Type II antithrombin deficiency caused by a large in‐frame insertion: structural,functional and pathological relevance

Summary. Background: The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences. Objective: To characterize the first antithrombin deficiency caused by a large in‐frame insertion. Patients/Methods: Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK‐EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin. Results: The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in‐frame insertion of 24 bp in SERPINC1, affecting strand 3 of β‐sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N‐terminal disulphide bonds, and was conformationally sensitive. The variant was non‐inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into β‐sheet A as the fourth strand, and maintained a native RCL. Conclusions: This is the first case of a large in frame‐insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non‐inhibitory variant, which acquires a hyperstable conformation with a native RCL.     

In-frame triplet deletions in RHD alter the D antigen phenotype

BACKGROUND: The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." STUDY DESIGN AND METHODS: Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. RESULTS: Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. CONCLUSION: Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.     

四川部分地区汉族献血者RhD阴性个体RHD基因多态性研究

目的了解四川部分地区汉族献血者中RhD阴性个体的基因多态性。方法用PCR-SSP及基因序列测定技术对经间接抗球蛋白试验(IAT)、吸收放散试验检测RhD均为阴性的标本分型。结果 69例RhD真正阴性的标本中,包括RHD基因完全缺失个体54例、13例携带RHD-CE(2-9)-D等位基因、1例携带RHD-CE(8-9)-D等位基因、1例携带RHD(711delC)等位基因。结论四川部分地区汉族献血者RhD阴性个体分子机制具有丰富的多态性和不同的遗传背景,主要为RHD全缺失为主,其次为RHD-CE(2-9)-D型,并存在其他稀有的基因型。     

Note from the Editor‐in‐Chief

Background: Rapid treatment of status epilepticus (SE) is associated with better outcomes. Diazepam and midazolam are commonly used, but the optimal agent and administration route is unclear. Objectives: The objective was to determine by systematic review if nonintravenous (non‐IV) midazolam is as effective as diazepam, by any route, in terminating SE seizures in children and adults. Time to seizure cessation and respiratory complications was examined. Methods: We performed a search of PubMed, Web of Knowledge, Embase, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, American College of Physicians Journal Club, Cochrane Central Register of Controlled Trials, the Cumulative Index to Nursing and Allied Health Literature, and International Pharmaceutical Abstracts for studies published January 1, 1950, through July 4, 2009. English language quasi‐experimental or randomized controlled trials comparing midazolam and diazepam as first‐line treatment for SE, and meeting the Consolidated Standards of Reporting Trials (CONSORT)‐based quality measures, were eligible. Two reviewers independently screened studies for inclusion and extracted outcomes data. Administration routes were stratified as non‐IV (buccal, intranasal, intramuscular, rectal) or IV. Fixed‐effects models generated pooled statistics. Results: Six studies with 774 subjects were included. For seizure cessation, midazolam, by any route, was superior to diazepam, by any route (relative risk [RR] = 1.52; 95% confidence interval [CI] = 1.27 to 1.82). Non‐IV midazolam is as effective as IV diazepam (RR = 0.79; 95% CI = 0.19 to 3.36), and buccal midazolam is superior to rectal diazepam in achieving seizure control (RR = 1.54; 95% CI = 1.29 to 1.85). Midazolam was administered faster than diazepam (mean difference = 2.46 minutes; 95% CI = 1.52 to 3.39 minutes) and had similar times between drug administration and seizure cessation. Respiratory complications requiring intervention were similar, regardless of administration route (RR = 1.49; 95% CI = 0.25 to 8.72). Conclusions: Non‐IV midazolam, compared to non‐IV or IV diazepam, is safe and effective in treating SE. Comparison to lorazepam, evaluation in adults, and prospective confirmation of safety and efficacy is needed. ACADEMIC EMERGENCY MEDICINE 2010; 17:575–582 © 2010 by the Society for Academic Emergency Medicine     

多重聚合酶链反应分析中国汉族人群RHD基因

目的 分析中国汉族人群RHD基因的构成。方法 采用2套多重PCR－SSCP扩增体系，对450例献血（328例RhD阳性、112例RhD阴性、10例D变异型）的基因组DNA进行RHD特异性外显子3、4、5、6、7、9和内含子4，RHDΨ及C、c等位基因扩增，并与血清学Rh定型结果进行对比。结果 328例RhD阳性献血中，326例具有全部的RHD基因特异性外显子，另有2例分别为D^Va和D^IVb；112例血清学RhD阴性献血中，35例存在全部的RHD基因特异性外显子（31．25％），但该35例样本中未检出RHDΨ基因37bp的插入片段。10例D变异型中3例为D^ⅥⅢ，1例D^Va,1例R0^Har,1例D^DFR;另外4例中，2例缺乏RHD所有特异性外显子，2例扩增出RHD所有特异性外显子。结论 中国汉族人群RHD基因的表达存在多种模式。多重PCR体系分析RHD基因既可诊断个体的RhD表型，又具有确认不完全D以及发现新的D变异型的优势。     

