Genotype -Phenotype Correlation of PKLR Variants with Pyruvate Kinase, 2,3-Diphosphglycerate and ATP Activities in Red Blood Cells of Patients with Sickle Cell Disease

镰状细胞病患者红细胞中 PKLR 变异体与丙酮酸激酶、2,3-二磷酸甘油酸和 ATP 活性的基因型-表型相关性

• 批准号: 10699742
• 负责人: Swee Lay Thein
• 金额: $ 79.04万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10699742
• 关键词: Acute; Adenosine Triphosphate; Affinity; African; African American population; Anemia; Binding; Biochemical; Biological Assay; Blood specimen; Cell membrane; Chronic; Clinical; Clinical Research; DNA; Deoxygenated Sickle Hemoglobin; Enzymes; Erythrocytes; Falciparum Malaria; Gene Frequency; Genes; Genetic Determinism; Genetic Variation; Genome; Genomic DNA; Genotype; Geographic Locations; Glycolysis; Hemoglobin; Hemolytic Anemia; Individual; Introns; Malaria; Mutation; National Heart, Lung, and Blood Institute; Oxygen; Pathology; Pathway interactions; Patients; Phase; Phenotype; Phosphoenolpyruvate; Plant Roots; Population; Production; Proteins; Protocols documentation; Pyruvate; Pyruvate Kinase; RNA Sequences; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Severity of illness; Sickle Cell; Sickle Cell Anemia; Sickle Cell Trait; Sickle Hemoglobin; Source; Syndrome; Variant; base; cohort; diphosphoglycerate; disease phenotype; disorder control; enzyme substrate; genetic variant; loss of function; orlistat; polymerization; pyruvate kinase deficiency; recruit; response; sickling; therapeutic target; trait; transcriptome; transcriptome sequencing

Polymerization of deoxy-sickle-hemoglobin (deoxy-HbS), the root cause of sickle cell disease (SCD) is influenced by a few factors, a key factor is 2,3-diphosphoglycerate (2,3-DPG) concentration in the red blood cells. 2,3-DPG is an allosteric effector on hemoglobin oxygen binding with a greater binding affinity to deoxygenated hemoglobin than to oxygenated hemoglobin, thus favoring polymerization of deoxy-HbS (Eaton and Bunn 2017). In addition, increased 2,3-DPG concentration decreases intracellular pH in red blood cells which further promotes HbS polymerization. 2,3-DPG is an intermediate substrate in the glycolytic pathway, the only source of ATP production in red blood cells (Rose and Warms 1966). Pyruvate kinase (PK) is a key enzyme in the final step of glycolysis; PK converts phosphoenolpyruvate (PEP) to pyruvate, creating 50% of the total red cell adenosine triphosphate (ATP) that is essential for maintaining integrity of the red cell membrane. Indeed, PK deficiency (PKD) caused by mutations in the PKLR gene that encodes red cell PK, leads to chronic hemolytic anemia (Grace et al, 2015). Reduced PK activity leads to accumulation of the upstream enzyme substrates, including 2,3-DPG. While increased 2,3-DPG concentration and reduction of hemoglobin oxygen affinity is beneficial in anemia caused by PKD, increased 2,3-DPG levels combined with decreased intracellular red cell pH can be detrimental in the presence of HbS, as it favors deoxy-HbS polymerisation, and thereby intravascular sickling (Charache et al, 1970). Indeed, the combination of PK deficiency and sickle cell trait causing an acute sickling syndrome has been previously reported in two cases (Alli et al, 2008; Cohen-Solal et al, 1998). PKLR mutations, however, are rare but intraerythrocytic PK enzyme levels form a spectrum which suggest that PKLR is likely to be a quantitative trait gene. A genetic diversity in PKLR with a range of SNPs, including several loss-of-function variants have been described in malaria-endemic populations, some of which have been associated with a significant reduction in attacks with Plasmodium falciparum malaria (Berghout et al, 2012; van Bruggen et al, 2015). These observations suggest that similar to HbS, malaria has led to positive selection of PKLR variants in the same geographic regions. This study seeks to determine the PKLR genetic diversity in our sickle cell cohort, and whether PKLR variants modify PK levels, and activities of 2,3-DPG and ATP, key players in the sickle pathology. If so, PKLR could be another genetic determinant of SCD severity and phenotype; and increasing PK-R activity, which leads to a decrease in intracellular 2,3-DPG concentration, presents an attractive therapeutic target for SCD. Enrolment for this protocol started on 11 Oct 2018, and has been actively accruing subjects - healthy African-Americans, Individuals who are carriers for SCD (AS) and individuals with SCD. Our accrual ceiling is 750 (total) with 250 in each cohort of AA, AS and SCD. An aggregate total of 590 has been recruited, 241 AA, 153 AS and 196 SS. DNA extraction, genotyping and assays for pyruvate kinase, 2,3-diphosphoglycerate and ATP are being performed as samples accrue.

