European Chromosome 6 Haplotypes Significantly Augment Fetal Hemoglobin Levels in Brazilian Sickle Cell Anemia Patients: Influence of Four HBS1L-MYB Intergenic Region SNPs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1002-1002
Author(s):  
Flávia Costa Leonardo ◽  
Stephan Menzel ◽  
Ana Flavia Brugnerotto ◽  
Kleber Yotsumoto Fertrin ◽  
Marcos André Cavalcanti Bezerra ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Intergenic Region ◽  
Conflicts Of Interest ◽  
Strong Association ◽  
Fetal Hemoglobin ◽  
Brazilian Population ◽  
Minor Allele ◽  
Chromosome 6 ◽  
Major Allele

Abstract Abstract 1002 Fetal hemoglobin (HbF) levels significantly modulate the severity of the 2 major β-hemoglobin disorders - sickle cell anemia (SCA) and β-thalassemia. Three major quantitative trait loci (QTLs; Xmn1-HBG2, the HBS1L-MYB [HMIP] intergenic region on chromosome 6q23, and BCL11A on chromosome 2p16) account for 20–50% of the common variation in HbF levels in SCA and β - thalassemia patients, and in healthy adults (Thein et al., Hum Mol Genet (2009) 18:R216). Lettre et al. (PNAS (2008) 105:11869) confirmed the influence of SNPs at the BCL11A and HBB loci in an African American cohort and a Brazilian cohort of SCA patients; as well as a significant influence of the HMIP region SNPs (rs7776054, rs9389268 and rs4895441) on HbF expression in the Brazilian SCA cohort. A strong association between HMIP polymorphisms that have a high frequency in the European population and modulation of F cell numbers has been reported (Creary et al., PLoS One (2009) 4:e4218). Given the unusually high admixture of the Brazilian population, the current study aimed to look at the influence of such HMIP markers on HbF production in SCA patients from this population (two regions, in the Northeast and Southeast of Brazil). We studied the influence and frequencies of the HMIP allele marker rs9376090 (that specifically tracks European chromosomes), as well as the rs9399137 marker (that has a much higher frequency in European descendents than in African descendents), as well as two HMIP markers (rs9389269 and rs9402686) that are also common in African descendents. Patients (220 HbSS, aged 12–68 years) were recruited at the Hematology Center, UNICAMP and at Fundação Hemope. The study was approved by the local Ethics Committees and informed consent was provided by all participants. Patients presenting the XmnI Gγ polymorphism (N = 2) were identified and excluded from further analysis, as this polymorphism has a known influence on HbF. The HMIP markers were genotyped by Taqman assays. Percentage HbF levels were determined by HPLC, using the Variant™ Bio-Rad kit, and were log transformed to normalize distribution for regression analyses. For those patients on hydroxyurea (HU) therapy, pre-HU HbF levels were used for analyses. Tests for associations between SNPs and HbF levels were conducted using linear regression models (SPSS v.15), including age and sex as covariates. High minor allele frequencies (MAF) for all four HMIP markers were observed in the population of patients studied (MAF; 0.09, 0.10, 0.12 and 0.12 for the rs9376090, rs9399137, rs9389269 and rs9402686 markers, respectively). For all four SNPs studied, higher levels of HbF were observed for the SCA individuals that were homozygotes for the minor allele, with strikingly higher levels of HbF presented by those individuals that were homozygotes for the rs9376090 and rs9399137 polymorphisms (see Table). The clinical courses of these patients were consistent with the higher levels of HbF observed (data not shown). Significantly higher HbF was also found in heterozygotes for the HMIP SNPs, compared to the major allele homozygotes. The variance in HbF levels due to rs9376090 was 7.1% (β= 0.270; p = 6.36 10−5), due to rs9399137 was 7.1% (β= 0.270; p = 9.59 10−5), due to rs9389269 was 8.3% (β= 0.287; p = 2.31 x10−5) and to rs9402686 was 8.3% (β=0.291; p = 2.18 x10−5). Our results confirm the HBS1L-MYB