阵发性睡眠性血红蛋白尿症患者粒细胞及血浆尿激酶受体的检测

目的了解阵发性睡眠性血红蛋白尿症(PNH)患者体内尿激酶受体(uPAR)的表达水平,并探讨uPAR检测在PNH诊断中的临床意义.方法用流式细胞仪检测20例PNH患者粒细胞表面uPAR、CD55、CD59的表达水平,并与21例正常人、59例其他贫血患者(18例自身免疫溶血性贫血、6例其他溶血性贫血、26例慢性再生障碍性贫血、9例缺铁性贫血)比较;同时采用免疫放射分析(IRMA)测定PNH患者与正常人血浆可溶性uPAR(suPAR)的水平.结果 20例PNH患者中uPAR 、CD55、CD59表达水平显著降低,且与正常人uPAR表达下限不重叠.在PNH患者中还存在峰型异常[双峰和(或)峰拖尾].而57例其他贫血患者中粒细胞表面uPAR的表达水平无改变.PNH患者血浆suPAR水平为(4.04±2.47 )μg/L,明显高于正常人的(1.73±0.96 )μg/L水平(P＜0.01).PNH患者血浆suPAR水平与粒细胞表面uPAR的表达呈负相关(r=-0.79,P＜0.01).结论 PNH患者粒细胞表面uPAR表达水平降低,而血浆suPAR水平增高,这一变化可作为诊断PNH新的特异性指标.     

阵发性睡眠性血红蛋白尿症与再生障碍性贫血患者干细胞因子和FLT3配体及其受体表达的研究

目的:检测干细胞因子(SCF)与FLT3配体(FL)及其受体在阵发性睡眠性血红蛋白尿(PNH)与再生障碍性贫血(AA)的表达情况,探讨2者在PNH与AA发病中作用。方法:用ELISA法检测PNH与AA患者骨髓SCF和可溶性FL的含量,用流式细胞术对AA与PNH骨髓单个核细胞c kit与FLT3的表达进行分析。结果:PNH、慢性AA患者SCF含量分别为(456.8±115.2)、(372.6±111.5),与正常对照组(389.2±123.3)比较差异无统计学意义(P>0.05);PNH、慢性AA患者c kit表达分别为(48.8±15.6)、(39.6±11.5),低于正常对照组(75.2±23.3)。PNH患者FL水平为226.3±50.6,明显高于正常对照组(89.4±20.8),但低于慢性AA(658.2±125.5)(P<0.01);PNH、慢性AA患者FLT3表达分别为(13.2±5.8)、(8.5±2.7),与正常对照相比差异无统计学意义(P>0.05)。结论:干细胞因子、FLT3配体及其受体参与PNH与AA的发病,2者共同的发病机制可能为干细胞因子受体缺陷、免疫紊乱。     

CD59在PNH患者CD+34细胞中的表达及意义

对5例阵发性睡眠性血红蛋白尿(PNH)、15例再生障碍性贫血(AA)及3例正常人的骨髓单核细胞采用直接免疫磁珠正选法富集CD 34细胞,并用CD34-PE标记后流式细胞仪测定其纯度;用FITC标记直接免疫荧光法通过流式细胞仪检测22例PNH、26例AA、5例AA-PNH、20例正常人血细胞和富集的CD 34干细胞中CD 59细胞的百分率.结果:PNH患者外周血功能细胞均有CD59部分或完全缺陷,骨髓单个核细胞中CD34细胞及CD 59细胞所占比例与正常人比较明显降低,骨髓CD 34 CD 59细胞表达率低于外周血粒细胞.认为对骨髓中CD 34细胞CD59表达进行检测,可预测病情变化并早期诊断PNH.     

流式细胞术检测CD55、CD59对阵发性睡眠性血红蛋白尿诊断价值的探讨

选取50例PNH患者,50例AA患者作为对照A组,50例缺铁性贫血(IDA)患者作为对照B组,50例骨髓增生异常综合征(MDS)患者作为对照C组,50例正常人作为对照D组,用流式细胞术检测衰变加速因子(CD55)和反应性溶血膜抑制物(CD59)的表达量。结果:研究组的CD55与CD59的表达缺失率高于对照组,P 0. 05。结论:使用流式细胞术检测CD55和CD59,能够特异且敏感地对PNH诊断,有效区分出PNH与AA、IDA、MDS。     

