Insights from Comparative Serum Proteomic Profiling of Children with Sickle Cell Disease: The Effect of Hydroxyurea and Genotype on Protein Abundance

Introduction: Sickle cell disease (SCD) is a multisystem disease, with substantial variation in presentation and clinical course. Many protein biomarkers have been described in serum and plasma of children with homozygous sickle cell disease. In order to understand the effect of hydroxyurea and the hemoglobin SC genotype on serum protein levels, we compared the relative abundance of serum proteins in healthy children and children with SCD.

Methods: The relative concentration of 140 different serum proteins was measured using liquid chromatography tandem mass spectrometry. Serum samples of healthy children and of children with SCD were collected at a baseline visit: 30 healthy African American children without sickle cell trait, 30 children with hemoglobin SS genotype, 30 children with hemoglobin SC (HbSC) genotype, and 30 children with hemoglobin SS (HbSS) genotype while adherent to hydroxyurea for at least one year. All groups were matched for age and gender. Type of SCD, hydroxyurea dose, laboratory values prior to starting hydroxyurea and at the time of sample collection were recorded. Disease and control samples were processed in a random block design stratified by disease, sex, and age to minimize the effect of sample preparation and technician processing. Samples were depleted of albumin and immunoglobulins. Tryptic peptides were analyzed on a linear ion-trap (LTQ) mass spectrometer. The acquired data was searched against the Human UniProt database using X!Tandem. Peptide identification and protein assignment were performed using the PeptideProphet and ProteinProphet in the Trans-Proteomic Pipeline. Alignment and quantification were done as described (Lai, X. et. al. 2011). Peptide-level quantitative information was analyzed for each protein using a mixed effects model fit with restricted maximum likelihood estimation to test the differential abundance of each protein. Within each comparison, the false discovery rate was controlled at 5% using the method of Benjamini and Hochberg.

Results: Seventy-one serum proteins were significantly different between the children with HbSS disease and the healthy children. In contrast, between children with HbSS disease treated with hydroxyurea and healthy children, only 56 serum proteins were significantly different. Furthermore, in children with HbSC disease, only 25 serum proteins were significantly different compared to healthy children. In assessing the effect of hydroxyurea treatment on serum protein abundance, we found a total of 50 proteins that were significantly different between the 30 children with HbSS disease not prescribed hydroxyurea and the 30 children who were adherent to hydroxyurea (average 24 mg/kg/day) for at least one year (average 3.3 years). We also found a total of 41 proteins that were significantly different in comparing the 30 children with HbSS disease and the 30 children with HbSC disease.

Conclusion: Due to the multisystem nature of sickle cell disease, proteins from many pathways are dysregulated, including inflammatory proteins, hemolysis-associated proteins, proteins involved in coagulation and complement regulation and apolipoproteins. Our findings indicate that the increase of HbF might not be the only beneficial effect of hydroxyurea treatment and that some of the clinical improvement might be due to decreased serum protein dysregulation.