Emma Drasar

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.

Association between hemolysis and albuminuria in adults with sickle cell anemia

Studies have questioned whether renal dysfunction in sickle cell disease is linked to hemolysis-associated vasculopathy. We have investigated renal function and markers of hemolysis in a cohort of 424 adult African-British patients with sickle cell disease. While significant associations were found in HbSS and HbSβ0 (sickle cell anemia) patients with and without controlling for covariates between hemolytic markers and albuminuria, the associations were not significant in patients with HbSC. Estimated glomerular filtration rate, a marker of renal function, correlated significantly with reticulocyte count and bilirubin. Alpha thalassemia, present in 34% of the sickle cell anaemia patients, had a protective effect against albuminuria in this group. Altogether, the incidence of hyperfiltration was 71% and microalbuminuria 37%, making nephropathy a common complication of sickle cell anemia.

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.

Survival in adults with sickle cell disease in a high-income setting

To the editor: Survival of patients with sickle cell disease (SCD) in high-income countries has improved greatly in the last 60 years. In 1960, it was described as a “disease of childhood”[1][1] whereas 25 years later, the Cooperative Study of Sickle Cell Disease reported that 85% of hemoglobin

Spectral domain optical coherence tomography in patients with sickle cell disease

Aim To evaluate spectral domain optical coherence tomography (SD-OCT) changes in central retina and choroid in patients with sickle cell disease (SCD). Methods Data on visual acuity, severity of sickle cell retinopathy, macular volume scans and choroidal thickness on SD-OCT were analysed from 208 eyes of 107 consecutive patients referred for screening for SCD. The retinal and choroidal thickness of eyes with SCD were also compared with age and ethnicity matched controls. Results 44% of the eyes of patients with SCD showed discrete areas of retinal thinning in the temporal macular area. Proliferative sickle cell retinopathy was more prevalent in these eyes compared with SCD eyes with normal macular morphology (67% vs 48%; p=0.0017). The temporal total and inner retinal thickness, macular volume and choroidal thickness were significantly lower in patients with SCD compared with age, gender and ethnicity matched controls. Macular splaying (widening of the macular contour) was noted in 30% of eyes in both groups. The choroidal and retinal thickness values showed good intergrader reliability using weighted κ statistics (0.550–0.9). Conclusions Quantitative and qualitative changes on SD-OCT are present in asymptomatic SCD eyes. Proliferative retinopathy is more prevalent in eyes with discrete areas of macular thinning.

Genetic determinants of haemolysis in sickle cell anaemia

Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome‐wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10−07). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)‐33, HS‐40 and HS‐48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r2 = 1) and in strong LD with rs7197554 (r2 = 0·75) and rs13336641 (r2 = 0·77); the latter is located between HS‐33 and HS‐40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the −∝3·7thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down‐regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.

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.

The clinical significance of K-Cl cotransport activity in red cells of patients with HbSC disease

HbSC disease is the second commonest form of sickle cell disease, with poorly understood pathophysiology and few treatments. We studied the role of K-Cl cotransport activity in determining clinical and laboratory features, and investigated its potential role as a biomarker. Samples were collected from 110 patients with HbSC disease and 41 with sickle cell anemia (HbSS). K-Cl cotransport activity was measured in the oxygenated (K-Cl cotransport100) and deoxygenated (K-Cl cotransport0) states, using radioactive tracer studies. K-Cl cotransport activity was high in HbSC and decreased significantly on deoxygenation. K-Cl cotransport activity correlated significantly and positively with the formation of sickle cells. On multiple regression analysis, K-Cl cotransport increased significantly and independently with increasing reticulocyte count and age. K-Cl cotransport activity was increased in patients who attended hospital with acute pain in 2011 compared to those who did not (K-Cl cotransport100: mean 3.87 versus 3.20, P=0.009, independent samples T-test; K-Cl cotransport0: mean 0.96 versus 0.68, P=0.037). On logistic regression only K-Cl cotransport was associated with hospital attendance. Increased K-Cl cotransport activity was associated with the presence of retinopathy, but this effect was confounded by age. This study links variability in a fundamental aspect of cellular pathology with a clinical outcome, suggesting that K-Cl cotransport is central to the pathology of HbSC disease. Increased K-Cl cotransport activity is associated with increasing age, which may be of pathophysiological significance. Effective inhibition of K-Cl cotransport activity is likely to be of therapeutic benefit.

