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: 10929188
• 负责人: Swee Lay Thein
• 金额: $ 98.68万
• 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/10929188
• 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; Ethnic Origin; Falciparum Malaria; Gene Frequency; Genes; Genetic; Genetic Determinism; Genetic Variation; Genome; Genomic DNA; Genotype; Geographic Locations; Glycolysis; Goals; Hemoglobin; Hemolytic Anemia; Individual; Introns; Malaria; Mutation; National Heart, Lung, and Blood Institute; Oxygen; Pathology; Pathway interactions; Patients; Phase; Phenotype; Phosphoenolpyruvate; Piezo 1 ion channel; Polymers; Population; Production; Proteins; Proteomics; Protocols documentation; Pyruvate; Pyruvate Kinase; Quantitative Reverse Transcriptase PCR; RNA Sequences; Reporting; SLC2A1 gene; Sampling; Severity of illness; Sickle Cell; Sickle Cell Anemia; Sickle Cell Trait; Sickle Hemoglobin; Source; Syndrome; Variant; cohort; diphosphoglycerate; disease phenotype; disorder control; enzyme substrate; genetic variant; loss of function; metabolomics; 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 780 (total) with the goal of completing 250 in each cohort of AA, AS and SCD. An aggregate total of 654 has been recruited, 251 AA, 177 AS and 226 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-HbS）的聚合，镰状细胞病（SCD）的根本原因受到几个因素的影响，其中一个关键因素是红色中的2,3-二磷酸甘油（2,3-DPG）浓度血细胞。 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高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 Bunn 2017)。此外，2,3-DPG 浓度的增加会降低红细胞内的 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，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，产生红细胞三磷酸腺苷 (ATP) 总量的 50%，这对于维持红细胞膜的完整性至关重要。事实上，由编码红细胞 PK 的 PKLR 基因突变引起的 PK 缺陷 (PKD) 会导致慢性溶血性贫血 (Grace et al, 2015)。 PK 活性降低会导致上游酶底物（包括 2,3-DPG）的积累。虽然增加 2,3-DPG 浓度和降低血红蛋白氧亲和力对于 PKD 引起的贫血有益，但增加 2,3-DPG 水平与降低细胞内红细胞 pH 值在 HbS 存在的情况下可能是有害的，因为它有利于脱氧 - HbS 聚合，从而导致血管内镰状化（Charache 等，1970）。事实上，之前已报道过两例 PK 缺陷和镰状细胞性状联合导致急性镰状综合征的病例（Alli 等，2008；Cohen-Solal 等，1998）。 然而，PKLR 突变很少见，但红细胞内 PK 酶水平形成的谱表明 PKLR 可能是数量性状基因。 PKLR 的遗传多样性具有一系列 SNP，包括几种功能丧失变异，已在疟疾流行人群中得到描述，其中一些变异与恶性疟原虫疟疾发作的显着减少有关（Berghout 等，2012）范布鲁根等人，2015）。这些观察结果表明，与 HbS 类似，疟疾导致同一地理区域中 PKLR 变异体的积极选择。 本研究旨在确定我们的镰状细胞群体中的 PKLR 遗传多样性，以及 PKLR 变异是否会改变 PK 水平以及 2,3-DPG 和 ATP（镰状细胞病理学中的关键参与者）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。 PK-R 活性的增加会导致细胞内 2,3-DPG 浓度降低，这为 SCD 提供了一个有吸引力的治疗靶点。 该方案的招募于 2018 年 10 月 11 日开始，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。我们的应计上限为 780 分（总计），目标是在 AA、AS 和 SCD 每组中完成 250 分。总共招募了 654 名士兵，其中 251 名 AA、177 名 AS 和 226 名 SS。随着样本的积累，正在进行 DNA 提取、基因分型以及丙酮酸激酶、2,3-二磷酸甘油酸和 ATP 的检测。