intergenic region as a key determinant of HbF levels in Brazilian SCA patients. The admixture of the Brazilian population has apparently led to a much higher incidence of European haplotypes at chromosome 6 in this population studied, when compared to the British and Tanzanian SCA populations. Importantly, the presence of these SNPs at the HMIP appears to have a very significant effect on HbF levels in the Brazilian SCA population, with probable clinical benefits. Table. HbF Levels in SCA Individuals, according to HMIP genotypes SNP (HBS1L-MYB locus) % HbF (Median ± S.D.) TT TC CC rs9376090 6.35 ± 4.24 8.45 ± 3.63 17.25 ± 1.63 N = 182 N = 32 N=2 p<0.01 p<0.05 rs9399137 6.47 ± 4.38 8.68 ± 3.63 17.25 ± 1.63 N = 167 N=37 N = 2 p<0.05 p< 0.05 rs9389269 6.40 ±4.29 8.59 ± 3.70 12.68 ± 2.45 N = 162 N = 42 N = 5 p< 0.01 p< 0.05 rs9402686 GG AG AA 6.26 ± 4.28 8.36 ± 4.67 12.68 ± 2.45 N =166 N = 41 N = 5 p<0.01 p<0.05 Significant differences for heterozygotes and homozygotes for the minor allele, compared to the homozygote major allele group are indicated by P values. Kruskal-Wallis test, Dunn's multiple comparison post test. Disclosures: No relevant conflicts of interest to declare.

Monocyte Shift to a Non-Classical CD14dim/CD16+ Phenotype Correlates with Fetal Hemoglobin Levels in Sickle Cell Anemia Patients Treated with Hydroxyurea

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 817-817 ◽  
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Dulcinéia Martins Albuquerque ◽  
Carolina Lanaro ◽  
Carla Fernanda Franco-Penteado ◽  
Flavia Rubia Pallis ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Pleiotropic Effect ◽  
Monocyte Subset ◽  
Classical Monocytes ◽  
Intermediate Monocytes

Abstract Abstract 817 Vaso-occlusion in sickle cell anemia (SCA) involves inflammation and cell activation; fetal hemoglobin (HbF) elevation by hydroxyurea (HU) remains the mainstay of SCA treatment. Monocytes are activated in SCA, and their contribution to the chronic inflammatory state includes the production of cytokines and reactive oxygen species (ROS). Monocytes are a heterogeneous group of leukocytes subdivided into distinct subsets: classical monocytes comprise over 80% of circulating monocytes, are highly positive for CD14 (CD14bright) and typically CD16-negative, while CD16-positive monocytes have been further subdivided into intermediate CD14bright/CD16+, and non-classical CD14dim/CD16+ monocytes. Intermediate monocytes are recognized as the main monocytic producers of ROS and are increased in inflammatory conditions such as atherosclerosis and sepsis. The less characterized non-classical subset is believed to have a patrolling behavior in blood vessels, does not produce ROS and constitutively produces IL-1 receptor antagonist (IL1-RA). Another relevant subgroup of monocytes expresses angiopoietin-2 receptor TIE2, and the role of TIE2-expressing monocytes (TEMs) has been investigated in angiogenesis in neoplastic diseases. TEMs usually correspond to intermediate monocytes, but their importance in inflammation is still unclear. We hypothesized that monocyte subsets in SCA patients would differ from controls, and that treatment with HU might also influence monocyte phenotypes, thus shedding light on the possible role of these subsets in an inflammatory condition not previously studied. EDTA-anticoagulated peripheral blood samples were collected upon written informed consent from 21 healthy controls (CON, ages 21–63 years) and 34 SCA patients (18 on HU, ages 16–58 years) in steady state, with no transfusion or acute sickling episode in the previous three months. Monocytes were immunophenotyped by flow cytometry on a multicolor FACSCalibur cytometer. Medical history of SCA-associated complications, HbF levels and dosage of HU in mg/kg/day were obtained from medical charts. Statistical analysis was performed on GraphPad Prism 5.0 software. As expected, we found that relative percentage and absolute count of CD16-positive monocytes were higher