Effects of co‐existing α‐thalassaemia in sickle cell disease on hydroxycarbamide therapy and circulating nucleic acids

Hydroxycarbamide (HC) is a key treatment option for sickle cell disease (SCD) (Charache et al, 1995; Ferster et al, 2001; Ware & Aygun, 2009; Voskaridou et al, 2010), but not all patients respond clinically, despite compliance (Steinberg et al, 1997). HC response is monitored by increases in fetal haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin. We have previously shown that such increases were muted in SCD patients with co-inherited a thalassaemia (a-SCD) compared to those without (SCD) (Vasavda et al, 2008). Cell-free DNA (cfDNA), a marker of tissue damage, is elevated during sickle acute pain (Vasavda et al, 2007) and reduced with HC therapy (Ulug et al, 2008). It is generally accepted that co-inherited a thalassaemia reduces haemolysis in SCD, subsequent to which the haematocrit is relatively increased with predisposition to vaso-occlusive complications (Ballas, 2001). We hypothesized that an increase in vaso-occlusive events would, in turn, have an effect on cfDNA levels in SCD. Here we describe the effect of co-inherited a thalassaemia on clinical response to HC, and on cfDNA levels. Fifty-two participants were recruited from the specialist haematology clinics at King’s College (n = 38) and Guy’s and St Thomas’ (n = 14) Hospitals, London. All participants from Guy’s and St Thomas’ hospital were treated with HC. The study was approved by the South London REC Office (08/ H0803/185); all participants gave informed, written consent. Inclusion criteria were regular attendance at the sickle cell outpatient clinics, and homozygous sickle cell disease (HbSS) or compound heterozygotes with b thalassaemia (HbSb). Exclusion criteria were: lack of data on a globin genotype, regular blood transfusion and poor compliance (in the treated group). Commencing or terminating HC therapy was a purely clinical decision, and dosing criteria were as previously described (Ulug et al, 2008). For patients treated with HC, clinical data were collected retrospectively for the year prior to starting HC therapy (‘Pre-HC’) and prospectively throughout the study (‘OnHC’). For patients commencing HC during the study, ‘OnHC’ data were collected from 6 months after starting HC therapy. Clinical response was assessed in terms of attendance at accident and emergency, hospital admissions per year, and days spent as an inpatient per year due to sickle-related pain (‘inpatient days’). Blood samples were collected at each ‘steady state’ clinic visit for cfDNA measurement, and plasma cfDNA extracted and quantified as previously described (Vasavda et al, 2007). Clinical data or blood samples were not collected for 3 months after any pro rata blood transfusion. Data was analysed using an unpaired Student’s t-test to compare data between the a-SCD and SCD groups (significance threshold P = 0Æ05). To avoid skewing of data in favour of patients with a greater number of samples and clinical data points, a ‘patient mean’ was calculated and used in further analyses. Clinical response was assessed for those patients treated with HC. Mean ‘on HC’ and ‘pre HC’ laboratory values were obtained, and response calculated as the magnitude change in each parameter; this change represents an intra-patient change in response to HC. An overall mean was then calculated for each group (SCD and a-SCD). For the cfDNA analysis data was not analysed intra-individually: results were stratified according to HC therapy, then further by a thalassaemia status. Laboratory (haematological) data were available for ‘pre-HC’ and ‘on-HC’, as these are routine care tests, whereas cfDNA is a research test, requiring a separate sample. Only a very small number of patients started HC during the study period, so sufficient pre and post intra-individual cfDNA values were not available for analysis. However, we did show in a previous study that HC therapy resulted in a significant change in cfDNA levels (Ulug et al, 2008). Patients with heterozygous (aa/-a) and homozygous (-a/-a) a thalassaemia were analysed as one group. Mean cfDNA levels in the a-SCD and SCD groups were compared separately for HC-treated and untreated patients. 33 patients were on HC therapy with a mean HC dose of 1151 mg (18Æ3 mg/kg/d); 19 were untreated during the study duration. Participants were followed for a mean of 10Æ4 months (range 3Æ6–19Æ2 months). Three participants required blood transfusions during the study (all in the ‘NoHC’ group). Further patient characterization is shown in Table I. Pre-HC data was not available for all participants; numbers used are shown with each result. HC therapy dramatically reduced the total number of hospital admissions and accident and emergency department (A&E) attendances per year in both groups (SCD and a-SCD) (Table I) but the difference between the groups was not significant. However, a-SCD patients had a significantly (P = 0Æ02) smaller reduction in the number of inpatient days (3Æ833 d ± 3Æ146 N = 7) compared to the SCD group (19Æ08 ± 3Æ932. N = 12) (Fig 1A); changes in haematological indices reflected our previous findings (Table I). correspondence