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.

脱氧镰状血红蛋白（deoxy-sickle-hemoglobin，deoxy-HbS）的聚合是镰状细胞病（sickle cell disease，SCD）的根本原因，其聚合受多种因素的影响，其中一个关键因素是红细胞中2，3-二磷酸甘油酸（2，3-diprophosphoglycerate，2，3-DPG）的浓度。2，3-DPG是血红蛋白氧结合的变构效应物，对脱氧血红蛋白的结合亲和力大于对氧合血红蛋白的结合亲和力，因此有利于脱氧HbS的聚合（Eaton和邦恩2017）。此外，增加的2，3-DPG浓度降低红细胞中的细胞内pH，这进一步促进HbS聚合。 2，3-DPG是糖酵解途径中的中间底物，是红细胞中ATP产生的唯一来源（Rose和Warms 1966）。丙酮酸激酶（PK）是糖酵解最后一步的关键酶; PK将磷酸烯醇丙酮酸（PEP）转化为丙酮酸，产生50%的总红细胞三磷酸腺苷（ATP），这是维持红细胞膜完整性所必需的。事实上，由编码红细胞PK的PKLR基因突变引起的PK缺乏症（PKD）可导致慢性溶血性贫血（Grace et al，2015）。PK活性降低导致上游酶底物（包括2，3-DPG）的蓄积。虽然增加的2，3-DPG浓度和血红蛋白氧亲和力的降低在PKD引起的贫血中是有益的，但是在存在HbS的情况下，增加的2，3-DPG水平结合降低的细胞内红细胞pH可能是有害的，因为它有利于脱氧-HbS聚合，从而促进血管内镰状化（Charache等人，1970）。事实上，之前在2例病例中报告了PK缺陷和镰状细胞性状联合导致急性镰状综合征（Alli et al，2008; Cohen-Solal et al，1998）。 然而，PKLR突变是罕见的，但红细胞内PK酶水平形成一个谱，这表明PKLR可能是一个数量性状基因。已在疟疾流行人群中描述了PKLR的遗传多样性（具有一系列SNP，包括几种功能丧失变体），其中一些与恶性疟原虫疟疾发作的显著减少相关（Berghout et al，2012;货车Bruggen et al，2015）。这些观察结果表明，与HbS类似，疟疾导致PKLR变体在相同地理区域中的阳性选择。 本研究旨在确定我们的镰状细胞队列中的PKLR遗传多样性，以及PKLR变体是否改变PK水平以及镰状细胞病理学中的关键参与者2，3-DPG和ATP的活性。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;增加PK-R活性，导致细胞内2，3-DPG浓度降低，为SCD提供了一个有吸引力的治疗靶点。 本方案的招募于2018年10月11日开始，并一直在积极招募受试者-健康的非裔美国人、SCD（AS）携带者和SCD患者。我们的累积上限是750（总数），AA，AS和SCD每个队列250。总共招募了590人，其中AA 241人，AS 153人，SS 196人。随着样本的增加，正在进行DNA提取、基因分型和丙酮酸激酶、2，3-二磷酸甘油酸和ATP测定。