Although Sickle Cell Anemia (SCA) is caused by a single nucleotide mutation in the beta globin gene, there is broad phenotypic variability in affected individuals. It would be highly advantageous to be able to predict which SCA patients are most likely to suffer severe complications and which are likely to benefit from specific treatments. Since two of the major pathologic mechanisms in SCA are erythrocyte dehydration and chronic inflammation, alterations in the expression and/or function of proteins affecting these processes may be responsible for heterogeneity in SCA phenotype. We have developed a 23-gene Next-Generation sequencing panel to identify variants in genes involved in erythrocyte hydration and in reactive oxygen species (ROS) generation and reduction in patients with SCA. We have collected blood samples from an initial cohort of 18 SCA patients with severe phenotype, defined by history of stroke or abnormal transcranial Doppler velocities, and 13 SCA patients with mild phenotype for DNA preparation and for specialized testing to evaluate erythrocyte hydration status and ROS generation.

Advia Automated Cell Counter results provided the clearest indications of erythrocyte dehydration in patient blood samples. The ability to examine reticulocyte parameters made it possible to evaluate hydration regardless of the frequency of transfusion. Reticulocyte CHCM, MCV, and percent hyperdense cells were significantly different between the two groups and indicative of a greater degree of dehydration in the severe phenotype group.

Osmoscans (using a LoRRca ektacytometer) demonstrated a highly significant left shift indicative of erythrocyte dehydration in both mild and severe groups relative to controls (O min and O hyper values significantly decreased). Both phenotypic groups also demonstrated a significantly lower EI max in relation to controls, indicating decreased deformability of sickle erythrocytes. Deformability scans revealed significant increases in SS ½ and decreases in EI max in both groups in relation to the controls. There were significant differences between mild and severe phenotype groups in osmoscan O min and O hyper values and in deformability assay SS ½ and EI max values. The severe group had values closer to the normal values, most likely due to the contribution of transfused blood in the severe phenotype group. There were no significant differences between the mild and severe SCA phenotype groups in intracellular cation levels (K+ and Na+) measured by flame emission spectroscopy, although both groups had significantly higher intracellular Na+ compared to normal controls.

Erythrocyte ROS detection was performed using a flow cytometry DCFDA assay. Both mild and severe phenotype groups demonstrated a highly significant increase in ROS compared to controls. However there was not a significant difference in erythrocyte ROS between the mild and severe phenotype groups.

DNA from patient blood samples has been sequenced using the Haloplex target enrichment system and Illumina high-throughput sequencing. We analyzed 23 genes that are likely candidates to be involved in erythrocyte hydration (ABCB6, ABCG5, ABCG8, AQP1, AQP3, ATP2B4, KCNN4, PIEZO1, RHAG, SLC9A1, SLC12A4, SLC12A6, SLC12A7, SLC2A1, SLC4A1, STOM, TRPC6, XK) or in ROS production or reduction (G6PD, NOX1, CYBB, NOX4, NOX5). At least 19 variants classified as possibly damaging have been detected in 10 of the genes. Future study will be directed toward exploring the phenotypic implications of identified variants.

Results of this study should further our understanding of the pathogenesis of SCD and help identify biomarkers of disease severity which may ultimately help guide clinical management for individual patients. They may also suggest novel targets for development of new therapies for SCD based on the modulation of erythrocyte hydration and inflammation.


Quinn:Amgen: Research Funding; Eli Lilly: Research Funding; Silver Lake Research Corporation: Consultancy. Joiner:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Author notes


Asterisk with author names denotes non-ASH members.

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