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Phenotypic markers for hereditary spherocytosis
and increased membrane loss, as determined by RBC vesi- cle numbers and a decrease in MPA; and (iii) heterogeneity in the RBC population reflected by differences in RDW and fractionation of RBC using a Percoll gradient.
We conclude that patients with moderate/severe HS have short-lived RBC of lower density and abnormally high intercellular heterogeneity, whereas patients with mild HS have a less pronounced reduction in RBC deformability resulting in the cells living longer and being subject to progressive loss of membrane. RBC from patients with mild HS thus become denser before they are taken up by the spleen.
Genotype to phenotype correlations in hereditary spherocytosis
While previous studies were limited to protein analysis by SDS-PAGE,12 we used next-generation sequencing to establish the cause of HS. This enabled us to define the primary genetic defect unequivocally, in contrast to other conventional techniques such as SDS-PAGE, which may lead to confounding results as it may be influenced by secondary protein defects in HS. Regardless of the under- lying mutation, all patients shared common features such as increases in reticulocyte counts and MCHC, dehydra- tion and increases of RBC density and heterogeneity and an overall reduction in deformability of the RBC due to destabilization of cytoskeletal structures.1,29 We also observed that red cell size, intracellular K+ content and reticulocyte counts did not differ between patients with SLC4A1, ANK1, SPTB and SPTA1 mutations (Figures 1H and 2B, Table 1). Within one group of patients with the same mutated protein we noted marked differences in severity and clinical manifestations of the disease, with the ANK1 group showing the greatest diversity. We also
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noted that patients with SPTA1 mutations had a less severe phenotype (based on hemoglobin level, reticulo- cyte count, RDW, intracellular K+, and EMA staining) compared to the other patients. This is in contrast with the more severe disease phenotype of patients with SPTA1 mutations reported in other studies.30 Similarly, our patients with SPTB mutations presented with nor- mal MCHC values, whereas in other studies MCHC was shown to be elevated in patients carrying SPTB muta- tions.31 Given the relatively small numbers of patients within each group we cannot draw firm conclusions on links between genotype and phenotypic expression.
Red blood cell density as a marker of clinical severity
Decreased hemoglobin, hematocrit and RBC counts associated with increased markers of hemolysis and ery- thropoietic activity were previously reported as markers of HS severity.9 Among the molecular mechanisms defin- ing severity of HS, a major role was assigned to RBC membrane instability and loss of membrane proteins28 (MCHC, EMA test, SDS-PAGE), along with decreased deformability as measured by osmotic gradient ektacy- tometry.5 In our cohort, increased disease severity was associated with membrane instability and extensive mem- brane and band 3 protein loss over a shorter time (Figure 4B, G-I). RBC turnover in patients with severe HS was reflected by a decrease in HbA1c levels, which were high- er than those in patients with mild HS (Figure 4A). The RBC in patients with moderate/severe HS had lower MCHC than those of patients with mild HS (Figures 3A and 4D). The average M fraction density of RBC from patients with moderate/severe HS did not differ from that of cells of healthy controls, whereas the cells from patients with mild HS showed an increase in density (Figure 4E)
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Figure 3. Red blood cell parameters and their relationship to clinical severity in unsplenectomized hereditary spherocytosis patients. (A) Mean corpuscular hemo- globin concentration (MCHC) (g/L), (B) red blood cell distribution width (RDW) (percent coefficient of variation, %CV), (C) reticulocytes (%), (D) intracellular potassium (mmol/L), (E) hypertonic osmolarity at 50% of maximal elongation (Ohyper) (mOsmol/L).
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