Am新等位基因分子遗传背景的研究

目的鉴定ABO新等位基因,分析Am亚型的分子基础。方法对1例血清学鉴定为Am亚型的标本,PCR扩增ABO等位基因的第6、7外显子及侧翼内含子,PCR产物采用直接测序和克隆测序的方法,确定其基因型。结果在血清学鉴定为Am亚型的标本中发现一个突变的A等位基因,它与ABO*A105等位基因相比只有1个点突变(595C>T)。结论 595位突变导致编码A糖基转移酶的199位氨基酸由精氨酸(Arg)转变为半胱氨酸(Cys),极大降低了酶的活性,表明199位氨基酸对决定ABO糖基转移酶活性是十分关键的。     

Bel亚型α1,3半乳糖基转移酶新等位基因的鉴定

目的 分析1例罕见B放散型的ABO血型分子背景,鉴定ABO新等位基因.方法 对1例正反定型未能鉴定其ABO血型的捐血者,分别采用ABO基因第6及第7外显子直接序列测定及单倍体克隆测序进行基因分型,以及检测ABO基因CBF/NF-Y微卫星增强子区域.结果 血清学鉴定为Bel的个体中发现了一个突变的B等位基因,该等位基因与B101等位基因相比,差异在nt905位A>G突变.该突变导致α1,3半乳糖基转移酶Asp302gly,定为Bel新基因,Genbank注册号为FJ009674.ABO基因微卫星增强区域序列测定为G/C型.结论 αl,3-D-半乳糖基转移酶基因中nt905A>G突变可能是Bel分子遗传机制之一,第302位氨基酸变异大大影响了糖基转移酶的活性.     

ABO变异型BW.12等位基因的鉴定及序列分析

目的研究ABO基因的α-1,3半乳糖基转移酶基因变异对于B抗原表达的影响。方法在标准血型血清学方法鉴定基础上,应用聚合酶链反应-序列特异性和ABO基因第1—7外显子PCR产物直接测序进行ABO基因分型及序列分析。结果先证者血清学检测为Bw亚型,PCR-SSP结果显示标本基因型为B/O2。直接测序分析发现c.278CT、c.297AG、c.526CG、 c.657CT、c.703AG、c.796CA、c.803GC和c.930GA 8个位点突变。该序列与B.01等位基因序列比对仅在278位发生CT的突变导致多肽链p.Pro93Leu替换。结论α-1,3半乳糖基转移酶基因278位碱基CT突变产生BW.12等位基因。     

α-1,3半乳糖基转移酶基因c.1055G＞A突变导致ABO亚型ABw07的分子生物学研究

目的 探讨1例ABO血型系统ABw07亚型的分子生物学机制.方法 选择2014年3月12日于青岛市中心血站献血的1例ABO正反定型不符的疑难血型无偿献血者为研究对象.采用试管法对其进行血型血清学检测;采用PCR-序列特异性引物(SSP)法对其ABO基因进行分型,并且对其ABO基因第6、7外显子扩增后,进行直接测序及克隆测序,确定其ABO基因型.结果 ①本例献血者血型血清学检测结果显示,正定型为AB型,反定型为A型.②本例献血者ABO基因第6、7外显子的直接测序结果显示,ABO基因第261位脱氧核糖核苷酸无缺失,存在c.297A＞G、c.467C＞T、c.526C＞G、c.657C＞T、c.703G＞A、c.796C＞A、c.803G＞C、c.930G＞A及c.1055G＞A杂合突变.③单克隆测序结果显示,本例献血者的ABO基因存在c.1055G＞A突变,导致密码子由CGG突变为CAG,所编码的第352位氨基酸精氨酸突变为谷氨酰胺(p.Arg352Gln),ABO基因型为A102/Bw07.结论 α-1,3半乳糖基转移酶基因(B基因)c.1055G＞A(p.Arg352Gln)突变可能导致产生ABw07亚型,该亚型血型个体的血清中存在抗-B.     