外周血细胞外囊泡中lncRNA H19对原发性胆汁性胆管炎患者肝纤维化的预测价值分析

目的 探讨原发性胆汁性胆管炎（PBC）患者外周血细胞外囊泡中长链非编码RNA(lncRNA)H19表达水平对肝纤维化的预测价值。方法 选择2021年8月至2022年8月在邢台市第三医院经肝穿刺活体组织病理检查确诊为PBC的84例患者设为PBC组,另选择同期来院的46名健康体检者设为对照组。在超声引导下行经皮肝穿刺获取PBC患者的肝脏病变组织,采用Scheuer评分系统进行肝纤维化分期及肝脏炎症反应分级。提取外周血细胞外囊泡,在透射电镜下观察其形态结构。采用纳米颗粒示踪分析法对外周血细胞外囊泡中颗粒数进行定量分析,蛋白质印迹法测定外周血细胞外囊泡的特异性标志物表达水平,实时荧光定量PCR法测定外周血细胞外囊泡中H19的表达水平。采用ROC曲线下面积（AUC）评估外周血细胞外囊泡中H19表达水平对PBC患者肝纤维化程度的预测价值。结果 PBC组和对照组的外周血细胞外囊泡直径约为100 nm,均有完整膜结构,囊泡表面均可检测出CD63、肿瘤易感基因101蛋白（TSG101）、ALG-2相互作用蛋白X(Alix)的表达,且PBC组的上述蛋白表达水平均显著高于对照组（P均<0.05）。PB...     

流式细胞术检测CD55和CD59在PNH诊断中的应用

目的探讨流式细胞术检测CD55和CD59在阵发性睡眠性血红蛋白尿（paroxysmal nocturnal hemoglobinuria,PNH）诊断中的应用价值。方法采用流式细胞术检测11例PNH患者、15例再生障碍性贫血（AA）患者、14例骨髓增生异常综合征（MDS）患者、20例健康对照者的红细胞和粒细胞膜CD55和CD59的表达。评判标准为红细胞、粒细胞膜CD55^-、CD59^-的百分率大于10%认定有PNH克隆。结果 11例PNH患者的粒细胞CD55^-、红细胞和粒细胞CD59^-表达均大于10%,2例PNH患者的红细胞CD55^-表达小于10%、其他9例大于10%;15例AA患者的红细胞、粒细胞CD55^-表达均小于10%,1例AA患者的红细胞、粒细胞CD59^-表达大于10%、其余14例均小于10%;14例MDS患者的红细胞、粒细胞CD55^-表达均小于10%,1例MDS患者的红细胞、粒细胞CD59^-表达大于10%、其余13例均小于10%。对照组20例红细胞、粒细胞CD55^-和CD59^-表达均小于10%。结论流式细胞术检测CD55和CD59在PNH诊断中有良好的应用价值,建议把流式细胞术检测红细胞和粒细胞膜CD55和CD59的表达作为诊断PNH的常规项目。     

早期胃癌外周血细胞免疫功能状况变化研究

目的检测早期胃癌患者外周血细胞免疫功能变化及意义。方法收集沈阳军区总医院2013年1-12月经手术确诊的胃癌患者58例,其中早期胃癌27例,进展期胃癌31例。采用流式细胞仪方法检测患者外周血辅助性T细胞(CD3+、CD4+、CD8+、CD4+/CD8+)和调节性T细胞(CD4CD25+Treg和CD4+CD25+Foxp3+Treg),并与24例健康志愿者(对照组)相同外周血T细胞亚群表达情况进行比较。结果早期胃癌患者外周血CD3+、CD4+表达浓度及CD4+/CD8+比值显著高于进展期胃癌组,低于健康对照组(P0.05);CD8+浓度则显著低于进展期胃癌组,高于健康对照组(P0.05);CD4CD25+Treg及CD4+CD25+Foxp3+Treg表达浓度均低于进展期胃癌组,高于健康对照组(P0.05)。结论早期胃癌患者外周血细胞免疫功能表达存在差异现象,应进一步研究。     

