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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.

Chronic hyperbilirubinemia is common in patients with sickle cell disease (SCD), frequently resulting in the formation of gallstones. This hyperbilirubinemia (predominantly unconjugated) is attributed to hemolysis that exceeds the conjugating capacity of the hepatic UDP-glucuronosyltransferase (UGT1A) enzyme. Previous studies have shown that genetic variants ([TA]n repeats) in the promoter region of the UGT1A gene have a major influence on the levels of bilirubin and gallstones. Alpha-thalassemia, which is associated with reduced hemolysis, has also been shown to affect bilirubin levels. Another potential modulating factor is heme-oxygenase, a rate-limiting enzyme in the heme catabolic pathway that results in the production of bilirubin. While the severity of jaundice and cholelithiasis in patients with SCD is predisposed by the inheritance of certain variants of the UGT1A gene, inconsistencies have been observed. We propose that some of these inconsistencies may be explained by the modulating effects of genetic variants of HO1 and α-thalassemia. A total of 263 patients with SCD attending specialist clinics in two hospitals were studied: King’s 116 SS, 5 Sβ0 and 59 SC; St Thomas’ 83 SS. 81 ethnically matched subjects were recruited as controls (HbAA). Groups were age and sex matched. Cholelithiasis data, ascertained by liver ultrasound, was available for a subset of SCD patients (76 SS, 4 SC). Samples were genotyped for variants of UGT1A, HO1 and α-thalassemia. The different genetic allele distributions were statistically similar between groups. Data was analysed according to the sum of [TA] repeats on both alleles of UGT1A. A range of 10–15 [TA] repeats was observed. For HO1, the median sum of repeats on both alleles was 63, so samples were grouped as <63 or ≥ 63. α-thalassemia genotypes were as follows: SS, 127 αα/αα, 55 αα/α-, 11 α-/α-; SC, 37 αα/αα, 20 αα/α-; controls 51 αα/αα, 22 αα/α-, 4 α-/α-. Median bilirubin levels varied as expected between groups according to β-globin genotype and were as follows: King’s SS 42 (15–269.5); St Thomas’ SS 52.5 (15.5–696.5); King’s SC 22 (10–81.8); AA Controls 10 (5–24), median (range) mmol/L. Regression analysis showed that serum bilirubin levels were strongly associated with UGT1A repeat length in all subjects (p<0.0001). Furthermore, the increase in serum bilirubin (21.3% mean for SS/Sβ0, 20.5% for SC) and cholelithiasis risk odds (86.5% mean for SS/Sβ0, 67.6% for SC) could be quantified per [TA] repeat. HO1 genotype did not affect serum bilirubin in SCD patients or the control cohort. The presence of α- thalassemia correlated (negatively) with serum bilirubin in SCD patients (p<0.0001) but not controls. This is the first time the relationship between UGT1A [TA] repeat length, serum bilirubin and cholelithiasis has been shown quantitatively. Additionally, co-existing α-thalassemia appears to moderate bilirubin levels but HO1 variants do not.

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/β0thalassemia) 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/β0thalassemia) during steady state; 970 (range 92 to 16070) in SS and S/β0thalassemia 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/β0thalassemia 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/β0thalassemia 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/Sβ0group 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.

Chronic hyperbilirubinemia is common in patients with sickle cell disease (SCD), frequently resulting in the formation of gallstones. This hyperbilirubinemia (predominantly unconjugated) is attributed to hemolysis that exceeds the conjugating capacity of the hepatic UDP-glucuronosyltransferase (UGT1A) enzyme. Previous studies have shown that genetic variants ([TA]nrepeats) in the promoter region of the UGT1A gene have a major influence on the levels of bilirubin and gallstones. Alpha-thalassemia, which is associated with reduced hemolysis, has also been shown to affect bilirubin levels. Another potential modulating factor is heme-oxygenase, a rate-limiting enzyme in the heme catabolic pathway that results in the production of bilirubin. While the severity of jaundice and cholelithiasis in patients with SCD is predisposed by the inheritance of certain variants of the UGT1A gene, inconsistencies have been observed. We propose that some of these inconsistencies may be explained by the modulating effects of genetic variants of HO1 and α-thalassemia. A total of 263 patients with SCD attending specialist clinics in two hospitals were studied: King's 116 SS, 5 Sβ0and 59 SC; St Thomas' 83 SS. 81 ethnically matched subjects were recruited as controls (HbAA). Groups were age and sex matched. Cholelithiasis data, ascertained by liver ultrasound, was available for a subset of SCD patients (76 SS, 4 SC). Samples were genotyped for variants of UGT1A, HO1 and α-thalassemia. The different genetic allele distributions were statistically similar between groups. Data was analysed according to the sum of [TA] repeats on both alleles of UGT1A. A range of 10–15 [TA] repeats was observed. For HO1, the median sum of repeats on both alleles was 63, so samples were grouped as <63 or ≥ 63. α-thalassemia genotypes were as follows: SS, 127 αα/αα, 55 αα/α-, 11 α-/α-; SC, 37 αα/αα, 20 αα/α-; controls 51 αα/αα, 22 αα/α-, 4 α-/α-. Median bilirubin levels varied as expected between groups according to β-globin genotype and were as follows: King's SS 42 (15–269.5); St Thomas' SS 52.5 (15.5–696.5); King's SC 22 (10–81.8); AA Controls 10 (5–24), median (range) mmol/L. Regression analysis showed that serum bilirubin levels were strongly associated with UGT1A repeat length in all subjects (p<0.0001). Furthermore, the increase in serum bilirubin (21.3% mean for SS/Sβ0, 20.5% for SC) and cholelithiasis risk odds (86.5% mean for SS/Sβ0, 67.6% for SC) could be quantified per [TA] repeat. HO1 genotype did not affect serum bilirubin in SCD patients or the control cohort. The presence of α- thalassemia correlated (negatively) with serum bilirubin in SCD patients (p<0.0001) but not controls. This is the first time the relationship between UGT1A [TA] repeat length, serum bilirubin and cholelithiasis has been shown quantitatively. Additionally, co-existing α-thalassemia appears to moderate bilirubin levels but HO1 variants do not.