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血型变异与B糖基转移酶关系研究

目的研究ABO血型B亚型系统Bw亚型与B糖基转移酶的关系。方法运用血型血清学鉴定方法鉴定1个家庭2例ABO血型的Bw亚型,运用聚合酶链式反应的方法扩增糖基转移酶1~7号外显子,送到试剂公司测序。结果直接测序发现2例ABO血型的Bw亚型,其中B糖基转移酶基因第721位C〉T的转变,导致糖基转移酶多肽链Arg241Trp的转变。结论糖基转移酶基因第721位的C〉T的突变引起糖基转移酶活性的消失或者减弱,导致Bw亚型。     

Bel亚型与α1,3半乳糖基转移酶基因G952A多态性关系的研究

目的 研究红细胞ABO血型系统中B放散型的ABO基因分子遗传基础.方法 通过标准血型血清学试验鉴定了3例Bel亚型及15例对照B型样本,采用ABO基因分型PCR序列特异性引物、ABO基因第6及第7外显子PCR产物直接测序及克隆测序等方法进行ABO基因及亚型的定型.结果 在1例血型血清学检测为Bel亚型标本中,发现一个新的B等位基因.该等位基因与B101标准等位基因相比,差异仅在于ABO基因的第7外显子上nt952位G>A突变,导致多肽链Val318Met,定为B放散型(Bel)新基因,GenBank注册号为EF117687.而其余2例Bel型样本及15例对照B型样本含正常标准B基因.结论 首次在ABO基因编码区核苷酸930位后的错义突变中发现并报道了新B等位基因,表明1,3半乳糖基转移酶基因G952A多态性可能是Bel分子遗传机制之一.     

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。     

罕见的AEL.05亚型等位基因的鉴定及家系分析

目的 A_(el)是中国人群中1种较为常见的ABO亚型,其分子机理尚未完全阐明。本文对1例A_(el)亚型家系进行分子机制研究。方法在常规血型血清学鉴定的基础上,应用吸收放散法鉴定弱血型抗原;PCR-SSP方法进行ABO基因初步分型;PCR方法扩增ABO基因的7个外显子;对扩增产物进行直接测序和克隆后测序分析。构建3D分子模型,并就突变对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。结果吸收放散试验证明患者红细胞上存在弱A抗原,血清学定型为典型的A_(el)表型;ABO基因初步分型为A1/O型,测序发现第7外显子767位碱基TC突变,导致A糖基转移酶第256位异亮氨酸被苏氨酸替换,血型基因型为ABO*AEL.05/O.01.01。分子模建与分析提示突变导致蛋白局部氢键作用力改变不显著,但突变导致ΔΔG值的升高,表明蛋白稳定性降低。结论α-1,3-N-乙酰半乳糖胺基转移酶p.I256T突变可能通过降低了酶的稳定性导致A_(el)表现型。     

1例A3B亚型的血清学和分子生物学结果分析及家系调查

目的 分析1例ABO血型A亚B型标本的血清学和分子生物学特征,探讨ABO亚型鉴定的准确方法。方法 采用常规血清学方法分析了1例标本ABO表型,首先使用PCR-SSP方法对样本进行ABO基因分型,再采用直接测序的方法对样本的第6、第7外显子测序确定其基因型。结果 血型血清学检测结果为A亚B型;PCR-SSP分型结果为A₁B型;测序发现标本ABO基因的第6和第7外显子存在突变：第6号外显子发生297A>G突变,第7号外显子发生467C>T、526C>G、657C>T、703G>A、745C>T、796C>A、803G>C及930G>A突变,该样本基因型为A307/B01。先证者母亲血型血清学检测结果疑似为A亚型;基因测序结果为A₃型。先证者父亲血型血清学检测结果为B型。结论 血清学实验结合分子生物学方法可准确鉴定ABO亚型,分子生物学方法还有助于探索ABO亚型的形成机制。     

