目标序列捕获测序检出红细胞丙酮酸激酶缺乏症PKLR基因新的复合突变

目的:探讨红细胞丙酮酸激酶缺乏症(pyruvate kinase deficiency,PKD)的分子发病机制。方法:应用目标序列捕获和高通量二代测序技术(next-generation sequencing,NGS)对临床拟诊PKD患儿的PKLR基因12个外显子及其侧翼序列进行测序,采用SIFT及PolyPhen-2数据库预测突变对蛋白质功能的影响,在确定患者致病基因型后,应用Sanger测序技术对此基因型进行验证。结果:NGS结果显示,患儿PKLR基因存在罕见的双重杂合突变:第5外显子661GA(Asp221Asn)和第10外显子1528CT(Arg510Ter),导致该基因的第221位氨基酸由天冬氨酸突变为天冬酰胺,第510位氨基酸由精氨酸突变为终止密码子,使PKLR基因氨基酸链编码提前终止;Sanger测序技术进一步验证了该双重突变的存在。检索相关文献及数据库显示,这两种突变在人群中的发生率极低,蛋白质功能预测均显示为有害。结论:PKLR基因661 GA与1528 CT双重杂合突变是该PKD患儿的分子发病机制,PKD患者同时存在上述复合突变的报道在国内外尚属首次。     

红细胞丙酮酸激酶缺乏症发病的分子机理

丙酮酸激酶(Pyruvate Kinase,PK)有四种同工酶:L、R、M_1和M_2。在成熟红细胞中以R型为主,由PKLR基因编码,现已分离并确定了人类L及R型cDNA全长核苷酸序列。到目前为止,已发现基因突变有35种,其中以点突变最常见。基因突变和PK缺乏之间的因果关系尚未肯定。随着对PK基因的全面了解和分子生物学技术的广泛应用,给本病的诊断,特别是产前诊断带来很大帮助。     

遗传性红细胞酶病的研究进展

已发现遗传性溶血性贫血至少可由19种红细胞酶缺乏和1种酶活性过高引起,发病率居前几位的遗传性红细胞酶缺乏症为葡萄糖6磷酸脱氢酶(G6PD)、丙酮酸激酶(PK)、嘧啶5′核苷酸酶(P5′N)和葡萄糖6磷酸异构酶(GPI)     

PK-LR基因新突变丙酮酸激酶缺乏症患儿经异基因造血干细胞移植治愈

目的 通过对2例丙酮酸激酶缺乏症(PKD)患儿临床症状及PK-LR基因新突变类型的报道,探讨PKD的PK-LR基因诊断方法及行异基因造血干细胞移植(allo-HSCT)的疗效.方法 选择2009年2月及2013年8月,上海交通大学医学院附属上海儿童医学中心收治的2例PKD患儿作为研究对象.本研究遵循的程序符合上海交通大学医学院附属上海儿童医学中心人体试验委员会所制定的伦理学标准,得到该委员会批准,征得受试对象监护人的知情同意,并与之签署临床研究知情同意书.通过上海儿童医学中心检验科采集2例患儿的血液标本,采用Sanger基因测序及全外显子捕获测序对2例患儿PK-LR基因进行检测分析.采用allo-HSCT对2例患儿进行治疗,并分别对2例患儿进行随访.回顾性分析该2例患儿的临床症状、诊断方法及治疗方案.结果 ①2例PKD患儿丙酮酸激酶(PK)活性均降低,分别为8.00 IU/gHb与7.61 IU/gHb.②2例PKD患儿PK-LR基因检测分析共发现4种PK LR基因错义突变.其中,PK-LR基因c.941T＞C(p.Ile314Thr)突变已有文献报道,c.119G＞A(p.Arg40Gln),c.1015G＞A(p.Asp339Asn)及c.848T＞C(p.Va1283Ala)突变为PK-LR基因新的突变类型.4种突变的SIFT功能预测结果分别为0.20,0.37,0及0.23.③2例PKD患儿均接受allo-HSCT.患儿1于移植后第14天粒系及红系造血功能获得恢复,移植后第19天血小板(PLT)计数＞5×101 0/L,移植后第22天造血干细胞(HSC) 100％嵌合,患儿血型亦转换为供者血型.患儿2于移植后第12天粒系及红系造血功能获得恢复,移植后第19天PLT计数＞3×1010/L,移植后第18天HSC 100％嵌合,患儿血型亦转换为供者血型.④对2例PKD患儿分别随访5年和1年,患儿生存状况良好,造血系统及其他系统指标均为正常,获得成功治愈.结论 全外显子捕获基因测序方法可用于临床PKD基因诊断及PK-LR基因新型突变类型的发现;PK-LR基因新突变类型c.G119G＞A(p.Arg40Gln),c.1015G＞A(p.Asp339Asn)及c.848T＞C(p.Va1283Ala)亦可导致PKD的发生;allo-HSCT治疗本研究中2例PKD患儿是有效、可行的.     

