Circulating DNA as a Prognostic Marker in Sickle Cell Disease.

Abstract

Free circulating DNA has been shown to be present in the plasma of healthy subjects and elevated in conditions characterised by increased cell death, such as cancer and physical trauma. In sickle cell disease (SCD) an increased cell turnover can be expected through hemolysis and recurrent episodes of vaso-occlusion and inflammation, leading to cell death and organ damage. We propose that circulating DNA levels would be higher in patients with SCD, that these elevations would increase with acute crises and that the extent of increase may serve as a prognostic marker of disease severity. Plasma samples were collected from patients with SCD attending the specialist clinic at King's College Hospital (KCH). Over a 2 year period (April 2003 - May 2005) a total of 442 samples from 154 patients (105 HbSS, 46 HbSC, 3 HbS/β0 thalassemia) in steady state at each visit to the KCH outpatient sickle clinic was collected. Samples were also obtained from 21 of these 154 patients during an acute crisis, as defined by hospital admission for sickle-related pain. Control subjects consisted of 55 healthy Afro-Caribbean and West African individuals (53 HbAA, 2 HbAS). Plasma DNA concentration was measured by real-time quantitative PCR using a probe specific for the β-globin gene. As the distribution of plasma DNA levels was not gaussian, data was normalised by log-transformation. The median plasma DNA levels in genome equivalents (GE)/ml were as follows: 933 (range 144 to 19370) in controls; 841 (range 60.1 to 16070) in all sickle patients (SS, HbSC and S/β0 thalassemia) during steady state; 970 (range 92 to 16070) in SS and S/β0 thalassemia during steady state; 719 (range 60.1 to 14650) in SC during steady state. Median DNA levels for crisis samples were 10070 (range 444 to 57910) in all SCD and 12000 (range 444–57910) in the SS and S/β0 thalassemia group. There was no significant difference in plasma DNA levels between controls and SCD patients during steady state. Differences between steady state and crisis did not reach significance in the SC group due to the small number of crisis samples (n=3). However, mean plasma DNA levels for sickle patients during steady state, and those in crisis were highly significantly different (by Student's t test) for all sickle patients (p<0.0001) and for the SS and S/β0 thalassemia group (p<0.0001). Circulating DNA levels correlated with CRP levels in the SS/Sβ0 (r = 0.24, p<0.005) and SC groups (r = 0.31, p <0.05) but not in the controls. DNA levels correlated with WBC in the SS/Sβ0 group only (r = 0.25, p<0.05). However, plasma DNA showed no correlations with hemoglobin, reticulocyte count, red blood cell count or LDH levels. Our preliminary studies show that, unexpectedly, circulating DNA levels are not elevated in steady state SCD despite ongoing organ damage and hemolysis. However, DNA concentration may be a reliable biomarker in SCD crisis. We are currently carrying out longitudinal studies to explore the value of serial measurement of plasma DNA levels and their association with organ damage in SCD.