无锡地区无偿献血者ABO亚型血型血清学及分子生物学特征研究

目的调查并分析无锡地区无偿献血者ABO亚型血型血清学及分子生物学特征。方法 2016年5月～2019年6月,有19例无偿献血者标本经血型血清学初步判定为ABO亚型。14/19例标本进行分子生物学检测,其中11例同时采用PCR-SSP(序列特异性引物聚合酶链反应)方法及DNA测序方法进行ABO基因检测,另3例仅采用了DNA测序方法检测。结果 120 118名无偿献血者中,用血型血清学方法检出ABO亚型的频率约为1.6/万,且B亚型数量多于A亚型;分子生物学检测获得Ael02、A201、Bw27、Bel03、Bw22、B310、A217、CisAB01、B(A)02九种ABO突变等位基因;3例标本在第1-7号外显子未见异常,发现1例新等位基因(GenBank:MT281528)。结论用血型血清学、PCR-SSP和DNA基因测序三种方法结合更能准确地鉴定ABO亚型。     

ABO血型系统B101-O02等位基因杂交导致的Bw亚型

目的 研究中国汉族个体红细胞ABO血型Bw亚型的分子遗传背景.方法 通过标准血型血清学方法 鉴定了1个家庭3例ABw亚型,PCR扩增样本基因组DNA的ABO基因增强子、启动子和外显子1～7及侧翼内含子序列,PCR产物经割胶纯化后直接测序,并将含有多态性位点的外显子7克隆到pcDNA3.1(-)质粒,转化DH5α后进行单倍体序列分析.结果 3例ABw亚型的直接序列分析发现其ABO基因均有A102、B101和002等3种等位基因部分序列特征,但无法直接确定其基因型.单倍体序列分析表明,其中1个等位基因为A102,另1个为B101-O02杂交等位基因,表现为B101等位基因基础上的646T>A,657T>C和681G>A变异.在110份随机样本中未发现该杂交等位基因.结论 B101和O02等位基因杂交可能是导致该Bw亚型的分子机制.     

1例ABw表型的血清学和基因型检测

目的 鉴定对1例ABO疑难血型标本的表型和基因型.方法 1例ABO疑难血型标本用血清学方法做ABO表型检测,用PCR-SSP法做ABO基因鉴定,用直接测序法和克隆测序法对ABO基因的第6、7外显子做序列分析.结果 血清学定型:ABw表型;PCR-SSP法鉴定:AB型;直接测序:A101/B101等位基因杂合,但伴有nt1055的G/A突变;克隆测序:G1055A突变发生在B101等位基因上,该突变产生Bw07等位基因.结论 在中国人群中发现A101/Bw07基因型.     

稀有B(A)02亚型标本的血型基因鉴定

目的 对1例ABO定型正反不符[血清学初定为B(A)]的标本进行PCR分型和测序分析,拟阐明其血清学的分子机制.方法 采用试管法进行血清学鉴定;用PCR-SSP方法对标本ABO基因亚型进行初筛,并测序分析标本ABO基因的第6和第7外显子.结果 血清学表现为AxB,且H抗原明显增强,反定与A1细胞和A2细胞皆有反应,初定为B(A)型;PCR-SSP分型定为B(A)02型.测序表明:标本的第6外显子存在261delG突变;第7外显子在B101序列的基础上,出现了B(A)02型700C/G的典型突变.结论 该标本的基因型为B(A)02型与O01型杂合.     

1例Bx亚型的分子生物学研究

目的研究Bx亚型的分子生物学特点。方法采用PCR-SSP方法,对3份血清学试验疑为Bx和ABx的标本,做ABO初步基因分型,明确其中的A、B、O基因;并进一步对其ABO基因第7外显子作深入的克隆测序分析。结果克隆测序结果显示3份标本均有C721T点突变,先证者及其妹均有正常的A基因,其父有正常的O基因。结论在中国汉族人中检测到Bw03等位基因。     

BW.03等位基因的鉴定及序列分析——附1例报告

目的对1例Bw血型的血清学及分子生物学特性进行分析。方法应用聚合酶链反应-序列特异性引物(polymerase chain reaction-sequence specific primer,PCR-SSP)和ABO等位基因第6、7外显子PCR产物直接测序方法进行ABO亚型基因分型。结果先证者红细胞未检测到B抗原,但血清中含有抗-A。PCR-SSP结果显示标本基因型为B/O2。直接测序分析发现c.297AG、c.526CG、 c.657CT、c.703GA、 c.721CT、c.796CA、 c.803GC和c.930GA 8个突变,可推断为BW.03/O.02.01基因型。该序列与B.01基因序列比对仅在721位发生CT的突变导致多肽链R241S替换。结论 B等位基因c.721CT突变产生BW.03等位基因,导致B抗原表达减弱。     