红细胞丙酮酸激酶缺陷的研究现况

丙酮酸激酶(Pyrurate Kinase,PK)是糖酵解途径中3个关链酶之一,此酶遗传性缺陷可引起遗传性非球形红细胞性溶血性贫血(Hereditary Nonspherecytic Hemolytic Anemia,HNSHA),它是糖酵解8种红细胞酶病中发现最早、最常见而研究最深入的酶病,本文就其研究现况作一综述。     

丙酮酸激酶缺陷症

丙酮酸激酶(PK)缺陷症是1961年Valentine等对7例先天性非球形细胞性溶血性贫血(CNHA)Ⅱ型患者作红细胞糖酵解诸酶的分析时发现的,它是一种常染色体隐性遗传病。过去认为罕见,随着酶分析技术的改进和发展,其检出率逐渐增高。至今,世界上已报道300多例,以北欧多见,分布较广,发生率仅次于葡萄糖6-磷酸脱氢酶缺陷,而在糖酵解通路中为最常见的一种遗传性红细胞酶病。丙酮酸激酶 PK为红细胞糖酵解通路末期的一个酶,催化如下反应: PK 磷酸烯醇式丙酮酸+ADP-→丙酮酸+ATP     

原发性和继发性丙酮酸激酶缺乏的先天性非球形红细胞溶血性贫血的变异型:Ⅰ.红细胞动力学类型

作者对先天性非球形红细胞溶血性贫血(CNSHA)的两种变异性——原发性丙酮酸激酶缺乏(PPKD)和继发性丙酮酸激酶缺乏(SDPKA),原发性红细胞ATP酶(Mg~(++))活力缺乏引起红细胞ATP水平增高和继发性丙酮酸激酶活力降低,进行了红细胞动力学类型的对比研究。作者所研究的4例CNSHA患者中,男2(L.W.和K.W.),女2(T.S.和O.M.),年龄19～65岁,其中1例(K.W.)为PPKD,另3例(O.M.T.S.和L.W.)为SDPKA。所有4例患者的无肝胆疾患,红细胞渗透脆性,红细胞乙酰胆硷酯酶活力正常。     

丙酮酸缺陷症研究现状

红细胞丙酮酸激酶（ｐｙｒｕｖａｔｅｋｉｎａｓｅ,ＰＫ．ＥＣ２．７．１．４０）是糖酵解途径中的３个关键酶之一。红细胞ＰＫ的遗传性缺陷可引起遗传性非球形红细胞溶血性贫血（ｈｅｒｅｄｉｔａｒｙｎｏｎｓｐｈｅｒｏｅｙｔｉｃｈｅｍｏｌｙｔｉｃａｎｅｍｉａ,...     

常染色体显性遗传性多囊肾的诊断与外科治疗

多囊肾病(polycystic kidney disease,PKD)指双侧肾脏发生多个囊肿且进行性增大而导致肾脏结构和功能损害的一种最常见的常染色体遗传性疾病.     

小儿异基因造血干细胞移植围手术期的护理

丙酮酸激酶缺乏症( pyruvate PK)指丙酮酸激酶基因缺陷,导致丙酮酸激酶活性降低或性质改变而引起的溶血性贫血,是仅次于葡萄糖-6-磷酸脱氢酶缺乏症的红细胞酶病.PK的临床表现多,表现为慢性溶血性贫血,极个别病患者由于溶血被完全代偿而不出现贫血,只有黄疸为唯一的临床表现.治疗方式包括对症支持治疗;药物治疗;手术治疗:脾切除有一定疗效;新生儿黄疸可用光疗,必要时输血治疗;根据情况可采用造血干细胞移植治疗.基因造血干细胞移植( all0-HsCT)是治疗骨髓增生异常综合征( MDS)的有效手段,亦为可能治愈PK的唯一方法[1].本文回顾性分析1例异基因造血干细胞移植治疗丙酮酸激酶缺乏症的临床资料,进一步探讨PK的治疗效果及护理.     