RHD mRNA剪接体与RhD抗原表达的相关性及其在输血及妊娠中的免疫应答研究

目的通过探讨RHD mRNA剪接体多态性与RhD抗原表达的强度在临床输血、妊娠中的异常免疫应答,为RhD血型定型标准与输血原则提供理论依据。方法选择案例1:RhD阳性(血清学凝集强度达4+)的男性患者因为多次输RhD阳性血合并自身免疫性疾病,血浆中产生了抗-D。案例2:RhD弱阳性孕妇怀孕RhD阳性胎儿产生了抗-D。案例3:Rhccdee表型的地贫患儿因长期(8年)输注常规定型为RhD阴性的血液后产生了抗-D。以上3例样本提取全血的mRNA通过反转录成cDNA,使用RT-PCR法通过自主设计的引物进行扩增测序与分析RHD mRNA剪接体的多态性结构。结果所选的3例患者中患者1、患者2均以3种剪接体的多态性表达了RHD基因。患者1的RHD mRNA剪接体形式:剪接体1与RHD基因参比序列全长一致,剪接体2与RHD基因参比序列相比缺失exon-7,并且在exon-3的第426位发生CA突变、exon-5的707位发生AG突变、713位发生TC突变,剪接体3与RHD基因参比序列相比缺失exon-7,8,9;患者2的RHD mRNA剪接体:剪接体1与RHD基因参比序列全长一致,剪接体2与RHD基因参比序列相比缺失exon-8,9,未发现其他核苷酸突变,剪接体3与RHD基因参比序列相比缺失exon-7,8,9;因为患者3是RhD阴性,缺失RHD基因全部外显子,因此未检测到RHD mRNA剪接体的多态性。结论 RhD抗原的表达直接受RH基因调控,RhD mRNA剪接体的多态性决定RhD抗原强度的差异。RhD抗原弱阳性或阴性的受血者在接受RhD阳性血液时也受同种异体免疫产生抗-D的风险。     

多重连接依赖的探针扩增(MLPA)技术在广州地区RhD阴性献血者基因分型研究中的应用

目的 采用新型Rh血型系统的MLPA检测试剂盒,对广州地区收集的200名RhD阴性献血者进行基因分型.方法 采用传统的血型血清学方法,对RhD阴性献血者进行RhD表型鉴定.提取基因组DNA,采用MLPA检测试剂盒对收集的200名RhD阴性献血者进行RHD基因分型.对于不能明确分型的标本,进行RHD基因直接测序分析.结果 在200名RhD阴性献血者中,采用MLPA方法对196名进行了明确的基因分型.其中,127名为RHD基因缺失(占63.5％);仅检测到包含RHD1227A突变的DEL等位基因有41例,其中38例包含杂合等位基因,其余3例包含纯合等位基因;仅检测到RHD (1-2)-CE (3-9)-D (10)等位基因的有23例,其中19例杂合,4例纯合;3例包含2种不同的RHD等位基因(1条为RHD (1-2)-CE (3-9)-D (10),另1条为RHD1227A);其余2例分别包含杂合的DFR-2(RHD-CE(4) -D)及弱D15型(RHD845A)等位基因.在其余4例直接测序分析的标本中,2例包含杂合的RHD711 delC突变;1例1条等位基因为MLPA检测到的包含RHD1227A突变的DEL等位基因,另1条为包含711delC突变的RHD等位基因;其余1例的1条等位基因为MLPA检测到的RHD (1-2)-CE (3-9)-D (10),另1条为包含新突变的RHD等位基因(1154G ＞T,385Gly＞ Val).结论 MLPA检测试剂盒是适用于中国人群的1种快速、有效的高通量基因分型方法,能够对中国人群常见的各种RHD基因型进行明确分型,适用于参比实验室相关疑难标本的基因分型检测.也可应用于中国人群常见的Del表型检测,使Del表型患者不必再输注RhD阴性血液,节约稀有的RhD阴性血液资源.     

两个家庭亲/子代RhD血型差异的RHD基因型分析

目的　为中国汉族人群RhD(- )个体遗传规律和RHD基因结构研究提供资料。方法　采用 3个厂家不同批号的ABO、Rh抗血清和HLA单克隆抗体血清 ,分别对两个家庭的 7位个体进行ABO、Rh血型检定和HLA A、B、DR、DQ抗原配型 ;并对其RhD(+)父母和RhD(- )子女共 6位个体 ,采用PCR 序列特异性引物 (SSP)试剂 ,直接鉴定Rh血型基因。结果　两例表型RhD(- )的子代个体 ,PCR SSP法显示例 1为RHD基因全部缺如 ;例 2为RHD基因部分携带。结论　本研究显示了 2个中国汉族家庭亲 /子代RHD基因遗传的个体情况。