Chimerism and mosaicism are important causes of ABO phenotype and genotype discrepancies

Discrepancies between blood group genotype and RBC phenotype are important to recognize when implementing DNA-based blood grouping techniques. This report describes two such cases involving the ABO blood group in the Korean population. Propositus #1 was a 22-year-old healthy man undergoing pretransfusion testing for minor surgery. Propositus #2 was a 23- year-old male blood donor. RBCs from both propositi were determined to be group AB and demonstrated unusual agglutination patterns on forward typing, which were inconsistent with their ABO genotype determined by allele-specific (AS) PCR. RBCs from propositus #1 demonstrated mixed field agglutination with both anti-A and -B, while RBCs from propositus #2 demonstrated mixed field only with anti-A reagents. Both had B/O genotypes by AS-PCR. Cloning and sequencing of ABO exons 6 and 7 revealed three alleles in both propositi: propositus #1: A102/B101/O04; propositus #2: A102/B101/O01. A panel of nine short-tandem repeat (STR) loci was tested on DNA extracted from blood, buccal mucosal cells, and hair from the propositi and on DNA isolated from their parents' blood. In all tissues tested from propositus #1, three loci demonstrated a double paternal and a single maternal DNA contribution, indicating that he was a chimera or a mosaic; in those from propositus # 2, one STR locus demonstrated a double paternal DNA contribution, indicating that he was a tetragametic chimera. Chimerism and mosaicism are uncommon but important causes of ABO genotype and phenotype discrepancies. The evaluation of patients and donors with unusual or unexpected serology in pretransfusion testing and consensus ABO alleles may include the evaluation of STR loci to detect these phenomena.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一种新的B3变异型相关的B糖基转移酶基因M142T突变研究

目的 研究中国人个体ABO血型系统中具有混合外观凝集特征的B3变异型的分子遗传背景.方法 血型血清学方法鉴定2例ABO血型疑难样本的红细胞表型,应用连续凝集方法和13个短串联重复序列(short tandem repeat,STR)位点检测法,排除外源性或内源性DNA嵌合的可能.对ABO基因第6、7外显子和部分内含子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定2个样本的ABO基因单倍型.结果 2个无关个体红细胞与抗-B和抗-AB发生混合外观凝集,连续凝集法和STR检测排除了样本的外源性DNA污染和内源性遗传嵌合子,根据血清学特征确定这2个个体红细胞均为A183血型.单倍型序列分析发现2个样本为A1B杂合子,其中B等位基因与B101相比,差异仅在第7外显子的425T>C错义突变,导致B糖基转移酶多肽链M142T替换.结论 在中国人群中发现一种新的可能导致B3变异型的ABO等位基因.     

一例ABO亚型CisAB个体的分子遗传学分析

目的研究1例ABO亚型的分子机制。方法先证者红细胞ABO表型鉴定采用常规血清学技术,ABO基因第6~7外显子序列采用PCR技术扩增并应用Sanger法双向进行测序分析。先证者ABO基因第6~7外显子单体型检测采用单链扩增测序技术。结果先证者红细胞与抗-A凝集强度4+、抗-A1不凝集,抗-B凝集强度3+,抗-H凝集强度4+;其血清与标准A细胞、O细胞和自身细胞不凝集,与标准B细胞在4℃呈现弱凝集,先证者血清学特性符合ABO亚型。ABO基因第6~7外显子双链测序分析显示先证者261 G/del、297AG、526CG、657CT、703GA、803GC、930GA杂合,796CC纯合。单体型测序显示先证者一个等位基因为ABO*O.01.01,另一个等位基因与ABO*B.01相比仅c.796A>C变异,导致266位蛋氨酸变成亮氨酸;比较国际输血协会ABO等位基因已命名的数据,发现该变异属于新等位基因。结论ABO*B.01等位基因c.796 A>C变异,导致266位蛋氨酸变成亮氨酸,可引起CisAB亚型。准确鉴定ABO亚型应结合血清学技术和分子生物学技术。     

Assay of alpha-D-galactosyltransferase of subgroup B sera

Carriers of weak B antigen were found in three generations of a family. The red cells of the propositus reacted with anti-A human serum and Dolichos biflorus lectin as strongly as normal A1B red cells, but they agglutinated at 8-fold dilution against anti-B human serum (1:128) and did not have a mixed-field agglutination. The red cells of her niece agglutinated at 32-fold dilution against the same anti-B serum and did not have a mixed-field pattern. Her red cells were provisionally designated B2, analogous to A2 of subgroup A. B antigen of the propositus appeared clearly depressed, and she was provisionally designated A1B2. When papain-treated O red cells were used as acceptors instead of untreated O red cells, group A1B2 sera could convert them into B-active cells, which were agglutinated by anti-B human serum. alpha-D-galactosyltransferase activity in A1B2 serum was about one-eighth that in normal B serum.     