Allogeneic hematopoietic stem cell transplantation in a 3-year-old boy with congenital pyruvate kinase deficiency: A case report

BACKGROUNDThe understanding regarding genetic variation, pathophysiology, and complications associated with pyruvate kinase deficiency (PKD) in red blood cells has been explained largely, and supportive treatment is currently the main management strategy. Etiotropic managements, including transplantation and genome editing, supplying for substitute dugs of the pyruvate kinase, are all under research.CASE SUMMARYWe herein report a 3-year-old boy with severe transfusion-dependent PKD cured by unrelated identical peripheral blood stem cell transplantation (PBSCT). Hemoglobin was corrected to a normal level by gene correction after PBSCT, with no complication related to the transplantation.CONCLUSIONHematopoietic stem cell transplantation could be a substitute for transfusion-dependent PKD.     

Rescue of Pyruvate Kinase Deficiency in Mice by Gene Therapy Using the Human Isoenzyme

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is a disorder caused by mutations in the PKLR gene that produces chronic nonspherocytic hemolytic anemia. Besides periodic blood transfusion and splenectomy, severe cases require bone marrow (BM) transplant, which makes this disease a good candidate for gene therapy. Here, the normal human R-type pyruvate kinase (hRPK) complementary (cDNA) was expressed in hematopoietic stem cells (HSCs) derived from pklr deficient mice, using a retroviral vector system. These mice show a similar red blood cell phenotype to that observed in human PKD. Transduced HSCs were transplanted into myeloablated adult PKD mice or in utero injected into nonconditioned PKD fetuses. In the myeloablated recipients, the hematological manifestations of PKD were completely resolved and normal percentages of late erythroid progenitors, reticulocyte and erythrocyte counts, hemoglobin levels and erythrocyte biochemistry were restored. Corrected cells preserved their rescuing capacity after secondary and tertiary transplant. When corrected cells were in utero transplanted, partial correction of the erythrocyte disease was obtained, although a very low number of corrected cells became engrafted, suggesting a different efficiency of cell therapy applied in utero. Our data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.     

Six novel variants in the PKLR gene associated with pyruvate kinase deficiency in Argentinian patients

BackgroundPyruvate kinase deficiency (PKD) is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. The disease shows a marked variability in clinical expression. We studied the molecular features of nine unrelated Argentinian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase deficiency. Design and Methods: Routine hematologic investigations were performed to rule out other causes of chronic hemolytic anemia. Sanger sequencing and in-sílico analysis were carried out to identify and characterize the genetics variants. Results: Six different novel missense variants were detected among the 18 studied alleles: c.661 G > C (Asp221His), c.956 G > T (Gly319Val), c.1595 G > C (Arg532Pro), c.347 G > A (Arg116Gln), c.1232 G > T (Gly411Val), c.1021G > A (Gly341Ser). Structural implications of amino-acid substitutions were correlated with the clinical phenotypes seen in the probands. Conclusions: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency in Argentina and the second from South America that would contribute to our knowledge on the distribution and frequency of PKLR variants in our population but also offer new insights into the interpretation of the effect of PKLR variants and phenotype.     

PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis

Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.     

靶向序列捕获联合高通量测序鉴定成人型多囊肾PKD1基因新突变

目的 探讨一个成人型多囊肾蛋白激酶D1(PKD1)基因新剪切位点突变及临床表型.方法 收集一个成人型多囊肾病家系临床资料,对家系内成员采用靶向序列捕获对PKD1基因行高通量测序,并结合文献加以分析.结果 该家系中先证者存在成人型多囊肾PKD1基因的c.12138+1G>A(IVS44)突变,而在家系内父亲因多囊肾引发尿...     

丙酮酸脱氢酶激酶4在膀胱癌化疗耐药中的作用

目的探讨丙酮酸脱氢酶激酶4(PKD4)在膀胱癌化疗耐药中的作用。方法通过实时荧光定量PCR(qRT-PCR)检测膀胱癌耐药细胞253J/DOX细胞株及人膀胱癌253J细胞系中PKD4的表达差异;通过siRNA转染降低253J/DOX细胞中PKD4的表达,噻唑蓝(MTT)法检测细胞半抑制浓度(IC50)的变化,流式细胞仪检测细胞凋亡率的变化,蛋白质印迹法(Western blot)检测细胞抗凋亡蛋白(Bcl-2蛋白)表达的变化情况。结果 qRT-PCR结果显示,PKD4在253J/DOX膀胱癌耐药细胞表达明显高于人膀胱癌253J细胞系,差异有统计学意义(P<0.05);MTT结果显示,相对于阴性对照组,干扰PKD4的253J/DOX细胞株的IC50明显降低,差异有统计学意义(P<0.05);凋亡检测结果显示,在相同剂量化疗药物作用下,干扰PKD4后253J/DOX细胞凋亡率明显高于阴性对照组,差异有统计学意义(P<0.05);Western blot结果显示,干扰PKD4表达可明显降低Bcl-2蛋白表达,差异有统计学意义(P<0.05)。结论 PKD4在膀胱癌化疗耐药细胞表达中明显升高,干扰PKD4表达可明显影响膀胱癌耐药,可考虑将PKD4作为治疗膀胱癌化疗耐药的重要分子靶标。     