in SCA patients than in controls. Surprisingly, a significantly higher percentage of non-classical CD14dim/CD16+ monocytes, rather than intermediate cells, was found in SCA patients on HU (SCA-HU) treatment (mean±SEM: CON 2.06±0.43%, SCA 2.91±0.50%, SCA-HU 6.42±0.80%, P<0.0001). TEMs were also increased in SCA patients compared to controls (CON 2.64±0.72%, SCA 20.48±5.40%, SCA-HU 32.97±5.92%, P<0.0001), but HU treatment did not significantly influence TEM counts. Mean TIE2 expression did not vary among the groups, and there was no correlation between TEMs and presence of SCA complications pathophysiologically associated with disturbed angiogenesis, such as pulmonary hypertension, osteonecrosis, leg ulcers and retinopathy. Higher percentages of non-classical monocytes in HU-treated patients were initially interpreted as a possible toxic effect of HU on monocytopoiesis, but the lack of correlation of monocytes subsets with the degree of relative monocytopenia made this hypothesis unlikely. Moreover, we found a significant positive correlation between percentages of non-classical monocytes and HbF levels (rS=0.4763, P=0.0068, see figure). This suggests that successful HU treatment with higher HbF could correlate with the expansion of this particular monocyte subset. During the study period, only one patient was available for comparison before and after HU, but the increase in HbF from 4.2% to 11.6% and in non-classical monocytes from 1.82% to 9.48%, in this case, corroborates that HU therapy may explain this phenotype shift in monocytes. Whether non-classical monocytes expansion represents yet another pleiotropic effect of HU, if these cells are less likely to take part in the vaso-occlusive process and have an antiinflammatory role or, furthermore, if a bone marrow counterpart of this monocyte subset could be involved in increasing HbF production, remains to be investigated. The correlation of the expansion of non-classical monocytes with HbF levels could prove to be an interesting biomarker of response to HU, and future studies may address its clinical usefulness. Disclosures: No relevant conflicts of interest to declare.

Absolute Reticulocyte Count Is Negatively Correlated with Fetal Hemoglobin during Infancy and Early Childhood in Patients with Sickle Cell Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4081-4081
Author(s):  
Emily R. Meier ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Maxine Weissman ◽  
Jeffery L. Miller
Keyword(s):  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Convenience Sample ◽  
Risk Of Death ◽  
Increased Risk ◽  
F Cells ◽  
Analytical Approaches

Abstract Hemoglobin switching is largely complete in healthy infants by 6 months of age. In infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Previous studies demonstrated that patients with an ARC greater than 200 K/uL during early infancy (60-196 days of age) were at the highest risk for SCA-associated events. 1,2 The objective of this study was to determine if ARC is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study [42 (39.6%) less than 1 year of age (28-362 days old), 46 (43.4%) between the ages of 1 and 10 years, and 18 (17.0%) between 10 and 20 years old]. After consent and assent were provided, discarded peripheral blood was obtained during routine clinic visits at steady state and analyzed within 48 hours of collection and storage at 40C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated by flow cytometry following intracellular staining with a fluorescent antibody directed against HbF. