Nisha Vasavda

Genetics of fetal hemoglobin in Tanzanian and British patients with sickle cell anemia

Fetal hemoglobin (HbF, α(2)γ(2)) is a major contributor to the remarkable phenotypic heterogeneity of sickle cell anemia (SCA). Genetic variation at 3 principal loci (HBB cluster on chromosome 11p, HBS1L-MYB region on chromosome 6q, and BCL11A on chromosome 2p) have been shown to influence HbF levels and disease severity in β-thalassemia and SCA. Previous studies in SCA, however, have been restricted to populations from the African diaspora, which include multiple genealogies. We have investigated the influence of these 3 loci on HbF levels in sickle cell patients from Tanzania and in a small group of African British sickle patients. All 3 loci have a significant impact on the trait in both patient groups. The results suggest the presence of HBS1L-MYB variants affecting HbF in patients who are not tracked well by European-derived markers, such as rs9399137. Additional loci may be identified through independent genome-wide association studies in African populations.

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.

Blood transfusion usage among adults with sickle cell disease - a single institution experience over ten years

Transfusion of red blood cells is a major therapeutic option in sickle cell disease (SCD). There is strong evidence for its efficacy, particularly in primary and secondary stroke prevention in children, however, its use in other areas remains controversial. This study assessed the patterns of transfusion in the adult cohort attending King’s College Hospital over a 10‐year period, from 2000 to 2009. Total blood usage has increased significantly (P = 0·006) during this time, with 78% of the blood received by only 6% of the patients. The increase is explained by increased automated red cell exchange and increased usage for planned and acute transfusions for sickle‐related complications.

Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples.

Quantitation of hepcidin in human urine by liquid chromatography–mass spectrometry

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.

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

The presence of α-thalassaemia trait blunts the response to hydroxycarbamide in patients with sickle cell disease

Hydroxycarbamide (HC), although a key drug therapy in sickle cell disease (SCD), does not result in a clinical response in all patients. Increases in fetal haemoglobin (HbF) and mean corpuscular volume of erythrocytes are standard clinical measures of HC efficacy in SCD. Genetic studies have determined that the majority of HbF regulation occurs outside the β‐globin locus. Approximately 30% of SCD patients have co‐inherited α‐thalassaemia resulting in hypochromic and microcytic erythrocytes. We provide data from 30 SCD patients (10 with α‐thalassaemia) demonstrating that co‐existing α‐thalassaemia significantly affects several standard measures of HC efficacy in SCD.

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.

Renal iron load in sickle cell disease is influenced by severity of haemolysis

Spin density projection‐assisted R2‐magnetic resonance imaging (R2‐MRI; FerriScan®) scans from 40 chelation‐naïve sickle cell patients were used to assess renal iron load by measuring renal R2 (R‐R2). Clinical data were collected retrospectively for the 2‐year period preceding the scan. R‐R2 showed no significant correlation with transfusional iron load (assessed by liver iron concentration), but correlated significantly with serum bilirubin (R = 0·61, P < 0·0001) and lactate dehydrogenase (R = 0·58, P < 0·0001). Mean (±standard deviation) R‐R2 was higher (P = 0·02) in patients with renal hyperfiltration (29·8 ± 10·3/s) than those without (23·11 ± 6·6/s). Five patients had significantly lower signal intensity in the renal cortex, as compared to the medulla. These patients had a significantly higher (P < 0·0001) mean R‐R2 than those showing no cortico‐medullary difference. We postulate that the increased R‐R2 is associated with haemolysis rather than transfusional iron load in sickle cell disease.

Global Genetic Architecture of an Erythroid Quantitative Trait Locus, HMIP-2

HMIP‐2 is a human quantitative trait locus affecting peripheral numbers, size and hemoglobin composition of red blood cells, with a marked effect on the persistence of the fetal form of hemoglobin, HbF, in adults. The locus consists of multiple common variants in an enhancer region for MYB (chr 6q23.3), which encodes the hematopoietic transcription factor cMYB. Studying a European population cohort and four African‐descended groups of patients with sickle cell anemia, we found that all share a set of two spatially separate HbF‐promoting alleles at HMIP‐2, termed “A” and “B.” These typically occurred together (“A–B”) on European chromosomes, but existed on separate homologous chromosomes in Africans. Using haplotype signatures for “A” and “B,” we interrogated public population datasets. Haplotypes carrying only “A” or “B” were typical for populations in Sub‐Saharan Africa. The “A–B” combination was frequent in European, Asian, and Amerindian populations. Both alleles were infrequent in tropical regions, possibly undergoing negative selection by geographical factors, as has been reported for malaria with other hematological traits. We propose that the ascertainment of worldwide distribution patterns for common, HbF‐promoting alleles can aid their further genetic characterization, including the investigation of gene–environment interaction during human migration and adaptation.