凋亡抑制基因Bcl-2表达与支气管哮喘关系的研究

目的 支气管哮喘的发病机制非常复杂,至今尚未完全阐明.目前认为细胞凋亡与支气管哮喘发病相关.Bcl-2基因是近年来广泛重视的一种凋亡抑制基因,可抑制多种细胞凋亡,与多种疾病的发生存在密切的关系.本研究测定支气管哮喘患者外周血白细胞Bcl-2基因mRNA的表达量及其血清中蛋白的含量,研究Bcl-2在支气管哮喘中的表达及其相互关系,初步探讨Bcl-2基因在支气管哮喘的发生、发展中的生物学意义. 方法 20例支气管哮喘中重度发作患者为实验组,20例无支气管哮喘、变态反应性疾病的健康人为对照组.采取抗凝静脉血标本5ml,立即用Dextron-PBS分离出血白细胞,采用TristarTM试剂按试剂盒说明提取总RNA,-80°超低温冰箱保存,采用TaKaRa试剂盒检测Bcl-2基因mRNA表达量.两步法第一步用ExScriptTM反转录酶,对Bcl-2mRNA反转录成cDNA;第二步用Premix Ex TaqTM酶对反转录后的cDNA进行扩增,10倍梯度稀释cDNA模板,分别为100、10-1、10-2、10-3、10-4倍梯度.用美国PE公司5700型实时荧光定量PCR仪进行检测,以GAPDH基因为对照基因,用仪器附带SDS软件自动算出Bcl-2基因与对照基因比值,确定Bcl-2基因的相对含量.同时抽取抗凝静脉血2ml,伊红染色,行外周血嗜酸性粒细胞计数.抽取不抗凝静脉血2ml,分离血清,采用澳大利亚Bender Medsystems公司人Bcl-2蛋白检测试剂盒,按试剂盒说明操作,通过酶联免疫吸附法(ELISA)检测Bcl-2蛋白含量.应用SAS 6.12计算机软件对实验结果进行统计学分析. 结果 支气管哮喘患者外周血嗜酸性粒细胞计数较正常对照组比较有非常显著性增加(P＜0.01),20例支气管哮喘患者外周血细胞Bcl-2基因mRNA表达量高于正常对照组,两组有非常显著性差异(P＜0.01),外周血清Bcl-2蛋白含量与正常对照组差异无显著性(P＞0.05). 结论 本研究证实支气管哮喘患者外周血嗜酸性粒细胞显著增多.支气管哮喘患者外周血细胞Bcl-2基因mRNA表达显著增多,揭示Bcl-2基因在支气管哮喘发病中具有一定的作用和意义,Bcl-2基因与支气管哮喘密切相关.而外周血清中Bcl-2蛋白含量与支气管哮喘的发病无直接相关.     

贫血患者外周血CD55-和CD59-细胞检测结果及其意义

目的检测贫血患者外周血中红细胞和中性粒细胞膜糖化磷脂酰肌醇(GPI)连接的补体调节蛋白CD55和CD59表达情况,探讨其临床意义.方法采用荧光素标记的CD55和CD59单克隆抗体,流式细胞术检测35例正常人、5例阵发性睡眠性血红蛋白尿症(PNH)、32例再生障碍性贫血、47例小细胞低色素性贫血、10例巨幼细胞性贫血、12例自身免疫溶血性贫血和15例造血系统肿瘤伴有贫血患者外周血中CD55和CD59-红细胞和中性粒细胞的百分率.结果正常人CD55-、CD59-红细胞和中性粒细胞的百分率均＜5%,PNH患者均＞40%,部分再障患者＞5%(均＜15%).约有50%的小细胞低色素贫血患者CD55-、CD59-红细胞＞5%(均＜15%),但中性粒细胞的结果全部正常.巨幼贫、自身免疫溶血性贫血和造血系统肿瘤伴有贫血患者的阴性红细胞和中性粒细胞比率均正常.结论利用流式细胞术同时检测外周血中CD55和CD59-红细胞和中性粒细胞是目前诊断PNH的最可靠,最敏感的方法,也可作为判断疗效的手段.本法也是临床鉴别诊断PNH与再障和小细胞低色素性贫血的较好方法.     

再生障碍性贫血-阵发性睡眠性血红蛋白尿综合征与典型阵发性睡眠性血红蛋白尿症的临床对照研究

目的　研究再生障碍性贫血 -阵发性睡眠性血红蛋白尿综合征 (AA PNH综合征 )与典型阵发性睡眠性血红蛋白尿症 (PNH)临床特征的异同 ,加深对AA PNH综合征的认识。方法　回顾分析了 2 8例AA PNH综合征和 5 1例典型PNH的临床表现、实验室检查及治疗反应 ,并进行了对照研究。结果　AA PNH综合征与典型PNH相比 :①血栓形成、黄疸、肝脾肿大等临床表现均较轻。②网织红细胞虽较低 ,但仍高于正常 ;骨髓涂片及活检多表现为增生减低 ,但红系比例不低 ;③各溶血指标检查阳性率均较低 ,但CD5 5、CD5 9表达异常的检出率为10 0 % ,且其在红细胞、粒细胞、淋巴细胞中表达的百分率在两组患者中无明显差异。④免疫球蛋白、T细胞亚群的检测 ,两组患者均无异常。⑤两组患者对肾上腺糖皮质激素为主的治疗均反应良好。结论　AA PNH综合征虽临床表现有别于典型PNH ,但与典型PNH无本质区别 ;CD5 5、CD5 9的检测有助于提高AA PNH综合征的检出率     

PIG-A mutations in normal hematopoiesis

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.     