脱氧 - 丝状血红蛋白（脱氧-HB）的聚合是镰状细胞疾病（SCD）的根本原因受到一些因素的影响，关键因素是红细胞中2,3-二磷酸甘油酸酯（2,3-DPG）浓度。 2,3-DPG是对血红蛋白氧结合的变构效应子，其与脱氧血红蛋白具有更大的结合亲和力，而不是对含氧血红蛋白的结合，因此有利于脱氧-HB的聚合（Eaton and Bunn和Bunn 2017）。此外，升高的2,3-DPG浓度可降低红细胞中细胞内pH值，从而进一步促进HBS聚合。 2,3-DPG是糖酵解途径中的中间底物，糖酵解途径是红细胞中ATP产生的唯一来源（Rose and Warms 1966）。丙酮酸激酶（PK）是糖酵解的最后一步的关键酶。 PK将磷酸烯醇丙酮酸（PEP）转化为丙酮酸，从而创造了三磷酸总红细胞腺苷的50％，这对于维持红细胞膜的完整性至关重要。实际上，由编码红细胞PK的PKLR基因突变引起的PK缺乏症（PKD）导致慢性溶血性贫血（Grace等，2015）。降低的PK活性导致上游酶底物的积累，包括2,3-DPG。虽然增加了2,3-DPG的浓度和血红蛋白氧亲和力的降低对PKD引起的贫血有益，但在HBS存在下，增加了2,3-DPG水平与降低的细胞内红色细胞pH值可能有害于HBS，因为它有利于Deoxy-Hbs HBS聚合，并因此有弹性化的Charcarecular Sickance（1970）。实际上，PK缺乏症和镰状细胞性状的结合以前在两种情况下已报道（Alli等，2008； Cohen-Solal等，1998）。 然而，PKLR突变很少见，但是肠内PK酶水平构成了A频谱，这表明PKLR可能是定量性状基因。 PKLR中具有一系列SNP的遗传多样性，包括疟疾流行种群中描述了几种功能丧失变体，其中一些与恶性疟原虫疟疾疟原虫的攻击显着降低有关（Berghout等人，2012; Van Bruggen等人，2015年）。这些观察结果表明，与HBS相似，疟疾导致了同一地理区域中PKLR变体的积极选择。 这项研究旨在确定我们镰状细胞队列中的PKLR遗传多样性，以及PKLR变体是否修改PK水平以及2,3-DPG和ATP的活动，这是镰状病理学的主要参与者。如果是这样，PKLR可能是SCD严重程度和表型的另一个遗传决定因素。 PK-R活性的增加，导致细胞内2,3-DPG浓度的降低，这是SCD的有吸引力的治疗靶标。 该协议的入学率始于2018年10月11日，一直积极地累积受试者 - 健康的非裔美国人，是SCD（AS）的载体和SCD的个人。我们的应计天花板为750（总计）为250个AA，AS和SCD。总共招募了590个总计，241 AA，153 AS和196 Ss。丙酮酸激酶，2,3-二磷酸甘油酸和ATP的DNA提取，基因分型和测定正在作为样品进行。