer capacity was undetectable in the AB_X father and son,the relative AEC%and MFI of the AB_x RBCs were 52%,59%and 3.4%,3.9%of normal B RBCs,respectively.There was significant reduction of GTB transfer capacity in cell supernatant（1:64 vs.1:1024）and B antigen expression on cell surfaces（AEC%and MFIwere 60%and 44%of those of wild type contrast）between HeLa cells transfected with B^x cDNA and B cDNA.Weak agglutination of ABx-RBCs can be mimicked by adding anti-B antibody to the HeLa-Bx cells.Mutant and wild type GTB have similar antigen concentration in HeLa cells.Molecular modeling analysis implies that amino acid substitution p.R180C may affect the interactions between R180 and the disordered amino acid residues in C-terminal,and then lead to a reduction of enzyme activity and cause B_x subgroup.