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

作为镰状细胞病(SCD)的根本原因之一的脱氧镰状血红蛋白(Deoxy-HBS)的聚合受多种因素的影响，其中一个关键因素是红细胞中2，3-二磷酸甘油酸酯(2，3-DPG)的浓度。2，3-DPG是血红蛋白氧结合的变构效应，与脱氧血红蛋白的结合亲和力大于与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浓度的增加降低了红细胞内的pH，从而进一步促进了HBS聚合。 2，3-DPG是糖酵解途径的中间底物，也是红细胞产生三磷酸腺苷的唯一来源(Rose和Warms 1966)。丙酮酸激酶(PK)是糖酵解最后一步的关键酶；PK将磷酸烯醇式丙酮酸(PEP)转化为丙酮酸，生成维持红细胞膜完整性所必需的总三磷酸腺苷(ATP)的50%。事实上，由编码红细胞PK的PKLR基因突变引起的PK缺乏(PKD)会导致慢性溶血性贫血(Grace等人，2015)。PK活性降低会导致上游酶底物的积累，包括2，3-DPG。虽然2，3-DPG浓度的增加和血红蛋白氧亲和力的降低对PKD引起的贫血是有益的，但在HBS存在的情况下，2，3-DPG水平的增加和细胞内红细胞pH的降低可能是有害的，因为它有利于脱氧HBS聚合，从而导致血管内镰刀状(Charache等人，1970)。事实上，PK缺乏和镰刀状细胞特征的组合导致急性镰状综合征之前已经在两个病例中被报道过(Alli等人，2008年；Cohen-Solal等人，1998年)。 然而，PKLR突变是罕见的，但红细胞内的PK酶水平形成了一种光谱，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中具有一系列SNP的遗传多样性，包括几个功能丧失的变种，其中一些与显著减少对恶性疟原虫疟疾的攻击有关(Berghout等人，2012年；van Bruggen等人，2015年)。这些观察表明，与HBS类似，疟疾在相同的地理区域导致了对PKLR变异体的积极选择。 本研究旨在确定在我们的镰刀细胞队列中PKLR的遗传多样性，以及PKLR变异是否改变了PK水平以及2，3-DPG和ATP的活性，这是镰刀病理中的关键角色。如果是这样的话，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浓度下降，是SCD的一个有吸引力的治疗靶点。 该议定书于2018年10月11日开始注册，并一直在积极收集研究对象--健康的非裔美国人、SCD(AS)携带者和SCD患者。我们的应计上限是750(总计)，AA、AS和SCD的每个队列中有250个。总共招募了590人，包括241名高级助理、153名高级助理和196名高级助理。随着样本的增加，正在进行DNA提取、基因分型和丙酮酸激酶、2，3-二磷酸甘油酸酯和ATP的检测。

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.