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals

In paroxysmal nocturnal hemoglobinuria (PNH), acquired somatic mutations in the PIG-A gene give rise to clonal populations of red blood cells unable to express proteins linked to the membrane by a glycosylphosphatidylinositol anchor. These proteins include the complement inhibitors CD55 and CD59, and this explains the hypersensitivity to complement of red cells in PNH patients, manifested by intravascular hemolysis. The factors that determine to what extent mutant clones expand have not yet been pinpointed; it has been suggested that existing PNH clones may have a conditional growth advantage depending on some factor (e.g., autoimmune) present in the marrow environment of PNH patients. Using flow cytometric analysis of granulocytes, we now have identified cells that have the PNH phenotype, at an average frequency of 22 per million (range 10-51 per million) in nine normal individuals. These rare cells were collected by flow sorting, and exons 2 and 6 of the PIG-A gene were amplified by nested PCR. We found PIG-A mutations in six cases: four missense, one frameshift, and one nonsense mutation. PNH red blood cells also were identified at a frequency of eight per million. Thus, small clones with PIG-A mutations exist commonly in normal individuals, showing clearly that PIG-A gene mutations are not sufficient for the development of PNH. Because PIG-A encodes an enzyme essential for the expression of a host of surface proteins, the PIG-A gene provides a highly sensitive system for the study of somatic mutations in hematopoietic cells.     

Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors. PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells. Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure. METHODS: We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis. Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed. RESULTS: From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated. The mean mutant frequency (M(f)) of aerolysin-resistant clones was 17.8 +/- 13.8 per 10(6) PBMC, range 5.0-59.6 per 10(6) cells. Clones had a Class A complementation defect and distinct PIG-A mutations. Patients with PNH had elevated aerolysin-resistant M(f) values averaging 19 x 10(-2), a 10,000-fold difference. Two patients with Fanconi anemia and two others with mild aplastic anemia had M(f) values less than 15 x 10(-6), but two with recovering aplastic anemia had M(f) values of 20 x 10(-4), representing an intermediate value between normal persons and PNH patients. CONCLUSION: Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.     

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia

Abstract:  Background:  Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI-APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI-AP deficient blood cells or PNH clone. Objective:  To explore the nature and mutation of a PNH clone in AA. Methods:  We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG-A) was also studied. Results:  Fifty-one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI-AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG-A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. Conclusions:  Our study indicates that PIG-A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.     

Clinical paroxysmal nocturnal hemoglobinuria is the result of expansion of glycosyl-phosphatidyl-inositol-anchored protein-deficient clone in the condition of Deficient Hematopoiesis

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, clonal hematopoietic stem cell disorder in which PIG-A, gene essential for the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor, is somatically mutated. Absence of GPI-linked proteins from the surface of blood cells is characteristic of the PIG-A mutant (PNH) clone and is also accountable fo certain manifestations, such as intravascular hemolysis. It is unclear how the PNH clone expands and comes to dominate hematopoiesis. In this study, CD34+ cells--committed progenitors (colony-forming cells) representing immature hematopoietic stem cells--and reticulocytes representing the differentiated erythroid cells were quantitated in peripheral blood of patients with PNH. Compared with normal controls (n = 29), CD34+ cell levels were significantly lower in PNH patients who did not have preexisting aplastic anemia (AA) (n = 12) (2.47+/-1.23 versus 4.68+/-1.05 x 106/L, mean +/- standard error; P = .022). PNH patients with precedent aplastic anemia (AA+/PNH) showed markedly low CD34+ cell levels compared with normal control subjects (0.6+/-0.29 versus 4.68+/-1.05 x 10(6)/L; P = .0001). In addition, colony-forming cells from PNH patients were significantly decreased compared with those from normal volunteers (erythroid burst-forming units, 2.8+/-1.2 versu 25.6+/-6.2/5 x 10(5) mononuclear cells; P = .0006; and granulocyte/macrophage colony-forming units, 1.2+/-0.5 versus 13.3+/-3.0/ 5 x 10(5) mononuclear cells; P = .0006). These findings occur in both aplastic and hemolytic types of PNH, suggesting hematopoietic failure in PNH. On the contrary, the numbers of reticulocytes and the reticulocyte production index of PNH patients were significantly higher than those of normal persons and comparable to those from patients with autoimmune hemolytic anemia, indicating accelerating erythropoiesis in PNH. The degree of reticulocytosis correlated well with the proportion of CD59- (PNH) reticulocytes. All of the findings suggest that in the condition of deficient hematopoiesis, the PNH clone arising from the mutated hematopoietic stem cell expands and maintains a substantial proportion of the patient's hematopoiesis.     

