一例类孟买型表型的分子机理研究

为了研究1例类孟买型的分子机理,在血清学方法初步鉴定先证者为A类孟买型的基础上,用PCR扩增先证者ABO基因第6、7外显子、FUT1基因（H）和FUT2基因（Se）的编码区序列,PCR产物经割胶纯化后直接测序分析,并应用PCR-SSP和基因扫描方法证实测序所发现的突变.FUT1基因突变通过克隆到质粒载体后测序分析.结果表明：直接测序发现先证者ABO血型基因型为A102A102,FUT1基因第682位A→G错义突变、第547-552位两碱基AG缺失（CAGAGAG→CAGAG）突变.克隆证实1条染色体上FUT1基因为A682G突变,导致M228V氨基酸突变;另一条染色体上第547-552位两碱基AG缺失,导致阅读框架发生移码,提前形成终止密码.FUT2基因为分泌基因Se^357和弱分泌基因Se^357,385杂合（第357位为T/T纯合,385位为A/T杂合）.结论：FUT1基因A682G和547-552 delAG双杂合突变是引起该例先证者表现为类孟买型的分子机理,A682G是一个新的引起类孟买型的FUT1基因突变.     

3例类孟买血型的分子遗传机制研究

目的:探讨类孟买血型个体的分子遗传机制。方法:采用常规血清学方法对先证者及其家系成员血液标本进行血型鉴定,对唾液标本进行ABH血型物质的检测;利用PCR扩增3例先证者及家系成员的FUT1基因编码区和ABO血型基因第6、7外显子编码区,对PCR产物进行基因分型和FUT1基因直接测序。结果:3例先证者均为类孟买血型。直接测序结果显示,先证者1的FUT1基因为第328位碱基G/A杂合突变、第547位碱基AG缺失突变的杂合型,FUT1基因分型为h1hnew2杂合;其父亲为单链第547位碱基AG缺失,其母亲为单链328位碱基G/A杂合突变,两者均不是类孟买血型;先证者2和3的FUT1基因为第547位碱基AG缺失突变,第880TT缺失突变的杂合型,FUT1基因分型为h1h2杂合;结论:通过分子生物学技术可以检测FUT1基因不同位点双碱基的缺失杂合和单链缺失突变杂合,进而探讨类孟买血型的分子遗传机制。     

类孟买血型的分子机制研究——附2例家系报告

目的对2例类孟买血型个体及其家系进行表型鉴定和分子机制研究。方法应用血清学方法检测ABO、H抗原;应用PCR技术扩增FUT1、FUT2编码区序列,并对PCR产物进行直接测序分析。结果发现2名先证者红细胞与抗H血清均不凝集,为血清学分型罕见的类孟买型Ahm和Bhm。直接测序发现2名先证者FUT1基因均存在658位C→T的纯合突变。先证者1父母、姑姑、弟弟和儿子FUT1基因均存在658位C→T杂合突变;先证者2父母、弟弟和2个胞姐FUT1基因也均存在658位C→T杂合突变。另外,所有被研究标本都是分泌型,其FUT2基因都具有357位C→T的同义突变。其中先证者1和2都为Se357等位基因纯合子。结论先证者1为Ahm型,先证者2为Bhm型,2例基因型均为h3/h3且均为分泌型。2名先证者的父母FUT1基因都是658位C→T杂合子,并都把突变的FUT1遗传给先证者,使其携带658位C→T的纯合突变,造成类孟买型的表型。     

类孟买血型FUT1新等位基因及其家系成员的分子遗传学分析——附1例报告

目的 研究1例FUT1基因236delG突变致类孟买血型及其家系成员的血清学和分子遗传学特征。方法 应用血型血清学方法对先证者及其家系成员进行ABO、H、Lewis血型鉴定,同时应用基因检测方法确认其ABO基因;用PCR方法扩增FUT1基因并对扩增产物进行测序分析;用SwissModel在线服务器对先证者FUT1 236delG酶结构进行3D模拟。结果 血清学结果显示：先证者为罕见的类孟买型ABhm, Le(a-b-),先证者父亲为A型,先证者母亲为B型;基因检测结果显示：先证者为AB型,先证者父亲为A型,先证者母亲为B型;测序结果显示先证者母亲FUT1基因有236delG杂合突变,先证者父亲FUT1基因有551＿552delAG杂合突变,先证者FUT1基因为236delG/551＿552delAG杂合突变;先证者FUT1 236delG酶结构3D模拟显示翻译产物为无实际功能的1个alpha螺旋结构。结论 236delG突变是FUT1基因型中的新发现的突变,它和551＿552delAG突变(FUT1^*01N.06基因型)的杂合型会导致类孟买血型的产生。     

