Journal club Dai Jianmin 2014-5-9

Background 从血清学到分子生物学 1900年→ → →至今 1990年 1985年

红细胞血型 -2010年，30个系统，308个抗原

人类红细胞膜模式图 参考文献：Williams hematology

ABO基因结构及染色体定位 Exon1－5序列: 编码糖基转移酶的穿膜区及柄部 Exon6－7序列: 编码酶的功能区（球形结构域） ABO位点 9q34.1－9q34.2 7个外显子，跨越基因组DNA 18kb 外显子1～7 ，28bp ～691bp不等 主要编码顺序：exon 6 和 exon 7 Exon1－5序列: 编码糖基转移酶的穿膜区及柄部 Exon6－7序列: 编码酶的功能区（球形结构域） A or B subgroups (with only few exceptions) result from a variety of nucleotide changes in exon 7 that cause alterations in the catalytic domain of the glycosyltransferase.

A和B抗原是红细胞膜表面糖蛋白、糖脂分子的糖类决定簇。 ABO基因 糖基转移酶 寡糖链末端糖基特异性 A和B抗原是红细胞膜表面糖蛋白、糖脂分子的糖类决定簇。 A抗原末端糖基:N-乙酰氨基半乳糖 B抗原末端糖基:D-半乳糖。 O基因：无有效的基因产物

A、B、O基因的核苷酸变异 ABO基因的参比序列：A*101等位基因 在A1基因的基础上核苷酸突变糖基转移酶改变影响催化活性抗原改变

单碱基缺失导致截短蛋白产物

单碱基缺失导致终止密码延后出现

ABO亚型 A抗原中主要为A1和A2 其它A亚型：A3、Ax、Aend、Am、Ay、和Ael B亚型一般比A亚型更少：B3、Bx、Bm和Bel等 ABO subgroups :the detection rate in Shanghai is approximately 0.015%.

Samples n = 2.1 million Chinese blood donors,2003 to 2011 322 ABO subgroup individuals 29 family members 120 healthy random Chinese donors included Aint, AintB, A3, A3B, Ax, AxB, AmB, Ael,AelB, AB3,B3, A3B3, Bx, AxBx, ABx, ABm, Bm, Bel, B(A), A2B, and A2Bx.

Methods I Serology: monoclonal anti-A, anti-B, anti-AB, anti-H; polyclonal anti-A, anti-B, anti-AB; an ABO red blood cell (RBC) kit Dolichos bijorus for anti-A1

各种A亚型血清学表现 Phenotype Red cells tested With anti-sera Serum tested with Known red cells Salivaof secretors Anti-A -B -AB -A1 -H A1 A2 B O V _ A and H Aint +++ ++ Some-times. + A3 ++mf Occa-sionally. + Am -/w+ Ax -/W+ +/++ Fre-quently + H

各种弱A亚型的鉴别程序 弱凝集 A3/ Ax/ Aend 混合外观(Mf）A60 A3 10％ 红细胞呈十分弱的mf凝集 只与抗-AB呈弱凝集 Ax 不凝集 吸附放散抗-A Am/ Ay / Ael 容易吸附放散抗-A Am 分泌型唾液中有一定量的A物质 不容易吸附放散抗-A Ay 分泌型唾液含少量的A物质 分泌型唾液只含H物质无A物质

Methods II Polymerase chain reaction ABO gene sequences : Exons 6 to 7 Exons 1 to 5 and their splice sites the promoter region DNA sequencing of subgroup PCR products gel-purified PCR products containing the mutation sites the pMD18-T vector Screening for subgroup-specific mutations by sequencing Nomenclature of mutations and ABO alleles

Methods III Analysis of ABO promoter activity pGL2-Basic vector K562 cells(Turbofect) renilla luciferase vector phRL-TK(cotransfected as an internal control) dual luciferases assay system: firefly and renilla luciferase activities

Results I Identification of 62 rare ABO alleles 29 novel subgroup alleles were newly linked to different kinds of ABO variations Promoter variant phenotype and genotype analysis

more than half occurred in conserved or invariant sites

Results II Identification of 62 rare ABO alleles Promoter variant phenotype and genotype analysis: the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes deletion at Positions -35 to -18 in their promoter region Ael and Bel phenotypes:the first naturally occurring ABO alleles with premature terminal codons in the 5′-region

decreased by more than 50%

Results III Identification of 62 rare ABO alleles Promoter variant phenotype and genotype analysis: Ael and Bel phenotypes:the first naturally occurring ABO alleles with premature terminal codons in the 5′-region the ABO mutations 52C>T and 7G>T, R18X and E3X in GTs

The end 谢谢！