The distribution of PIG-A gene abnormalities in paroxysmal nocturnal hemoglobinuria granulocytes and cultured erythroblasts

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia that is characterized by a deficiency of glycosylphosphatidylinositol-anchored membrane proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities in various lineages of peripheral blood cells and hematopoietic precursors. The purpose of our study was to clarify the distribution of PIG-A gene abnormalities among various cell lineages during differentiation and maturation in PNH patients.The expression of CD16b or CD59 in peripheral blood granulocytes or cultured erythroblasts from three Japanese PNH patients was analyzed using flow cytometry. PIG-A gene abnormalities in both cell types, including glycophorin A(+) bone marrow erythroblasts, were examined using nucleotide sequence analysis. The expression study of PIG-A genes from each patient was also performed using JY-5 cells.Flow cytometry revealed that the erythroblasts consisted of negative, intermediate, and positive populations in Cases 1 and 3 and negative and intermediate populations in Case 2. The granulocytes consisted of negative and positive populations in all three cases. DNA sequence analysis indicated that all the PNH cases had two or three types of PIG-A gene abnormalities, and that a predominant clone with an abnormal PIG-A gene was different in granulocytes and erythroblasts from Cases 2 and 3. Expression studies showed that all the mutations from the patients were responsible for the null phenotype.PIG-A gene abnormalities result in deficiencies of glycosylphosphatidylinositol-anchored proteins in PNH erythroblasts and granulocytes. The distribution of predominant PNH clones with PIG-A gene abnormalities is often heterogeneous between the cell types, suggesting that a clonal selection of PIG-A gene abnormalities occurs independently among various cell lineages during differentiation and maturation.     

Paroxysmal nocturnal hemoglobinuria: An acquired genetic disease.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by an intravascular hemolytic anemia. Abnormal blood cells lack a series of glycosylphosphatidylinositol (GPI)-anchored proteins. The lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria. In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective. At least four genes are involved in this reaction step, and one of them, an X-linked gene termed PIG-A, is mutated in affected cells. The PIG-A gene is mutated in all patients with PNH reported to date. Here, we review recent advances in the understanding of the molecular pathogenesis of PNH.     

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.     

Frequent detection of T cells with mutations of the hypoxanthine-guanine phosphoribosyl transferase gene in patients with paroxysmal nocturnal hemoglobinuria.

Acquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions have been suggested by the coexistence of multiple clones with different mutations of PIG-A and by the appearance of leukemic clones in patients with PNH. This study sought to test this hypothesis by examining the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations, identified by both resistance to 6-thioguanine (6-TG) and gene analysis. T-cell colonies resistant to 6-TG formed in methylcellulose culture were found in 8 (67%) of 12 PNH patients and 3 (18%) of 17 age-matched healthy volunteers (P <.02, Fisher exact probability test). The incidence of resistant colonies ranged from 40 to 367 (mean 149, x 10(-7)) in the 8 patients and from 1 to 16 (mean 7, x 10(-7)) in the 3 healthy donors. Thus, the HRPT gene mutated more frequently in patients with PNH than in healthy controls (P <.02, Mann-Whitney test). Analysis of bone marrow cells supported these findings. Like the PIG-A mutations in PNH, the HPRT mutations were widely distributed in the coding regions and consisted primarily of base deletions. Unlike PNH cells, 6-TG-resistant cells expressed CD59, indicating that the HPRT mutations did not occur in PNH clones. No correlation was noted between HPRT mutation frequency and content of therapy received by the patients. It is concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.     

Frequent HPRT mutations in paroxysmal nocturnal haemoglobinuria reflect T cell clonal expansion, not genomic instability

Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the Vbeta2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH.