目的：以一个血型Ax亚型家系为研究对象,探讨我国人群血型中产生Ax亚型的潜在分子机制。方法：采用血清学方法鉴定该家系成员的ABO血型,测定血浆α-1,3-N-乙酰半乳糖胺基转移酶活性,行DNA直接测序和克隆后测序分析ABO基因序列,并构建3D分子模型,就发现的突变对α-1,3-N-乙酰半乳糖胺基转移酶稳定性改变 (ΔΔG)的影响进行预测。结果：血清学鉴定和DNA测序分析显示,先证者的血型为AxB亚型,其2个女儿分别为A型和AB型,其ABO基因型分别为AW.38/B.01、A1.02B.01、A1.02/AW.38。先证者和其A型女儿的第7外显子均存在c.426G>C杂合突变,导致α-1,3-N-乙酰半乳糖胺基转移酶的氨基酸发生p.M142I改变。分子模建分析提示,该基因突变改变了蛋白局部氨基酸间氢键的数目,从而导致氨基酸间作用力发生改变,而动力学改变,ΔΔG值升高表明其蛋白稳定性降低。结论：α-1,3-N-乙酰半乳糖胺基转移酶p.M142I突变可能通过降低了酶的稳定性导致Ax表现型,而其深入机制有待体外实验进一步研究。
Objective: To explore the potential molecular mechanism of Ax subtypes in Chinese population based on an Ax subtype family. Methods: The ABO blood group serological tests and detection of the total plasma activity of alpha α-1,3-N-acetylgalactosaminyltransferase were conducted. ABO gene was sequenced directly and sequenced after cloning to construct 3D molecular model, and then the effect of identified mutation on protein stability changes (ΔΔG) of alpha α-1,3-N-acetylgalactosaminyltransferase was predicted. Results: The proband was diagnosed as AxB subgroup byserological analysis, and AW.38/B.01 was identified by DNA analysis. His two daughters were identified as A and AB blood group, and their ABO genotypes were A1.02/B.01, A1.02/AW.38, respectively.There was a c.426G>C heterozygous mutation in ABO exon 7 of the proband and his A type daughter, leading to p.M142I change in the amino acid of α-1,3-N-acetylgalactosaminyltransferase. Molecular modeling and analysis suggested that the mutation may lead to changes in the number of hydrogen bonds formed. The increase of ΔΔG value of thermodynamic stability changes indicated that the protein stability was reduced. Conclusions: The mutation of p.M142I in α-1,3-N-acetylgalactosaminyltransferase may lead to Ax phenotype by reducing the stability of the enzyme. In vitro study is needed for further investigation.
[1] Yamamoto F, McNeill PD, Yamamoto M, et al. Molecular genetic analysis of the ABO blood group system: 3. A(X) and B(A) alleles[J]. Vox Sang,1993,64(3):171-174.
[2] Olsson ML, Chester MA. Heterogeneity of the blood group Ax allele: genetic recombination of common alleles can result in the Ax phenotype[J]. Transfus Med,1998 Sep,8(3):231-238.
[3] Ogasawara K, Yabe R, Uchikawa M, et al.Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes[J]. Immunogenetics,2001,53(3):190-199.
[4] Olsson ML, Irshaid NM, Hosseini-Maaf B, et al.Genomic analysis of clinical samples with serologic ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles[J]. Blood,2001,98(5):1585-1593.
[5] Seltsam A, Hallensleben M, Kollmann A, et al.The nature of diversity and diversification at the ABO locus[J]. Blood,2003,102(8):3035-3042.
[6] Deng ZH, Yu Q, Wu GG, et al.Molecular genetic analysis for Ax phenotype of the ABO blood group system in Chinese[J]. Vox Sang,2005 ,89(4):251-256.
[7] Cai X, Jin S, Liu X, et al.Molecular genetic analysis of ABO blood group variations reveals 29 novel ABO subgroup alleles[J]. Transfusion,2013,53(11 Suppl 2):2910-2916.
[8] Campbell AJ, Lamb ML, Joseph-McCarthy D.Ensemble-based docking using biased molecular dynamics[J]. J Chem Inf Model,2014,54(7):2127-2138.
[9] Cai XH, Jin S, Liu X, et al.Molecular genetic analysis for the B subgroup revealing two novel alleles in the ABO gene[J]. Transfusion,2008,48(11):2442-2447.
[10] Pettersen EF, Goddard TD, Huang CC, et al.UCSF Chimera--a visualization system for exploratory research and analysis[J]. J Comput Chem,2004,25(13):1605-1612.
[11] Reumers J, Schymkowitz J, Ferkinghoff-Borg J, et al.SNPeffect: a database mapping molecular phenotypic effects of human non-synonymous coding SNPs[J]. Nucleic Acids Res,2005,33:D527-D532.
[12] de Baets G, van Durme J, Reumers J, et al. SNPeffect 4.0: on-line prediction of molecular and structural effects of protein-coding variants[J]. Nucleic Acids Res,2012,40(Database issue):D935-D939.
[13] Guerois R, Nielsen JE, Serrano L.Predicting changes in the stability of proteins and protein complexes: a study of more than 1 000 mutations[J]. J Mol Biol, 2002,320(2):369-387.
[14] Yu Q, Deng ZH, Wu GG, et al.Molecular genetic analysis for a novel Ael allele of the ABO blood group system[J]. J Hum Genet, 2005, 50(12):671-673.
[15] Zhu F, Xu X, Hong X, et al.A 425T>C mutation in the B allele for the ABO transferase is associated with the B3 phenotype in Han Chinese persons[J]. Transfusion,2008, 48(11):2476-2477.
[16] DANIELS G.Human blood groups[M]. Oxford: Blackwell Scientific, Second edition, 2002.
[17] 蔡晓红, 金沙, 刘曦, 等. 中国人群ABO 亚型中一个新的Ax等位基因的鉴定[J]. 临床血液学杂志, 2009,22(6):309-312.
[18] Lee S Y, Ihm C, Shin D J, et al.The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase[J]. Transfusion, 2013,54(5):1298-1304.
[19] Patenaude SI, Seto NO, Borisova SN, et al.The structural basis for specificity in human ABO(H) blood group biosynthesis[J]. Nat Struct Biol,2002,9(9):685-690.
[20] Cho D, Shin DJ, Yazer MH, et al.The M142T mutation causes B3 phenotype: three cases and an in vitro expression study[J]. Korean J Lab Med,2010,30(1):65-69.
