PS1111 QUANTIFICATION OF METABOLITES IN DRIED BLOOD SPOTS OF DIAMOND BLACKFAN ANEMIA PATIENTS REVEALS NOVEL INSIGHTS IN DEREGULATED CELLULAR PATHWAYS INVOLVED IN HYPOPLASTIC ANEMIA
Authors: van Dooijeweert B. , Broeks M. , Jans J. , van Wijk R. , Bartels M. 
Published: June 2019

Background:

Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder, characterized by severe hypoplastic anemia, associated congenital anomalies, and an increased risk of developing cancer. Based on the underlying mutations in ribosomal protein (RP) genes, DBA is classified as a ribosomopathy. While disruptions in ribosomal biogenesis would be expected to have global effects, hematological in DBA are primarely of the erythroid lineage. Moreover, since cellular determinants following ribosomal stress are still not well understood, and most functional studies are performed in models representing only the most frequently affected RP‐genes, the development of novel therapeutic strategies for all DBA patients has been been hampered. Here we propose, for the first time, a novel untargeted metabolomics approach to study anemia in DBA patient cells. Metabolomics is the large‐scale, unbiased study of metabolites and their interactions within a biological system, and a powerful functional tool, directly reflecting the underlying biochemical activity and the state of cells. The DBA‐metabolome can be used to study commonly deregulated cellular pathways during erythroid development, irrespective of the underlying molecular defect.

Aims:

Defining a metabolic signature of RP‐deficiency in DBA, will 1. Increase our understanding of erythroid‐specific defects in DBA, and 2. Contribute to the identification of potential novel therapeutic targets.

Methods:

Quantification of metabolites was performed on dried blood spots from 6 DBA patients (RPS26, RPL5, RPL9, unknown) by direct infusion high resolution mass spectrometry (DI‐HRMS) followed by an untargeted metabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with dried blood spots samples of 10 healthy controls.

Results:

Preliminary data from 6 DBA patients with different genotypes demonstrate a significant increase in intermediates of the pentose phosphate pathway (PPP) (Figure 1A). This indicates that the PPP is upregulated in red blood cells from DBA patients, regardless of the underlying molecular defect. In line with this we observed a significant increase in erythrocyte reduced glutathione (GSH) content in 9 DBA patients (RPS26, RPL5, RPL11, unknown), compared to 12 controls (Figure 1B). In red blood cells the PPP mainly serves to provide the cell with sufficient amounts of NADPH to maintain high intracellular levels of GSH, which is needed to protect the cell against oxidative damage. Our results therefore support a role for oxidative stress response in DBA pathophysiology irrespective of the underlying genotype.

Figure 1. (A) Increase in metabolites involved in PPP in DBA patient cells. (B) Increased GSH concentration in DBA patient erythrocytes.

Summary/Conclusion:

DBS metabolomics can be used as a novel functional tool to investigate deregulated cellular pathways in DBA irrespective of the underlying molecular defect. This will contribute to increased insights in cell type specific phenomena and DBA pathophysiology in general.

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