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t tests were used to compare means. Initial studies compared groups based upon ARC greater than or equal to 200 K/uL (ARC≥200) during infancy because of the previously reported utility of this threshold as a predictive marker for SCA severity.1 Over one third of the infants less than 1 year of age (n=16) had an ARC≥200. Mean HbF and F-cell levels were significantly lower in the ARC≥200 group when compared to the ARC<200 group (HbF: 29.9±10.9% vs. 53.5±17.6%, respectively, p=2.2E-05; F-cells: 83.5±13.2% vs. 96.6±5.7%, p=6.2E-05). Mean hemoglobin levels were also lower in the ARC≥200 group [8.1±1.4 g/dL vs. 9.5±1.6 g/dL (ARC<200), p=0.005]. Of the 22 (52.4%) infants who had a HbF level greater than 40%, only 2 (9.1%) had an ARC greater than 200K/uL. Enrolled patients were also grouped according to age and comparisons were made between ARC and HbF or F-cell levels. HbF and F-cell levels were negatively correlated to ARC in the infant subgroup (r=-0.696, p=3.1E-07 and r=-0.795, p=0.000, respectively). HbF and F-cell levels from children between the ages of 1 and 10 years were inversely related to the ARC, but the correlation was less significant (r=-0.626, p=3.3E-06 and r=-0.538, p=1.2E-04, respectively). The inverse relationship was no longer present in the oldest group of patients (HbF vs. ARC r=-0.203, p=0.420 and F-cells vs. ARC, r=-0.258, p=0.302). According to both analytical approaches described here, increased ARC is associated with decreased HbF and F-cell levels in infants with SCA. Less robust negative correlations are maintained through age 10 years, but no significant correlation was identified in adolescence and young adulthood. Overall, the data suggest that increased ARC levels may identify SCA infants who manifest a more rapid or greater loss of fetal hemoglobin during the later stages of the HbF-to-HbS switching phenomenon. Meier ER, Byrnes C, Lee YT, et al. Increased reticulocytosis during infancy is associated with increased hospitalizations in sickle cell anemia patients during the first three years of life. PLoS One 2013; 8(8):e70794. doi: 10.1371/journal.pone.0070794.Meier ER, Wright EC, Miller JL. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. Am J Hematol 2014 May 31; doi: 10.1002/ajh.23777. [Epub ahead of print] Disclosures No relevant conflicts of interest to declare.

Fetal Hemoglobin in Sickle Cell Anemia: A Novel Method for High-Resolution Discovery of Associated Genomic Copy Number Variations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2491-2491
Author(s):  
Daniel Dworkis ◽  
Paola Sebastiani ◽  
Efthymia Melista ◽  
Jason Parente ◽  
Griffin Lester ◽  
...  
Keyword(s):  
High Resolution ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Copy Number ◽  
Fetal Hemoglobin ◽  
Copy Number Variations ◽  
Chromosome 6 ◽  
Genomic Copy Number ◽  
Genomic Copy ◽  
Novel Method

Abstract Fetal hemoglobin (HbF) can inhibit the polymerization of sickle hemoglobin, and the HbF level is an important modulator of the severity and course of sickle cell anemia. Genetic regulation of HbF levels is complex and under active investigation. Although multiple quantitative trait loci have been discovered, it is estimated that half of the genetic variance of HbF levels remains unaccounted for. Genomic copy number variations (CNVs), defined as inherited duplications or deletions of kilo-to mega-base lengths of DNA, represent a significant source of genetic heterogeneity among humans that might be involved in HbF regulation. Additionally, CNVs can significantly alter assumptions about genotype frequencies in their genomic region, and are therefore important to locate for multiple types of genetic association studies. Here, we present a novel method for the high-resolution discovery of CVNs related to HbF levels in sickle cell anemia, using genome-wide association study (GWAS) data. We used the Illumina 610K single nucleotide polymorphism (SNP) genotyping array to examine 727 adult subjects with sickle cell anemia, with or without a thalassemia, who were enrolled in the Cooperative Study of Sickle Cell Disease (CSSCD; aged 18 to 69 years, mean age 31 years; 44% male; not on hydroxyurea therapy). The Illumina array consisted of ~610K probes spread across the entire genome. At each locus, the relative amount of DNA detected was compared to a reference and expressed as the log R ratio