ABO*BEL.11 等位基因突变致红细胞B 抗原弱表达的家系血型血清学及分子机制研究

目的 探讨由ABO*BEL.11 等位基因导致的ABO 正反定型不符样本及家系的血清学和分子生物学特性,研究其遗传方式。方法 常规血型血清学方法和吸收放散试验鉴定样本的ABO 血型表型;PCR 方法扩增ABO 基因7 个外显子及其侧翼内含子,对扩增产物进行直接测序和克隆测序分析,并对先证者的亲属进行家系调查。结果 先证者血型血清学结果为B弱;测序显示:存在ABO*B.01/O.01.01 杂合且伴c.586C/T 突变;克隆测序:c.261delG,297A>G,c.526C>G,c.586T>C,c.657C>T,c.703G>A,c.796C>A,c.803G>C 和c.930G>A 共9 个突变,基因型结果为ABO*BEL.11/O.01.01。家系调查显示先证者父亲、母亲、妻子和女儿血型血清学结果分别为B 型、O 型、A 型及B 弱,其中先证者父亲及女儿的ABO 基因存在ABO*BEL.11 等位基因。结论 ABO*B.01 等位基因上c.586T>C 的突变产生ABO*BEL.11 等位基因,从而导致红细胞上B 抗原的弱表达,且能够稳定遗传突变。     

Bx亚型1例血清学与分子生物学分析

正输血是现代医疗中重要的救治手段之一,正确的血型鉴定在输血工作中有极其重要的意义。ABO血型有多个亚型~[1-2]。若血清学显示ABO血型正反定型不符及抗原性减弱,很容易造成忽视和漏检,给临床血型鉴定、交叉配血及器官移植等带来极大的困扰,给临床输血带来危险。最近我们在临床送检样本中发现了1例正反定型不符的样本,并对其进一步做血清学以及基因测序分析,最终确定为Bx亚型,现报告如下。     

A unique 502C>T mutation in exon 7 of ABO gene associated with the Bel phenotype in Taiwan

BACKGROUND: The ABO system includes many variant subgroups. Some of them are difficult to identify serologically, leading to mistyping of blood groups. For example, Bel is often typed as O blood group. STUDY DESIGN AND METHODS: DNA sequencing and a molecular approach were explored to accurately determine the genotypes of Bel subgroups. Seven Bel blood donors and 106 individuals with other blood groups were analyzed serologically and molecularly. RESULTS: The serologic results of these seven Bel blood donors showed that their RBCs do not react with anti-B or anti-A,B, and their B antigen was detected by adsorption and elution methods. Sequencing results for exons 6 and 7 of ABO genes showed a new Bel allele with a C>T substitution at nucleotide position 502 in exon 7 of the ABO gene in all seven cases but not in other blood groups. Consequently, an amplification-created restriction site protocol was designed to detect the 502C>T genotype in Bel subgroup cases. CONCLUSION: A novel 502C>T mutation was found in the Bel subgroup in Taiwan and successfully developed a rapid and accurate molecular protocol to detect this mutation. To our knowledge, the new Bel allele that was found is unique in Taiwanese residents.     

Serology and gene sequence analysis of the B(A) subtype of patients in Hunan

IntroductionB(A) is a relatively rare ABO subtype. The frequency of B(A) in European Caucasians is low, but is higher in China. B(A) is common in northern China, gradually decreasing from north to south. The frequency of B(A) has been researched in northern China, but not in southern China. This study aimed to investigate the serological characteristics and molecular mechanism of the B(A) subtype in Hunan.MethodsThe phenotypes of ABO blood group were analyzed by blood group serology for the patients in the Third Xiangya Hospital from 2016 to 2020; Exons 1–7 of the ABO gene were screened by PCR-SSP and sequencing.ResultsNine cases were suspected as belonging to the B(A) subtype by serological tests. The activity of D-galactosyltransferase in these patients' serum was significantly higher than that of group B plasma; meanwhile, there was no activity of Nacetylgalactosyltransferase. The ABO genotype was ABO*B.01/O.01.01 or ABO*B.01/O.01.02, and the gene sequencing results confirmed the phenotype as B(A), whose gene frequence was 1/26,000. Allele ABO*BA.02 (1/38,000) had the highest frequency, followed by ABO*BA.04 (1/77,000).ConclusionAmong the patients in Hunan, the most common allele encoding the B(A) phenotype was ABO*BA.02. The identification of the B(A) blood group required serological methods combined with genotyping.     