Molecular study of pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia.

DNA analysis was performed on 30 unrelated patients with hereditary nonspherocytic hemolytic anemia (HNSHA) who had been found to be pyruvate kinase (PK) deficient by enzyme assay. 19 different mutations were identified among 58 of the 60 alleles at risk. 13 of these were missense mutations that caused single amino acid changes. Included were the following nucleotide substitutions: 401A, 464C, 993A, 1022C, 1076A, 1178G, 1179A, 1373A, 1378A, 1456T, 1484T, 1493A, 1529A. The remaining six mutations were as follows: two nonsense mutations, 721T and 808T; a nucleotide deletion, 307C; a nucleotide insertion, 1089GG; a three nucleotide in frame deletion, 391-392-393 and a deletion of 1149 bp from the PKLR gene that resulted in the loss of exon 11. All the patients were studied for two polymorphic sites, nucleotide (nt) 1705 A/C and a microsatellite in intron 11, to better understand the origin of the mutations. The 1529A mutation, which is the most common mutation in the European population, was found in 25 alleles. With a single exception this mutation was in linkage disequilibrium with both of the polymorphic markers, i.e., found with 1705C and 14 repeats in the microsatellite. This finding is consistent with a single origin of this common mutation. Other mutations occurring more than once were of much lower frequency than the 1529A mutation.     

Novel PKLR missense mutation (A300P) causing pyruvate kinase deficiency in an Omani Kindred—PK deficiency masquerading as congenital dyserythropoietic anemia

We report herein a child with transfusion‐dependent chronic anemia, the cause of which was difficult to establish because of his transfusion dependency. The clinical and laboratory features suggested a chronic nonspherocytic hemolytic anemia (CNSHA) with bone marrow features suggestive of congenital dyserythropoietic anemia (CDA). DNA studies, however, revealed the underlying condition to be due to a novel mutation in the PKLR gene responsible for pyruvate kinase deficiency (PKD). Molecular investigations by a targeted next‐generation sequencing (t‐NGS) using a custom panel of 71 genes involved in the red blood cell (RBC) disorders revealed that the patient was homozygous for a novel missense mutation c.898G>C, p.Ala300Pro, whereas both his parents were heterozygous for the same mutation.     

ADAPTATIONS OF ENERGY METABOLISM IN THE CULTIVATED MACROPHAGE

Adaptive changes in energy metabolism, as reflected by pyruvate kinase and cytochrome oxidase activities, were examined during in vitro differentiation of the cultivated macrophage. Serum concentrations of tissue culture media, which directly influence endocytic activity, and ambient oxygen tension were both shown to influence pyruvate kinase and cytochrome oxidase activities. Cells maintained in high serum concentrations (30% newborn calf serum [NBCS]) exhibited a 300–400% increase in pyruvate kinase activity and a 40% increase in cytochrome oxidase activity, whereas cells maintained in low serum concentrations (2% NBCS) exhibited a lesser increase (65%) in pyruvate kinase activity and no change in cytochrome oxidase activity. Anaerobiosis resulted in additional alterations in pyruvate kinase and cytochrome oxidase activities. Cells maintained for 48–72 h under anaerobic conditions exhibited a 500–600% increase in pyruvate kinase activity and a 40% decrease in cytochrome oxidase activity. Increased pyruvate kinase activity was dependent on continued protein synthesis. Enzyme increases occurred in anaerobically cultured cells despite an overall reduction in cell protein synthesis. It is suggested that adaptive changes in pyruvate kinase and cytochrome oxidase activity resulting from alterations in either serum concentration or ambient oxygen tension are regulated by two independent mechanisms. One mechanism is aimed at providing energy for endocytic activity and the other in compensating for impaired oxidative metabolism during anaerobiosis.