PS1205 META‐ANALYSIS USING DATA OF RED BLOOD CELL ENZYME ASSAY AND PROTEOME IN PATIENTS WITH DIAMOND‐BLACKFAN ANEMIA
Authors: T. Utsugisawa, T. Uchiyama, T. Aoki, A. Kinoshita, Y. Okamoto, T. Kawakami, H. Ogura, T. Toki, T. Yamamoto, A. Ohara, S. Ohga, E. Ito, H. Kanno
Journal: HemaSphere (2019)

Background: Diamond‐Blackfan anemia (DBA) is a form of congenital bone marrow failure syndrome, which is characterized by RBC aplasia with varied malformation in infants. Elevated enzymatic activity of adenosine deaminase (ADA) is known to be a valuable biomarker of DBA. The ADA assay has been shown to be more sensitive than the gene test of ribosome subunit genes. We recently reported that reduced glutathione (GSH) in red blood cells (RBCs) is another biomarker of DBA, and simultaneous assessment of ADA and GSH enabled us to discriminate DBA patients from normal subjects as well as unaffected family members (Blood Cells Mol Dis 59:31–36, 2016). Aims: This study was performed to investigate the detailed changes in enzymatic activity and enzyme protein mass in DBA erythrocytes. We reanalyzed the enzymatic activity and GSH in 14 DBA patients and compared the results with those obtained from simultaneously assayed 14 normal subjects. Furthermore, we performed a meta‐analysis using proteome data as recently reported by Pesciotta, et al. (PLoS ONE 10:e0140036, 2015). Methods: The gene mutations of the enrolled patients were previously confirmed. We estimated the activity of RBC enzymes and GSH content by a standard method using a spectrophotometer. We calculated the relative expression of both enzymatic activity and protein content. The statistical analyses were performed by Wilcoxon rank‐sum test and a P value of &lt;0.001 was considered statistically significant. Results: The combined analyses of proteomic and enzymatic data revealed that ADA, PGD, and HK‐1 (proteome)/HK (activity) were elevated, while PFKL (proteome)/PFK (activity) and PKLR (proteome)/PK (activity) were decreased. The G6PD enzyme activity, but not its protein level, was characteristically increased. Summary/Conclusion: We speculate that the discrepancy between the activity and protein level of G6PD was attributable to the decreased inhibition of G6PD activity by NADPH. In other words, G6PD activity was supposedly activated by a lower NADPH level in DBA. To maintain the affordable level of GSH in RBCs, reduction of GSSG was necessarily maximized through the reaction catalyzed by glutathione reductase, which used NADPH as a cofactor. As a result, NADPH content was presumably exhausted, resulting in decreased inhibition of G6PD. In this study, we found that HK activity and HK‐1 content were elevated significantly. On the other hand, the activity and protein content of two important rate‐limiting enzymes (PFK/PK and PFKL/PKLR) involved in glycolysis were found to be decreased. Higher demand for NADPH production through the pentose phosphate pathway leads to a reduction in glucose consumption in the glycolytic pathway, and/or activation of p53 leads to inhibition of glycolysis, as previously reported. The present study shows the inhibition of glycolysis in the RBCs in DBA. It needs to be elucidated whether impaired glycolysis partly accounts for premature cell death of erythroblasts in DBA. Abbreviations: DBA: Diamond‐Blackfan anemia; RBC: red blood cell; ADA: adenosine deaminase; GSH: reduced glutathione; PGD: 6‐phosphogluconate dehydrogenase; HK: hexokinase; PFK (L): 6‐phosphofructokinase (liver type); PK (LR): pyruvate kinase (liver and erythrocytes type); G6PD: glucose‐6‐phosphate dehydrogenase; NADPH: reduced nicotinamide adenine dinucleotide phosphate; GSSG: glutathione disulfide.