2个新的ABO血型等位基因的发现和序列分析

目的 探讨4例ABO血型正反定型不符标本的遗传背景。方法 采用血型血清学方法对ABO正反定型不符标本进行亚型鉴定;采用PCR方法扩增ABO基因的7个外显子;采用测序方法对7个外显子进行直接测序。结果 血型血清学试验证实4例标本为ABO亚型,分别为:Bx(1例);ABx(1例);Ax(2例)。基因直接测序发现Bx和ABx在ABO~*B.01基础上1～6外显子未发生改变,仅在B等位基因第7外显子发生了449位核苷酸AG的突变;2例Ax在ABO~*A1.01基础上1～6外显子未发生改变,仅在A等位基因第7外显子发生了467位核苷酸CT的突变和798位插入了一个碱基T,以上的基因位点改变均导致了ABO血型的血清学表型变化,表现为ABO亚型。结论 研究揭示了4例ABO亚型的分子遗传背景。发现2个新的ABO血型等位基因,即:c.449AG单基因位点突变,c.467CT的突变和c.798insT单碱基插入。     

ABO疑难血型的基因型鉴定与序列分析

目的 研究20例ABO疑难血型标本的基因型并鉴定其分子生物学特性。方法 应用标准血型血清学鉴定技术分析标本的血清学表型。应用聚合酶链反应(polymerase chain reaction, PCR)扩增标本ABO基因7个外显子，对PCR产物进行直接测序，分析ABO疑难血型的基因分型和序列。结果 20例ABO疑难血型标本的血清学表型分别为：A抗原减弱5例、A抗原减弱合并抗-A15例、A抗原正常合并抗-A12例、B抗原减弱8例，基因型分别为：Ax02/O01 3例、Ael07/O01 2例、B313/O01 2例、A204/O02、A220/O01、Ael07/O02、Ael02/O01、Ael02/O02、Ax03/O01、Ax03/O02、B313/O02、B302/O01、B302/O02、Bw19/O02、A102/B313、A101/Bw37各1例。结论 ABO基因分型技术可精准鉴定疑难标本的血型，提供明确的血型遗传学信息，保障临床用血安全。     

B(A)04亚型血清学与分子生物学鉴定及与其他AB亚型的鉴别要点

目的对3例血清学定性为A_(weak)B亚型的血标本进行ABO基因检测,探讨其分子遗传学特点,并探讨B(A)亚型与CisAB、A亚B型的鉴别要点。方法用PCR-SSP对常规血清学方法定型困难的3例标本进行ABO基因分型,并对其ABO基因第6、7外显子扩增后进行直接测序及单倍型测序,确定其基因型。结果 3例标本血型血清学结果均为A_(weak)B型,存在不规则抗A抗体。基因分型均为B(A)04型,其B等位基因在第7外显子存在nt640AG突变,标本1为B(A)04/O02,标本2、3为B(A)04/O01。结论当血清学检测结果正反定型不一致时,应采用分子诊断技术进行辅助诊断,并采用配血相合输血策略,可确保临床输血安全。     

3例ABO血型鉴定正反不符患者基因序列分析——附1个新的ABO等位基因突变点位的发现

目的 探讨导致3例ABO血型正反定型不符的分子机制。方法 采用血型血清学方法对ABO正反定型不符标本进行亚型鉴定;采用PCR方法扩增ABO基因的6、7外显子;采用测序方法对ABO6、7外显子进行直接测序。结果 血型血清学试验结合基因测序证实3例标本为分别为：AwB(1例)、Ael(2例)。基因直接测序发现先证者1在A等位基因第7外显子发生了476C>T、595C>T突变,在B等位基因第7外显子526C>G、657C>T、703G>A、796C>A、803G>C、930G>A突变;先证者2、3在A等位基因第7外显子发生了476C>T和767C>T突变,两标本突变点符合Ael₀₅。以上的基因位点改变均导致了ABO血型的血清学表型变化,表现为ABO亚型。先证者3的母亲及女儿都携带Ael₀₅等位基因。结论 本研究揭示了3例ABO亚型的分子遗传背景,并发现1个新的ABO血型等位基因,即：c.595C>T单基因位点突变。     

