Juliette Cunningham

The linear effects of alpha-thalassaemia, the UGT1A1 and HMOX1 polymorphisms on cholelithiasis in sickle cell disease

Serum bilirubin levels and predisposition to gallstones in sickle cell disease (SCD) are influenced by genetic variation in the hepatic uridine diphosphate (UDP)‐glucuronosyltransferase (UGT1A1) gene, but the association is not consistent. This study investigated whether variation in the gene encoding haem oxygenase (HMOX1), a rate‐limiting enzyme upstream of UGT1A in the haem catabolic pathway, and α‐thalassaemia could explain some of the inconsistent effects. The UGT1A1 [TA]n and HMOX1 [GT]n promoter polymorphisms and α globin genotypes were determined in 263 SCD patients (199 HbSS, 5 HbS/β0, 59 HbSC). Detection of gallstones was based on ultrasound of the liver/biliary tree. Regression analysis showed that serum bilirubin levels and the incidence of gallstones were strongly associated with the number of UGT1A1 [TA] repeats in all subjects (P < 0·0001 and P < 0·01, respectively). While HMOX1 genotype had no effect, co‐inheritance of α‐thalassaemia reduced serum bilirubin levels in all SCD patients independently of the number of UGT1A1 [TA] repeats. Each additional [TA] repeat is associated with an increase in mean serum bilirubin levels of 21% and cholelithiasis risk of 87% in SCD.

Association of sickle avascular necrosis with bone morphogenic protein 6

Dear Editor, Avascular necrosis (AVN) of femoral and humeral heads is a frequent and debilitating complication in patients with sickle cell disease (SCD), its prevalence being highest in individuals with SCD-Hb SS and coincidental α-thalassemia. Family and sibling studies suggest a genetic predisposition, and recently, variation in genes involved in bone modeling or the vasculature have been proposed as significant factors in the development of AVN [1]. Baldwin et al. [1] investigated single nucleotide polymorphisms (SNPs) in candidate genes involved in vascular function, inflammation, oxidant stress, and endothelial cell biology for association with AVN. SNPs in bone morphogenic protein 6 (BMP6), annexin A2 (ANXA2), and klotho (KL) genes were suggested to be associated with sickle cell AVN. We attempted to replicate this association in 244 adult patients with SCD, 39 of whom had joint symptoms with radiological and/or magnetic resonance imaging (MRI) evidence of AVN (AVN group; cases), whereas the remaining 205 had no clinical symptoms of AVN (nonAVN group; controls). Those with AVN, on average, were 3 years older than the controls (Table 1). In addition, individuals with AVN had a higher prevalence of coincidental α-thalassemia, higher hematocrit levels, and lower fetal hemoglobin (Hb F) levels when compared to the nonAVN group, though these observations were not statistically significant (Table 1). All patients were of West African or African Caribbean descent. The study was approved by the King’s College Hospital Research Ethics Committee (LREC 01-083). Based on the visual inspection of phased haplotypes in the Yoruba population from Ibadan, Nigeria (YRI), two haplotype blocks were defined in KL, one block in ANXA2, and three blocks in BMP6. Tag SNPs with population frequency information were then selected using the HapMap Project data provided for the Yoruba community, as this ethnic group relates most closely to our sample population. Of the selected tag SNPs, one per gene was identical to that used by Baldwin et al. (Table 2). The selected tag SNPs were genotyped using the TaqMan® allelic discrimination assay. Most assays performed well, but ANXA2-2 failed repeatedly. Genotype data generated from the SNP assays for the remaining six markers ranged from 95.2% to 100% complete, and markers displayed no deviation from the Hardy–Weinberg equilibrium. To test for trait association with the candidate SNPs, allele frequencies between cases (AVN group) and controls (non-AVN group) were compared using Pearson’s chisquare statistic with a significance threshold of p≤0.05 (Table 2). Characterization of the six SNP markers revealed that only one (BMP6-3 or rs3812163) showed significant evidence of association (p=0.021) with AVN. Ann Hematol (2009) 88:803–805 DOI 10.1007/s00277-008-0659-5

Circulating DNA: a potential marker of sickle cell crisis

Free circulating DNA is present in the plasma of healthy subjects, and is elevated in conditions characterized by increased cell death, such as cancer and physical trauma. Analysis of circulating DNA in plasma could provide a useful biomarker in sickle cell disease (SCD) in view of the increased cell turnover through chronic ongoing haemolysis, recurrent vaso‐occlusion and inflammation. Plasma DNA was determined by real‐time quantitative polymerase chain reaction (PCR) amplification of the β‐globin gene (HBB) in 154 patients with SCD [105 haemoglobin (Hb)SS, 46 HbSC and three HbS/β0 thalassaemia] and 53 ethnically matched controls. Blood samples were obtained from all patients in steady state; 21 of the 154 patients were also sampled during admission to hospital for acute pain. Median concentration of circulating plasma DNA in acute pain was more than 10‐fold that in steady state and in controls – 10070 vs. 841 and 10070 vs. 933 genome equivalents/ml respectively (P < 0·0001, in both cases). During steady state, patients had plasma DNA levels similar to controls. Plasma DNA levels in SCD correlated with C‐reactive protein levels (P < 0·005) and total white cell counts (P < 0·05) in steady state. The study shows that plasma DNA concentration may have potential as a biomarker in sickle cell patients.

Hydroxyurea therapy lowers circulating DNA levels in sickle cell anemia

Hydroxyurea reduces the frequency of acute pain in sickle cell disease (SCD). We sought to determine if hydroxyurea therapy affects cell free DNA (cfDNA) levels in SCD. cfDNA levels fell in all 10 patients studied; before hydroxyurea, mean was 1,879 (95% CI 1,104–3,199) GE/mL; after hydroxyurea, mean was 780 (95% CI, 634–959) GE/mL (P = 0.002). Mean cfDNA level in the 10 HbSS adults prior to starting hydroxyurea was also significantly higher than that in 115 HbSS case controls who had never taken hydroxyurea (1,879 vs 975 GE/mL, P = 0.02). cfDNA levels may be useful in monitoring response to hydroxyurea therapy in SCD. Am. J. Hematol., 2008.