score (LRR). Normal diploid regions of DNA have LRRs close to zero, whereas regions with CNVs have LRRs that are either higher for areas of duplication or lower for areas of deletion. Using LRR information in the context of a GWAS, we developed a novel, two-step signal-processing technique that combines CNV discovery with subsequent phenotypical association analysis. First, the distribution of LRR values at each locus is stratified using a +/− 1.5 standard deviation band-pass filter. This created three groups: a central major group comprised of people with diploid amounts of DNA, and two minor variant groups, one composed of people with elevated LRRs, suggesting &gt;2 DNA copies, and one of people with decreased LRRs, suggesting &lt;2 DNA copies at that locus. To reduce noise, loci without at least one minor group containing &gt;5% of the sample were excluded from further analysis. In the second step, a two-sample Student’s t-test was used at each locus to examine the variation in distributions of HbF between the major, diploid group and any variant groups with &gt;5% of the population. Using this method, we examined chromosomes 2, 6, and 11, which include regions known to modulate HbF in patients with sickle cell anemia, individuals with β thalassemia, and in the normal population. We successfully detected multiple clear duplications and deletions (approx. 1 per 6–22 mbp, depending on the chromosome) that showed typical CNV LRR distributions with &gt;10% of the population exhibiting the polymorphism. Several of these were mildly related to HbF levels (p&lt;0.05), including deletions in ASB1 on chromosome 2, and HACE1 on chromosome 6, both ankyrin motif containing proteins involved in the ubiquitin ligase system, as well as an upstream duplication and intragenic deletion involving HLA-DRB5 on chromosome 6. None of these clear CNVs, however, overlapped regions known to affect HbF concentration. Additional potential CNVs were detected throughout each chromosome, many exhibiting atypical LRR distributions not easily classified as either a normal diploid or clear CNV region. Further studies are required to confirm the presence of a CNV at these atypical loci. With this method, we were able to detect CNVs and CNV breakpoints across a population with a single-probe resolution, to within &lt;1kb in some cases. This resolution offers a distinct advantage over other detection methods that utilize a multiple-probe, sliding-window approach to detect LRR deviations in an individual sample. In conclusion, this two-step method of high-resolution detection of CNVs followed by analysis of phenotypical associations shows promise for explaining variations in expressed protein levels, such as those typical of HbF in sickle cell anemia, and possibly for future exploration of differences in HbF responses to therapeutics in sickle cell anemia.

Elevated Plasma Levels and Platelet-Associated Expression of the Pro-Thrombotic and Pro-Inflammatory Protein, LIGHT (TNFSF14), In Sickle Cell Anemia (SCA)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2651-2651
Author(s):  
Vanessa T. Garrido ◽  
Venina M. Dominical ◽  
Renata Proença-Ferreira ◽  
Marcos André Cavalcanti Bezerra ◽  
Mariana R. B. Mello ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Therapeutic Target ◽  
Plasma Levels ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Endothelial Activation ◽  
Potential Therapeutic Target ◽  
Activation Markers ◽  
Inflammatory Protein