A Novel Variant B Allele of the ABO Blood Group Gene Associated with Lack of B Antigen Expression

SUMMARY: BACKGROUND: The gene locus for the ABO blood group system encodes a glycosyltransferase. Alterations in the DNA sequence are associated with the blood groups and the expression levels of antigens on red blood cells. A number of ABO alleles have been described as the molecular basis of weak A or B antigens. PATIENTS AND METHODS: Here, we describe a novel variant B allele in a blood donor with discrepant results in routine forward (group A) and reverse (very weak anti-B isoagglutinins) ABO blood grouping. RESULTS: Determination of the ABO genotype using polymerase chain reaction-sequence-specific primers (PCR-SSP) indicated blood group A(2)B. Sequencing of the ABO gene exons 6 and 7 showed for 1 allele a G insertion into the GGGGGG sequence at position 811-816 of exon 7. The 816insG mutation (designated ABO*Bw20) led to a frame shift of the coding sequence and subsequent alteration of the protein sequence. The location of the mutation on a B allele was proven by PCR-SSP. Screening for the novel mutation in 211 blood donors with regular ABO phenotypes indicated that *Bw20 is a rare variant. CONCLUSIONS: The low levels of anti-B isoagglutinins associated with this novel variant indicate that residual undetectable amounts of B antigen may be present on red blood cells. The serological and molecular analysis of members of the blood donor's family further proved the phenotype-genotype correlation of the *Bw20 allele with antigen O and individually variable levels of anti-B isoagglutinins. The characterization of novel alleles associated with ABO subgroups may ensure the correct determination of blood groups in which serological methods are combined with molecular genetic approaches.     

A novel cis-AB allele derived from a unique 796C>A mutation in exon 7 of ABO gene

BACKGROUND: The cis-AB phenotype is very rare, and only three genotypes that correspond to specific ABO allele changes have been reported. Cis-AB01 involves the A102 allele with a nonsynonymous substitution G803C in exon 7, whereas cis-AB02 and cis-AB03 involve different nonsynonymous substitutions A796C and C700T, respectively, on the B101 allele background. The nucleotide substitutions give rise to a change of the respective glycosyltransferase, resulting in varying bifunctional AB transferase activities. STUDY DESIGN AND METHODS: Two cis-AB phenotypes were identified in a Taiwanese C. family and two unrelated individuals, respectively. Serologic studies, molecular cloning, and sequencing of exon 6 and exon 7 were carried out to determine their respective phenotypic characteristics and cis-AB alleles. A cohort of 300 AB-phenotype, healthy random individuals served as controls. RESULTS: A novel cis-AB allele is uncovered out of the three family members, of which a 796C>A substitution occurs predicting an amino acid change at residue 266 of leucine to methionine on the background of A102 allele. It is serologically like cis-AB03, an A2B phenotype, but molecularly different. Both of the two unrelated individuals are of cis-AB01 allele, and all of the 300 AB blood group controls are excluded cis-AB phenotype. CONCLUSION: The C. family described carries a novel cis-AB allele that differs molecularly from all previously reported cis-AB alleles.     

Evolution of the O alleles of the human ABO blood group gene

BACKGROUND: To date, at least 40 different alleles O have been characterized on the basis of exon 6 and exon 7 sequences but not always for intron 6. STUDY DESIGN AND METHODS: Among 415 individuals, from four continents (Africa, Europe, South America, and Asia), studied for exon 6 and exon 7 sequences, we selected 46 individuals (of respective phenotypes O [39], AB [3], B [3], or A [1]) for sequencing 1800-bp amplicons spanning exon 6, intron 6, and exon 7. The amplicons were characterized either by direct sequencing or after cloning when required. RESULTS: We defined 14 new intron 6 O allele sequences, including four recombinant alleles. Based on sequence comparison, a phylogenetic network was constructed. It confirmed recombinant allele origins and that most O alleles are derived by point mutations from the two worldwide distributed alleles O01 and O02. CONCLUSION: Allele O phylogenetic analysis suggests that the most frequent silencing mutation (deletion of a G in exon 6) appeared once in human evolution in the ancient O02 allele lineage and that allele O01 resulted from an interallele exchange between O02 and A101. Assuming constancy of evolutionary rate, diversification of the representative alleles of the three human ABO lineages (A101, B101, and O02) was estimated at 4.5 to 6 million years ago.     