2例类孟买血型的鉴定及基因突变分析

目的探讨类孟买表型形成的分子机制和输血策略。方法采用常规血型血清学方法对ABO正反定型不一致的患者进行ABO、H血型检测,采用PCR扩增技术检测ɑ-1,2-岩藻糖基转移酶(α-1,2-fucosyl transfer,FUT1)基因并测序。结果 2例均为类孟买血型Bbm。例1 FUT1基因测序显示为第547~552del AG碱基缺失,即CAGAGAG突变为CAGAG;其2个儿子血型为B型、H表型正常。例2 FUT1基因测序显示547~552del AG和814AG复合突变,母亲的FUT1基因为814A/G和547~552del AG杂合,复合突变遗传于母亲。结论 2例FUT1基因纯合和杂合突变致类孟买表型形成。     

类孟买型在两个近亲与随机婚配家系中的遗传特点

目的 调查分析近亲与随机婚配的两个家系成员个体间的类孟买型分布状态,了解类孟买表现型在该两家系中的遗传特点.方法 以血型血清学试验作为检测基础,用PCR-SSP法进行ABO基因分型,扩增FUT1,FUT2基因,对PCR扩增产物进行直接测序或分子克隆测序后,分别鉴定两家系成员的类孟买表现型.结果 近亲婚配的家系1中,先证者与其兄、弟均从父母中得到了FUT1基因的547～548位两碱基AG缺失(h1),880～881位两个T碱基缺失的突变点(h2),表现为h1,h2类孟买型;父亲为h2基因携带者;母亲与先证者的女儿均为h1基因携带者.随机婚配的家系2中先证者与兄弟姐妹共4人,只有先证者本人的两条单倍体均遗传了父、母亲的FUT1基因的547～548位两碱基AG缺失(h1),表现为h1h1类孟买型;其他兄弟姐妹与父亲、母亲、先证者的儿子均为h1基因携带者,血型为正常的表现型.两家系成员的FUT2位点均为正常的野生型.近亲婚配家系Ⅰ中hh纯合子遗传发生率为100%;随机婚配家系Ⅱ中hh纯合子遗传发生率为25%.结论 该两个家系调查发现,类孟买表现型的遗传方式符合常染色体隐性遗传规律,近亲婚配比随机婚配可大大增加类孟买表现型的遗传概率.     

中国福建地区类孟买血型个体Fut1基因突变研究

本研究旨在探讨中国福建地区类孟买血型的分子机制。采用血清学方法鉴定研究对象为类孟买血型,采用PCR扩增研究对象的α1,2岩藻糖基转移酶(FUT1)基因编码区序列,并将PCR产物经TA后进行测序,分析fut1基因编码区序列,以证实类孟买血型的发生机制。结果表明,PCR扩增产物电泳分析证实成功扩增fut1基因编码区序列;克隆后测序分析发现本例先证者2条染色体上fut1基因均为第547-552位AG两碱基缺失(CAGAGAG→CAGAG),导致阅读框架发生移码,提前形成终止密码。结论:本例类孟买血型个体fut1基因为h1h1型纯合突变。     

FUT1 基因杂合或纯合突变致类孟买血型形成研究简

本研究旨在探讨类孟买血型表型形成的分子机制.采用血清学方法鉴定个体红细胞H抗原;采用高保真PCR扩增检测个体的α1,2岩藻糖基转移酶（FUT1）基因编码区序列,并进行测序、比对分析,以查明个体类孟买血型的发生机制.结果表明：凝胶电泳分析证实成功扩增FUT1基因编码区全长序列;克隆后测序、比对发现：个体1的1条染色体上FUT1基因为h1 （547-552delAG）,另1条染色体上为h4（35C＞ T）,为h1h4杂合子;个体2,3的2条染色体上FUT1基因均为h1 （547-552delAG）,为h1h1纯合子.结论：FUT1基因杂合或纯合突变均可致类孟买血型的发生.     

1例类孟买型家系的分子遗传学研究

目的分析攀枝花地区1例类孟买型家系的分子遗传学基础。方法采用PCR扩增产物直接测序法,对1例类孟买型家系的FUT1基因编码序列进行序列检测。结果该先证者的FUT1基因有1处纯合突变,第547-552位的3个重复连续的AG中缺失1个AG;其父母FUT1基因则含有第547-552位AG缺失的杂合突变。结论 FUT1基因第547-552位AG缺失使阅读框架发生移码,提前在804-806位核苷酸形成终止密码,导致H抗原缺乏,是形成类孟买型的机制之一。     