Abstract Abstract 2651 Background: LIGHT (TNFSF14; CD258), a recently-identified member of the TNF superfamily, is found associated with and produced from platelets, amongst other immune cells. Increased circulating levels of this protein have been observed in patients with myocardial infarction and acute atherothrombotic stroke and LIGHT has been proposed as a potential therapeutic target in atherosclerosis, due to its reported pro-thrombotic and pro-inflammatory effects upon human endothelial cells. This study evaluated whether production of LIGHT is altered in patients with sickle cell anemia (SCA), and the participation that the platelets (PLTs) may have in this production, since SCA is characterized by a significant chronic inflammation and endothelial activation that may initiate the vaso-occlusive process. Patients and Methods: Soluble LIGHT (sLIGHT) was determined in PLT-free plasma (obtained by sequential centrifugations and ultrafiltration) from healthy control individuals (CON), SCA patients in steady state (SCA) and SCA patients on hydroxyurea therapy (SCAHU; 20–30 mg/kg/day HU) by ELISA. Expressions of PLT-membrane LIGHT and PLT activation markers were evaluated by flow cytometry using anti-CD258-PE, anti-CD62P-FITC or anti-PAC1-FITC. Subjects had not taken ASA during the previous 14 days. Results: sLIGHT was significantly elevated in the plasma of SCA and SCAHU individuals, compared to CONs (SCA; 35.4 ± 9.4 pg/ml; SCAHU, 37.3 ± 7.5 pg/ml; CON, 9.7 ± 1.5 pg/ml, n=27, 27, 19, resp.; P<0.01 for SCA/SCAHU, compared to CON; Kruskal-Wallis/Dunn's). Plasma sLIGHT in SCA/SCAHU individuals presented no correlation with hematological variables, such as PLT counts (rs=-0.079, P=0.65) and fetal hemoglobin (rs=0.107, P=0.51). In contrast, plasma sLIGHT levels in SCA patients presented an impressive correlation with plasma levels of CD40L, another important PLT-derived inflammatory protein (rs=0.817, P<0.0001 for SCA group and rs=0.651, P<0.0001 for SCA+SCAHU) and correlated with IL-8, an endothelium-derived inflammatory mediator (rs=0.900, P<0.05 for SCA and rs=0.455, P=0.06 for SCA+ SCAHU). Expression of LIGHT protein (CD258) was significantly higher on the membrane of PLTs from SCA and SCAHU individuals, compared to CON PLTs (SCA, 27.1 ± 4.5 %; SCAHU, 33.7 ± 5.8 %; CON, 5.5 ± 1.6 % positive PLTs, n=15, 20, 15, resp.; P<0.001 for SCA/SCAHU comp. CON). Notably, when PLTs were activated by incubation with ADP (20 μM, 30 min), PLTs from SCA/SCAHU individuals still expressed significantly more surface LIGHT than CON PLT (SCA, 41.8 ± 4.5 %; SCAHU, 41.3 ± 5.4 %; CON, 11.1 ± 2.3 % positive PLTs; n=15, 20, 14, resp. P<0.001 for SCA/SCAHU comp. CON); successful activation of PLTs from all groups was confirmed by increased PAC-1 (anti-activated αIIbß3 integrin) binding and increased P-selectin (CD62P, a PLT activation marker) expression (data not shown); furthermore LIGHT expression on ADP-activated cells correlated significantly with both PAC-1 binding (rs=0.483, P=0.03) and P-selectin expression (rs=0.502, P=0.02, n=20) on SCA PLTs. sLIGHT release from SCA PLTs during 90 min (37°C, 5% CO2 in Krebs) was evaluated and demonstrated that PLTs of SCA individuals may be an important source of circulating sLIGHT, releasing 22.2 ± 4.7 pg LIGHT/108 PLTs (n=10); release of sLIGHT from SCA PLT was significantly augmented by incubation with collagen (P<0.01), but not ADP (P>0.05); basal and collagen-stimulated sLIGHT release correlated significantly with CD40L release (rp=0.654; rp=0.764, resp. P<0.05). Conclusion: The pro-inflammatory and atherogenic protein, LIGHT, is found significantly elevated in the plasma of SCA patients. The correlation of plasma LIGHT with IL-8 indicates that LIGHT may participate in, or reflect, endothelial activation, whilst the correlation of circulating LIGHT and PLT release of LIGHT with CD40L indicates that the production of these two proteins may be tightly coupled. LIGHT protein is highly expressed on the PLT surface in SCA and this expression appears to be associated with PLT activation, rather than PLT number. Interestingly, HU therapy was not associated with any significant alteration in circulating LIGHT, nor PLT surface LIGHT. Future investigations will determine the extent of the contribution of LIGHT to endothelial activation, inflammation and vaso-occlusion in SCA and whether this protein holds promise as a potential therapeutic target for the disease. Disclosures: No relevant conflicts of interest to declare.