ABO血型基因转录结构的研究及应用

目的 建立研究ABO血型系统基因转录结构的方法.方法 使用两套RNA逆转录-巢式PCR-序列测定的反应策略对中国人群6种常见ABO基因型的11个血液标本转录结构进行分析.应用所建立的方法对3例ABO血型正反定型不符的标本进行ABO基因转录结构的分析.结果 未能检测到O基因的转录结构;两套PCR策略均发现有2种转录结构,一种是完整的ABO基因编码区,另一种是缺失了整个第6外显子的ABO基因编码序列.一个变异的ABO等位基因在Bx表型个体被发现.结论 ABO基因转录结构的剪切方式是复杂的,不同剪切位点的ABO转录子被观察到.O等位基因的转录结构在RT-PCR分析未检测出来.O型基因转录结构这个特点的发现,可以应用于ABO基因单倍体序列测定中.     

B亚型中Bx新等位基因的鉴定

目的对ABO血型中B亚型分子遗传背景的研究,发现并鉴定ABO新等位基因。方法对5份血型血清学鉴定为B亚型的样本,采用PCR-SSP、ABO基因第6及第7外显子直接测序方法进行基因定型;并对目的B亚型样本进行RT-PCR、巢式PCR扩增、测序,分析其cDNA结构的差异。结果5份血清学为B亚型的样本中,血清学鉴定为Bx的个体发现了一个新的B等位基因。该等位基因与B101等位基因相比,差异仅在于第7外显子的B基因序列中nt721位C>T突变。其余4份B亚型样本的ABO基因第6、7外显子都未发现新的点突变。结论首次在中国汉族人中发现一个721C>T新变异的B等位基因,该等位基因nt721位由C转变为T,241位氨基酸由精氨酸转变为色氨酸,可导致糖基转移酶活性的降低,表明ABO基因的第241位氨基酸对决定糖基转移酶活性是至关重要的。     

B抗原减弱表型的表型频率与ABO基因分析

目的对血清学表现为B抗原减弱的血液样本进行筛查和ABO基因分析,了解其分布特征和分子遗传学基础。方法筛查并收集B抗原减弱（与抗-B血清试管法凝集强度中等）的样本,采用血型血清学方法进行鉴定分析,采用直接测序的方法对ABO基因的第6、7外显子及第6内含子进行序列分析,对可追踪家庭进行家系分析。结果从241952份样本中检出13例B抗原减弱表型,其中B型9例、AB型4例;所有样本的红细胞与抗-H反应均增强;其中2例可检出不规则抗-B;ABO基因型分别为A102／Bw12（1例）、BIOI／B101（2例）、B10I／002（3例）、A102／B101（3例）、B101／001（4例）。在1例基因型为B101／001个体的家系成员（父亲）中检出相同表型。结论该类表型在B型人群中的频率约为1：7000;除1例样本的B等位基因存在278C〉T突变（Bw12）外,其余样本在第6、7外显子和第6内含子中均未检出突变;ABO基因酶催化活性区域编码序列以外的基因变异,可能是导致B抗原减弱的原因之一。     

ABO blood group in Kuwaitis: detailed allele frequency distribution and identification of novel alleles

BACKGROUND: The ABO blood group is clinically the most important blood group system and can now be genotyped easily by DNA-based methods without family studies. STUDY DESIGN AND METHODS: Samples (n = 166) from a Kuwaiti population were phenotyped by standard serologic techniques for the ABO blood group and genotyped for the ABO locus by an established multiplex polymerase chain reaction protocol followed by single-strand conformation polymorphism (SSCP) analysis. Nonstandard SSCP patterns were investigated by DNA sequencing of exons 6 and 7 and, if necessary intron 6. RESULTS: Standard SSCP patterns identified six classical alleles in this population: A101 (0.1115), A102 (0.0181), A201 (0.0301), B101 (0.1627), O101 (0.3103), and O201 (0.2500). One A, 1 B, and 8 O variant alleles were identified (total frequency, 0.1175). All variant alleles were each present in one or two chromosomes (< or =0.0060) in our samples except O109 (0.0813). Three of these 10 variant alleles were novel alleles defined by newly identified single-nucleotide polymorphisms in exon 7 (527G>A, 687C>T, and 1116G>A). One new base substitution result in amino acid change. CONCLUSIONS: This is the first study reporting the detailed distribution of ABO alleles and genotypes in Kuwaitis. Sixteen alleles were identified, including 3 novel alleles.