类孟买血型鉴定与输血策略研究

目的 研究类孟买血型的鉴定方法、血清学特点和输血策略。方法 对先证者及其四妹采用凝胶微柱法和试管法正反定型,吸收放散试验证实红细胞上弱表达的ABO抗原,测定唾液中的ABH物质,筛查血清中不规则抗体,应用PCR-SSP技术进行ABO基因和FUT1基因分型,并扩增ABO基因的第6、第7外显子和FUT1编码区序列后对PCR产物进行直接测序分析。采用血清学方法为先证者筛选适宜输注的红细胞。结果 先证者和其四妹血清学鉴定为Bm~h和Om~h,血清中有抗-H,ABO基因分型结果为BO2、0102,FUT1基因分型结果均为h1h1型。先证者ABO基因测序结果为B101/002,FUT1基因测序结果为第547-552位AG 2碱基缺失的纯合突变。结论 血型血清学联合分子生物学方法可准确鉴定类孟买血型;类孟买血型个体输血时应采取特殊输血策略,以避免输血不良反应的发生。     

Molecular basis for para-Bombay phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele

BACKGROUND: The para-Bombay phenotype is characterized by H-deficient or H-partially deficient red blood cells (RBCs) in persons who secrete ABH antigens in their saliva. The studies that determined the genotypes for two Chinese individuals with the para-Bombay phenotype are described. STUDY DESIGN AND METHODS: RBC phenotypes were characterized by conventional serologic methods. Exons 6 and 7 of the ABO gene were amplified, as well as the entire coding region for FUT1 and FUT2, with four independence polymerase chain reactions (PCRs) from genomic DNA. PCR products were excised, purified from agarose gels, and sequenced directly. Mutations of FUT1 were identified by TOPO cloning sequencing. RESULTS: For both individuals, RBC ABO genotypes correlated with ABH substances in their saliva. One individual (a patient) had two heterozygous mutations of FUT1 by direct DNA sequencing, namely, a C-->T heterozygous mutation at position 293(C293T) and AG heterozygous deletion (CAGAGAG-->CAGAG) at position 547 to 552. These two mutations were confirmed to be compound heterozygotes; that is, each mutation was determined to be on a separate homologous chromosome by TOPO cloning sequencing. The FUT2 genotype was Se(357)Se(357). The other individual (a blood donor) had an AG deletion at position 547 to 552 homozygous allele in FUT1. The FUT2 genotype was Se(357)Se(357,385). C293T mutation can cause Thr/Met at amino acid position 98. AG deletion at position 547 to 552 caused a reading frameshift and a premature stop codon. CONCLUSION: A novel nonfunctional FUT1 allele C293T was identified in a person with the para-Bombay phenotype. This rare H-deficient phenotype may result from different nonfunctional alleles.     

类孟买型的FUT1和FUT2等位基因突变的分析

目的分析类孟买型个体的FUT1和FUT2位点基因突变的分子生物学基础。方法采用PCR扩增产物直接测序法,对2例类孟买型个体的FUT1和FUT2位点的等位基因进行序列检测。结果1例类孟买型个体FUT1位点547～552位的3个重复连续的AG中缺失1个AG,使得氨基酸序列发生182框码移位;另1例FUT1位点880～882位3个重复连续的T中缺失2个T,使得氨基酸序列发生294框码移位,而2例类孟买型的FUT2位点均为正常野生型。结论FUT1位点的移码突变是导致H抗原缺乏,形成类孟买型的原因之一。     

双碱基缺失型等位基因复合杂合导致的类孟买型个体1例

本研究探索1例类孟买型血型的分子机制,为稀有血型的筛选和鉴定提供理论基础。以血型血清学方法鉴定该个体红细胞的ABO和H表型,利用聚合酶链反应扩增类孟买表型个体的abo基因第6、7外显子和α1,2岩藻糖基转移酶基因(fut1)编码区,并对PCR产物直接测序分析。对纯化的fut1扩增产物进行TOPO克隆和测序,对2处变异位点进行单倍体序列分析。结果表明:血清学分析确认该个体为罕见的类孟买表型;直接测序发现先证者fut1基因第547位和880位附近存在碱基缺失或插入变异;TOPO克隆测序法证实,fut1基因1条单倍体存在第547-552位两碱基AG缺失,另1条单倍体存在880-882位两碱基TT缺失。这2种变异均导致移码突变,并提前形成终止密码。结论:在献血人群中发现1例罕见的类孟买表型,其分子机制为双碱基缺失型fut1等位基因复合杂合所致的α1,2岩藻糖基转移酶活性减弱。     

类孟买血型基因分析及1种FUT1新无效等位基因的发现

目的 分析类孟买血型基因及H抗原缺乏血型的分子机制,了解FUT1与FUT2基因间的连锁遗传关系.方法 用血清学方法鉴定H抗原缺乏的12名献血者及4名患者的类孟买血型,采用直接测序和克隆测序的方法分析类孟买血型个体的FUT1和FUT2基因.结果 在16名类孟买血型个体者中检出3种已知的FUT1无效等位基因(h1,nt547-552△ag;h2,nt880-882△tt;h3,nt658c→t)和1种新的FUT1无效等位基因(h9,nt424c→t).FUT1基因型分别为h1/h19例,h1/h2 4例,h3/h2、h2/h2及h1/h9各1例.在检出的FUT1无效等位基因中,h1和h2等位基因分别为68.75％(23/32)和21.87％ (7/32).所有FUT2等位基因均存在纯合的nt357c→t同义突变,在具有h2等位基因的个体中,都检出nt716g→a突变(Se357.716);未发现FUT2无效等位基因.结论 发现1种引起H抗原缺乏血型的新FUT1无效等位基因;证实h1和h2为福建地区类孟买血型个体的主要流行基因,支持FUT1和FUT2基因存在连锁遗传的观点.     