Co-Inheritance of Delta Thalassemia Might Contribute to the High Fetal Hemoglobin in Sickle Cell Anemia Patients with the Saudi-Indian Haplotype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1056-1056
Author(s):  
Abdulrahman Alsultan ◽  
Duyen A. Ngo ◽  
John J. Farrell ◽  
Hazem Ghabbour ◽  
Idowu Akinsheye ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
African Americans ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Transcriptional Level ◽  
Eastern Province ◽  
Hbf Level ◽  
Positively Charged

Abstract Abstract 1056 Most sickle cell anemia patients (HBB glu6val homozygotes) indigenous to the Eastern Province of Saudi Arabia have a fetal hemoglobin (HbF) level of about 20% that is associated with a mild clinical phenotype. Their HbS gene is on the Saudi-Indian (SI) haplotype characterized by an Xmn1 restriction site at position −158 5' to HBG2 (rs7482144), a Hinc2 site 5' to HBE (rs3834466) and other polymorphisms in the HBB gene-like cluster. However, the functional elements within the HBB gene-like cluster and elsewhere in the genome causing high HbF are yet to be determined. In a previous study we found that Saudi HbS homozygotes with the SI haplotype had a common region of autozygosity that spanned about 126 kb and included the complete HBB cluster. We have sequenced 13.6 kb in the HBD-HBG1 intergenic region (chr11:5255683-5269326, HG19), the region of the Corfu deletion. We found a SNP at position −68 bp 5' to HBD (c.-118 C>T) only in individuals with a SI haplotype. This SNP was not present in dbSNP build 132 or the 1000 Genomes databases. No other mutation in HBD was identified. This same SNP was recently associated with δ thalassemia (Phylipsen et al. Int. J. Lab. Hematol. 2011, 33: 85–91). Homozygotes for the −68 HBD SNP, who were not on hydroxyurea, had a mean HbF of 23%, range 12.1%-33.4% and mean HbA2 of 2.1%, range 1.2%-3%. We did not find the −68 HBD SNP in 15 African Americans with sickle cell anemia selected because of their unusually high HbF (mean HbF 17.2%, range 11%-28.9%). Parents and sickle cell trait carriers from the families of Saudi Eastern Province patients were heterozygous for the −68 HBD mutation (mean HbF 1.6%, range 0–4.2% and mean HbA2 2.7%, range 2.4%-3.2%) and one normal sibling did not carry this mutation (HbF 0 and HbA2 2.9%). Thirty patients with sickle cell disease indigenous to the Southwestern Province of Saudi Arabia, with the HbS gene on an African origin haplotype, (mean HbF 15.5%, range 4.5%-23% and mean HbA2 2.9%, range 2.1%-3.4% in HbS homozygotes) and 13 normal Saudi controls were examined and none carried the −68 HBD mutation. We also sequenced HBD and its promoter in 8 Southwestern Province HbS homozygotes with a Benin haplotype, 4 with HbF <5% and 4 with HbF >15%, and none had the −68 HBD SNP or any other mutation in HBD. Inverse relationships between percent HbF level and percent HbA2 was seen in 3 groups of HbS homozygotes:1) SI haplotype homozygotes; 2) Benin haplotype homozygotes from the Southwestern Province; 3) African Americans with diverse HbS haplotypes. The increased HbF in sickle cell anemia with δ thalassemia might involve both post-translational and transcriptional mechanisms. Increased HbF levels might in part be due to the preferential post-translational assembly of αγ dimers compared with αδ and αβS dimers. When δ thalassemia reduces available δ-globin chains, this might further favor preferential binding of less positively charged γ-globin subunits to positively charged α-globin compared with more positively charged δ-globin subunits. Perhaps more importantly, at the transcriptional level, the −68 HBD δ+-thalassemia promoter mutation might favor the interactions of transcription complexes with HBG promoters. The stimulus of hemolytic anemia and expanded erythropoiesis might be required before the favorable genetic milieu for increased HbF production can be fully utilized with the achievement of clinically significant increases in HbF that modulates the course of disease. For example, increased HbF levels of only 3.3%-4.7% have been reported in some hematologically normal individuals with homozygous δ0 thalassemia (Ohta et al. Hemoglobin 1980, 4: 417–425). High HbF levels in SI haplotype HbS homozygotes might involve the interactions of one or more HBG regulatory regions linked to the HBB gene-like cluster, like the −68 HBD SNP, perhaps with trans acting elements. Although the −68 HBD δ+-thalassemia mutation is associated with the SI haplotype and high HbF, functional studies are needed to establish causation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Longitudinal Analysis of Hydroxyurea and Fetal Hemoglobin in Adult Patients with Sickle Cell Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2054-2054