Molecular genetic mechanism analysis and pedigree investigation of rare Bweak subgroup

ObjectiveTo analyze the molecular mechanism of rare B_weak subgroup in the ABO blood group system and conduct pedigree investigations.MethodsThe blood group was detected by conventional serological method, and ABO gene of proband and her family was amplified and sequenced by polymerase chain reaction method.ResultsThe study showed that the proband was a B_weak phenotype by conventional serological method. Her family’s serological results were as follows, her father and eldest brother were B_weak subgroup while her mother and second eldest brother were O group. The proband’s ABO gene sequencing result was ABO*BW.27/ABO*O.01.02. Her father, mother and two elder brothers were ABO*BW.27/ABO*O.01.01, ABO*O.01.01/ABO*O.01.02, ABO*BW.27/ABO*O.01.02, ABO*O.01.01/ABO*O.01.02.ConclusionConventional blood group serology combined with molecular diagnostic technology can accurately identify the B_weak subgroup, and the pedigree investigation analysis showed that the proband's allelic mutation came from her father. She has gained a point mutation of c.905A>G on the basis of ABO*B.01.     

α-1,3-N-乙酰半乳糖胺基转移酶基因742C>T突变导致A2亚型

本研究探讨2例ABO亚型A2B的分子机制。利用单克隆抗体检测先证者红细胞ABO血型抗原,用标准A、B、O细胞检测先证者血清中的ABO抗体,采用聚合酶链反应(polymerase chain reaction,PCR)技术扩增先证者ABO基因的第5至7外显子序列,其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基因第6、7外显子双向测序分析。结果表明,先证者红细胞有A、B抗原,同时其血清中存在抗A1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、742C/T、796C/A、803G/C、930G/A杂合。克隆测序得到B101和1个新的A等位基因。与A102相比,新A等位基因仅在第742位C→T突变,导致第248位精氨酸变成色氨酸,新等位基因已被正式命名为A213。结论 :α-1,3-N-乙酰半乳糖胺基转移酶基因第742位C→T突变导致产生A2表型,表型个体血清中可含有抗A1抗体。     

Different inactivating mutations in the LU genes of three individuals with the Lutheran-null phenotype

BACKGROUND: The null phenotype of the Lutheran blood group system, Lu(null) or Lu(a-b-), is characterized by the lack of all Lutheran system antigens. It can arise from three genetic backgrounds: recessive, dominant, or X-linked. Lu(null) of the recessive type appears to result from homozygosity for an inactive LU gene. STUDY DESIGN AND METHODS: Three unrelated recessive Lu(null) individuals were assessed by standard serologic tests. All exons of the LU gene were directly sequenced from amplified genomic DNA. The validity of the observed mutations within the LU gene was confirmed by the use of either restriction enzymes or allele-specific primers. RESULTS: All three individuals had the serologic characteristics of recessive Lu(null). One individual was doubly heterozygous for a nonsense mutation 691C>T in exon 6 (Arg231STOP) and a deletion of LU exons 3 and 4. The other two samples showed homozygous nonsense mutations: one had 711C>A in exon 6 (Cys237STOP) and the other 361C>T in exon 3 (Arg121STOP). CONCLUSIONS: The results revealed four unique genetic backgrounds from three examples of the rare recessive Lu(null) phenotype, each encoding Lutheran glycoproteins with a disrupted structure.     

一例B(A)血型的血清学及分子生物学鉴定

摘要：目的?对1例血型血清学正反定型不符的标本进行ABO血型鉴定。方法?运用血型血清学方法鉴定标本ABO血型；用PCR-序列特异性引物(SSP)基因定型和ABO基因直接测序的方法确定该标本的基因型。结果?血型血清学检测结果表现为B(A)亚型的特点；PCR-SSP分型结果定为B(A)04/O1型；测序发现标本ABO基因的第6和第7外显子存在变异：第6外显子存在261delG、297A>G；第7外显子存在526C>G、640A>G、657C>T、703G>A、796C>A、803G>C、930G>A，判断该样本血型为B(A)04/O01血型。结论?该血型血清学初步鉴定为B(A)的标本用PCR-SSP法和ABO基因直接测序法确认为B(A)04/O01型。