Identification of two novel FUT1 mutations in people with Bombay phenotype from Iran

Background and purposeBombay and Para-Bombay phenotypes are characterized by FUT1 gene mutation and lack of H antigen expression in red blood cells. ABH antigens are not present in the body secretions of Bombay individuals, while they are expressed in the secretions of para-Bombay. The aim of this study was to investigate the molecular basis of FUT1 and FUT2 genes in Iranians with the Bombay or Para-Bombay phenotype.Materials and MethodsABO phenotype analysis and routine serological tests were performed on 11 people with Bombay and Para-Bombay phenotypes. The coding regions of FUT1 and FUT2 genes were amplified by PCR followed by sequencing. The ABO genotypes were also determined by sequencing exons 6 and 7 of the ABO gene. Results: Serological investigations confirmed the Bombay phenotype in 8 samples and the Para-Bombay phenotype in 3 samples. Family members with the Bombay phenotype had the classic c 0.725 T > G mutation in the FUT1 gene, accompanied by deletion of the FUT2 gene. Other samples had c.653 A>G, c 0.661 C>T, c 0.652 C>G, and c.722 A>C mutations in the FUT1 while FUT2 was silenced by c 0.461 G>A. Conclusion: In this research, we identified two novel mutations in the FUT1 gene in individuals with the Bombay phenotype. This and previous works confirm the variety of FUT1 mutations.     

A Para-Bombay Blood Group Case Associated with a Novel FUT1 Mutation c.361G>A

BackgroundHere we report a case of para-Bombay phenotype due to a novel mutation FUT1 c.361G>A p.(Ala121Thr) and a nonfunctional allele FUT1*01N.13(c.881_882delTT) which showed a discrepancy in the routine ABO blood group typing.Materials and MethodsThe ABO phenotype and the Lewis blood group were typed with serological methods. The ABH antigens in saliva were determined by a hemagglutination inhibition test. The CDS region of ABO, FUT1and FUT2 were amplified with polymerase chain reaction and then directly sequenced. The novel mutation was confirmed by cloning and sequencing. Three-dimensional (3-D) structural analysis of the mutant and wild-type Fut1 were performed by the Chimera software.ResultsA, B and H antigens were not detected on the surface of red blood cells (RBCs) by the serological technique, and the B and H blood group substances were detected in the saliva, while the Lewis phenotype was Le(a–b+). Sequencing and cloning analysis showed the presence of a novel FUT1 mutation c.361G>A and a nonfunctional allele FUT1*01N.13(c.881_882delTT). The ABO genotype was ABO*B.01/ABO*O.01.01. The in silico analysis showed that the mutation p.(Ala121Thr) of FUT1did not change the 3-D structure of the whole enzyme but caused a certain amplitude of turnover in the loop region where Ala121 was located.ConclusionsA novel FUT1 allele (FUT1*c.361G>A) was identified in a Chinese individual with para-Bombay B phenotype. The FUT1c.361G>A mutation may significantly downregulate the expression of H antigens on RBCs by damaging the enzyme conformation.     

中国浙江人群中Lewis血型相关的FUT3基因多态性初步研究

为研究中国浙江人群中Lewis血型相关的FUT3基因多态性,以血清学方法鉴定183例随机样本的Lewis表型,将其中39例Le（a-b-）以及选取的Le（a＋b-）、Le（a-b＋）和Le（a＋b＋）各3例进行FUT3基因全编码区序列分析,并对各种可能的复合杂合基因型进行单倍体序列分析.结果显示,真正的Lewis阴性表型在中国浙江人群中比例约为10.4%,在48例直接测序样本中发现59T＞G、202T＞C、314C＞T、508G＞A和1067T＞A等5个变异位点;单倍体分析显示,除功能性的Le等位基因外,发现5种非功能性的le等位基因,分别为le^59（59T＞G）;le^1067（1067T＞A）;le^59,1067（59T＞G和1067T＞A）;le^59,508（59T＞G和508G＞A）和le^202,314（202T＞C和314C＞T）.结论：在浙江人群中,FUT3非功能性等位基因具有较为广泛的遗传多态性.