Biomarker Discovery: Novel Insights into Red Blood Cell Metabolomics in Patients with Sickle Cell Disease at Risk for Early Mortality

Background : In adults with sickle cell disease (SCD), an elevated tricuspid regurgitant jet velocity (TRV) by Doppler echocardiography is associated with increased morbidity and mortality. A TRV>2.9 m/s occurs in approximately 10% of patients with SCD, has a 67-75% positive predictive value for pulmonary hypertension identified by right heart cardiac catheterization, and is the most robust non-invasive biomarker of early mortality risk for patients with SCD identified to date. Metabolomics is an integrated biosystems approach to molecular fingerprinting that may be able to differentiate individual metabolic characteristics of patients at high vs. low risk of mortality based on TRV elevation.

Objective: To compare the metabolic differences between patients with SCD at high risk for early mortality defined by a TRV>2.9 m/s on Doppler echocardiogram compared to a low risk group with a TRV<2.5 m/s.

Methods: A liquid chromatography-high resolution mass spectrometry method was used to determine metabolites in sickle erythrocytes from 90 case-control selected participants from the walk-PHaSST screening cohort, matched for sickle genotype (Hb-SS), age, gender, hydroxyurea use and alpha-thalassemia trait, divided equally into patients with a TRV>2.9 m/s compared to those with a TRV<2.5 m/s. Statistical and bioinformatics analyses used one-way analysis of variance (ANOVA) and partial least-squares discriminant analysis (PLS-DA) to identify groupwise discriminatory factors contributing to 95% separation of high vs. normal TRV. Significant features with one-way ANOVA p-value<0.05 were used to perform pathway analysis.

Results: Patient characteristics are summarized in Table 1. Differential expression analysis showed that 145 accurate mass m/z features extracted from C18 column with positive ionization mode were significantly different between the high TRV and normal TRV subjects based on one-way ANOVA with p<0.005 (FDR<0.2) and PLS-DA with VIP>2 but no significant metabolism pathways were identified. With less stringent threshold p<0.05, one-way ANOVA revealed 1702 significant features which can discriminate the samples according to TRV status. Pathway enrichment analysis using Mummichog and the 1702 significant features with p-value<0.05 revealed 9 significant pathways (Figure 1).

Conclusions: Metabolic profiles in sickle erythrocytes differentiate SCD patients with a normal TRV and low mortality risk from those at high risk for early mortality with a TRV>2.9 m/s through unsupervised pathway enrichment analysis. The pathways identified are intriguing and warrant further study. Identification of the porphyrin metabolic pathway may be related to higher hemolytic rates, while carnitine shuttle holds importance in mitochondrial function. Cholecalciferol (vitamin D3) metabolism is of interest given this common vitamin deficiency in SCD and studies linking vitamin D to vaso-occlusive pain, while omega 3 fatty acid metabolism is critical to normal cell membrane function. This preliminary work identifies metabolic pathways for future targeted analysis, and may lead to novel mechanistic insight and therapies that target these pathways.

View largeDownload slide

View largeDownload slide

Close modal