A FAMILY CASE OF BLOOD GROUP B VARIANTS

Carriers of weak B variant were found in two generations of a family. The saliva of the father contained a small amount of B substance, but the saliva of his two daughters contained only H but not B. The erythrocytes of the propositus reacted only with potent anti-B and anti-(A + B) sera, and though the erythrocytes were not agglutinated by the weaker anti-B sera, the eluates showed that they combined with anti-B antibody. The eluates from normal human B and AB erythrocytes, and guinea-pig's erythrocytes which had adsorbed human anti-B agglutinin, agglutinated normal B erythrocytes but not the weak B. The eluates from the weak B erythrocytes and rabbit's erythrocytes both of which had adsorbed anti-B, agglutinated the weak B erythrocytes as well as the normal B erythrocytes. These weak B erythrocytes showed agglutinability as strong as that of O erythrocytes against anti-H eel serum. The sera of these weak B variants contained normal anti-A agglutinin and a weak anti-B cold agglutinin which was reactive at 5°C. As the saliva of the propositus contained as much H substance as was found in group O secretor saliva, but not B substance, her blood group was denoted by B_x. Her sister has same characters of B_x. in the erythrocytes and in the saliva. Her father's blood group was AB, and his erythrocytes showed weak B antigenicity, and his saliva contained rather weak B substance as well as the normal amounts of A and H substances.     

Paternity pseudo-exclusion caused by tetragametic chimerism in a gestational surrogacy case

Chimerism is a condition when an organism is composed of two or more populations of genetically distinct cells. Chimerism often produces curious results of medical and genetic investigations and could be a major cause of false negative conclusions in parentage testing. Here we describe a paternity pseudo-exclusion due to tetragametic chimerism in a gestational surrogacy case originated in a fertility clinic. Initial analysis using a buccal swab from the child and a peripheral blood sample from the father showed paternity exclusion at 6 STR loci. To find out the reason for observed paternal discrepancy father's semen sample used for IVF and samples from other tissues were genotyped. Buccal swabs, semen, hair follicles, nail clippings and cerumen showed identical mixed autosomal STR profiles originated from two genetically different cell lines and for all the 24 informative loci contain paternal obligate alleles. Results of Y-STR profiling of all paternal sample types showed a DNA profile originated from a single man. The mixed profiles obtained for different tissue types suggest that two genetically different cell lines contributed to formation of both the endoderm and the ectoderm of the father. The mesoderm seems to be monoclonal having originated from a genetically homogenous cell line as evidenced by the STR profile of peripheral blood. Such allelic pattern for various tissues suggests that the clonal origin happened at the very early stage of embryonic development. Approaches to minimise the rate of false exclusions in DNA parentage tests due to chimerism are discussed.     

一例ABO血型Aw33亚型新等位基因的分子生物学鉴定

目的研究1例ABO血型A亚型的分子生物学机制。方法患者ABO血型正反定型采用卡式法和试管法分别进行鉴定。利用PCR序列特异性引物检测该患者所含ABO基因。PCR扩增该患者ABO基因1~7外显子的全部编码序列并进行测序分析,通过克隆测序进行ABO基因单倍型分析。结果患者红细胞与抗A呈现弱凝集,与抗B不凝集,其血清与Ac凝集1+,与Bc呈现4+凝集,血清学特性可定义为Aw亚型。ABO基因测序分析显示患者存在c.106G>T、c.188G>A、c.189C>T、c.220C>T、c.297A>G、c.467C>T、c.543G>C、c.646T>A、c.681G>A、c.771C>T、c.829G>A杂合变异和c.261delG缺失。结合克隆测序结果,推测患者ABO基因型为ABO*Aw.33.new/O.01.02;与ABO*A1.01相比存在c.467C>T和c.543G>C变异,与ABO*A1.02相比存在c.543G>C变异,该新等位基因序列已提交GenBank,序列号为MK302122。结论发现1例Aw33亚型新的等位基因,其GTA转移酶基因存在c.467C>T和c.543G>C变异。     

