| ObjectiveThis study was designed to explore the phenotype of ABO blood group and the distribution of homozygotes and heterozygotes among blood donors in Lianyungang,Jiangsu,Chian,and the feasibility of sequence specific primer polymerase chain reaction(PCR-SSP)was also verified for subsequent molecular genetic analysis.The polymorphism of ABO subtypes in this region was studied and its genetic characteristics were further analyzed.The present study might be beneficial to findings could obtain evidence for establishment of blood group sample information bank,blood product reserve and emergency safeguard mechanism based on the genetic analysis of ABO subtype.MethodsSerological testing was performed according to the instructions of the microcolumn gel card reagent.After getting serum blood type,sequence specific primers were designed for PCR-SSP reaction of 1000 blood donors and 17 difficult blood group samples.The amplified products were analyzed by gel electrophoresis.The exons 6 and 7 of ABO gene were amplified by gel card identification,and the amplified products were sequenced by Sanger assay.The sequencing results were then compared with Snap Gene software.The results of exon 6 and exon 7 sequencing were normal or the unexplainable serological phenotype or the FUT1 gene were amplified in the samples suspected of Bombay-like blood group,and the amplified products were also sequenced by Sanger assay.For the samples with difficulty in judging the alleles of the heterozygous peaks,the haploids were isolated and the genotypes were determined by TA cloning and sequencing.ResultsPart I: the positive phenotypic distribution of 1000 unpaid blood donors in Lianyungang,Jiangsu was as follows: Type A in 330 cases(33%),Type B in 300 cases(30%),Type O in 330 cases(33%)and Type AB in 40 cases(4%).The results of PCR-SSP in 998 samples were consistent with those of serological test.The genotypes of A and B phenotypes were as follows: 60 Genotype A/A cases e in accounting for 18% of Phenotype A.270 Genetype A/O cases,accounting for 82% of Phenotype A;.Among Phenotype B,Genotype B/B was accounted for 14%(42/300),and Genotype B/O accounting for 86%(259/300).Among them,2 cases of unknown serological samples were detected by PCR-SSP,and the genotypes were A /B and B / O,respectively.The final genotypes of exons 6 and 7 were identified as A201/B101 and B(A)04/O02 by amplification,Sanger sequencing and result comparison.Part II: Among 17 cases with forward and reverse ABO typing discrepancy,there were 5 A alleles(A102,A223,A215,Aw43 and Ael07),4 B alleles(B101,B103,B(A)04,Bx02),4 O alleles(O01,O02,O04,O12)and 1 Bombay blood type H3.it should be noted that A223 is a newly discovered subtype for A alleles and its c DNA has C > T and A > G base mutations at positions 467 and 608,respectively,which are non-synonymous mutations.C.608 A > G mutation is the first time to be identified assigned MK988574 as the unique recognition number in the gene bank.ConclusionThrough the serological analysis of ABO phenotype in Lianyungang area,it was shown that the distribution of ABO blood group in Lianyungang area did not fully accord with the distribution characteristics of ABO blood group in Chinese Han population.The regional characteristics of the same distribution ratio of type A and type O are also different from Zhejiang,Sichuan and other area.The genetic basic study of ABO subtype found that 5 of 17 subtype samples were B(A)04,accounting for 29.4% of the total,and the frequency of occurrence of this blood group was high.Some studies have shown that the majority of B(A)blood groups in Han population are B(A)04 and B(A)02,and the results are basically consistent with that.And a new mutation site was found for the first time in this study(c.608A>G,MK988574). |
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