ABO亚型ABw07一家系的分子生物学研究

目的 研究一个ABO亚型ABw07家系的分子机制.方法 用单克隆抗体检测先证者红细胞ABO血型抗原.标准A、B、O红细胞检测先证者血清中的ABO抗体,采用聚合酶链反应技术扩增先证者ABO基因的第6和7外显子序列,PCR产物经酶切后直接测序分析.同时PCR产物经TOPO TA克隆到质粒载体中获得单链,对所得克隆进行ABO基因双向测序分析.家系调查采集先证者父母和姐姐的标本进行血型血清学实验和ABO基因第6和7外显子直接测序分析.结果 先证者红细胞有A、B抗原,同时血清中存在抗B抗体.直接测序分析发现第261位无缺失,第297位A/G、467C/T、526C/G、657C/T、703G/A、796C/A、803G/C、930G/A、1055C/A、1096A/G杂合,可推断为A102Bw07基因型.克隆测序得到两个等位基因A102和Bw07.与B101相比,Bw07第1055位G→A,导致1个氨基酸改变:第352位氨基酸精氨酸变成谷氨酰胺.家系调查显示先证者Bw07基因从母亲遗传所得,母亲血液标本ABO血型血清学特性和测序分析结果与先证者完全一致.结论 α-1,3-半乳糖基转移酶基因(B基因)第1055位G→A突变导致产生Bw07表型,其血清中可含有抗B抗体.     

Fetal ABO blood group typing using amniotic fluid

Amniotic fluids (AF) obtained by second trimester amniocentesis were examined using a hemagglutination-inhibition test. In the anti-A and anti-B dilution series three different patterns of agglutination were found. One pattern, indicating the presence of high levels of antigen, is typical of secretors (Se/-) of the blood group antigen. The second pattern is indicative of total absence of soluble antigen and is produced in secretor (Se/-) and non-secretor (se/se) fetuses lacking the I^A or I^B allele at the ABO locus. The third pattern shows the presence of trace amounts of soluble antigen, about one-tenth of that found in secretors, and it appears in non-secretor fetuses bearing the corresponding ABO allele. These trace quantities, if a constant feature of AFs from these non-secretor fetuses, could allow blood group typing information to be obtained on all non-secretor as well as secretor fetuses. A total of 157 amniotic fluid samples were surveyed in this and a previous study. Of these, 31 were from non-secretor fetuses. Blood group type for the 31 non-secretor fetuses was predicted (“blindly”) on the basis of the presence or absence of trace amounts of soluble antigen. These predictions were verified by standard ABO typing of the infants afterbirth. In each case the (“blind”) typing result agreed with the predictions made from the titration curves. This agreement establishes that the production of trace amounts of soluble antigen(s) is a consistent feature of non-secretors with the I^A and/or I^B allele. The soluble blood group substances are of fetal origin and are independent of maternal blood type and secretor status. Type O non-secretors produce no soluble A or B antigen and also lack detectable soluble H antigen. Thus it appears that the nature of the detectable amounts of soluble blood group antigen(s) present in second-trimester AF can be used to accurately assess the ABO blood type of all fetuses.     

A novel a subtype allele that demonstrates allelic competition

A 68-year-old man with an ABO discrepancy discovered during a routine check up and 3 members of his family were referred for ABO analysis. A novel allele (A(w)14; 699 C>A, H233Q) was found by sequence analysis of exons 6&7 and in those with the A(w)14 allele, complete ABO analysis was performed. The propositus and his son had an A(w) phenotype with the A(w)14/O01 genotype. His daughter had an A(w)14/B101 genotype with a normal B phenotype. Three-dimensional molecular modelling was used to predict the effect of the H233Q amino acid change on the resulting A glycosyltransferase. The A(w)14 produced a weak A phenotype, and its ability to produce A antigens was further reduced when it was co-inherited with a B101 enzyme.