Author(s):  
Aryeh Pelcovits ◽  
Giancarlo Riotto ◽  
Katie Cherenzia ◽  
Patrick T. McGann ◽  
John L Reagan
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Surrogate Marker ◽  
Fetal Hemoglobin ◽  
P Value ◽  
Significant Difference ◽  
Dosing Strategies ◽  
Over Time ◽  
Painful Crisis

Abstract Introduction: Hydroxyurea (HU) was the first FDA-approved therapy for sickle cell anemia (SCA) and remains the most well-established disease-modifying therapy, reducing painful crisis and improving morbidity and mortality. HU improves outcomes through the upregulation of fetal hemoglobin (HbF). Dosing is highly variable with doses ranging from &lt;10-35 mg/kg/day. Most dosing strategies escalate with a goal of mild myelosuppression, commonly defined using an absolute neutrophil count (ANC) between 2,000-4,000 and platelets &gt;80,000. Macrocytosis is often used as a surrogate marker for compliance and effect. Despite its well-established effectiveness, clinical use remains limited with only 12% of adults with SCA taking HU. The reasons for this are multifactorial but include skepticism by both providers and patients regarding its effectiveness in the adult population. Some experts suggest up to 30% of adults are non-responders with no significant HbF change, and many believe that the effect of HU fades with time. Dosing strategies are usually quite conservative to prevent excessive myelosuppression, though dose is highly correlated with clinical effect and ultimate %HbF. There is very limited real-world data regarding long term effectiveness of HU in adults with SCA over time. Methods: We retrospectively analyzed the records of the 109 adults with SCA currently treated in our multidisciplinary sickle cell clinic. Data from 1/1/2011-6/30/2021 was collected, including genotype, HU prescription status, current and maximum laboratory values (HbF, MCV, ANC), and number of admissions for painful crisis. We performed Wilcoxon rank sum testing between pts prescribed and not prescribed HU and measures of HbF (peak, average and current) and number of admissions for painful crisis over the study period. Results: Among 106 pts (3 excluded from analysis, 2 for lack of data and 1 for post-transplant status), 79 are currently prescribed HU (75%). Among our 63 pts with HbSS genotype 58 are prescribed HU (92%). Average HbF over time for all pts prescribed HU was 11.9%. Average peak HbF was 18.1% and average current HbF is 12.4%. In the pts not prescribed HU HbF average, peak and current levels were 5.5%, 6.7%, and 5.0% respectively. HU prescription was significantly associated with increased HbF at peak, average, and current levels (p-value &lt;0.001, Figure A). HU prescription was also significantly associated with decreased number of admissions for painful crisis (p-value 0.001). Among pts prescribed HU, MCV levels &gt;100 at time of peak HbF were associated with higher peak levels than pts with MCV &lt;100 (p-value &lt;0.001, Figure B). Larger peak HU doses were also correlated with higher MCVs at the time of peak HbF levels (Figure C). Larger current HU doses were also significantly associated with higher current HbF levels (Figure, D). Doses &gt;9.9m/kg were associated with significantly higher HbF levels however there was no significant difference between dose levels 10-19.9mg/kg and 20-29.9 or 20-29.9 and 30-39.9mg/kg (p-values 0.02, 0.68 and 0.84 respectively). Among pts prescribed HU, 34 obtained ANC levels &lt;4000 at the times of peak HbF and 24 at the current dosing level (43% and 30% respectively). 24 pts obtained both MCV&gt;100 and ANC &lt;4000 at time of peak HbF and only 11 achieved both at current dosing levels (30% and 14% respectively). Conclusion: In our adult multidisciplinary sickle cell clinic prescribing rates are well above current HU usage for adults with SCA. Our data notes that elevated HbF levels can be maintained over long periods of time. HU prescription was significantly associated with increased HbF levels and reduced admissions for painful crisis. This was despite a majority of pts not meeting target prescribing levels of ANC &lt;4000 and MCV &gt;100. While higher peak HbF levels were significantly associated with higher HU doses, this was only true for doses above 9.9 mg/kg. Further investigation is needed to explore the factors contributing to suboptimal HbF and MCV response, including careful assessment with medication adherence and dose selection. Overall, these data illustrate the importance of dosing and suggest that one size dosing of HU for adults with SCA does not fit all. We hypothesize that there may be a role individualized dosing strategies in adults with SCA, incorporating factors such as pharmacokinetics and renal function to achieve the optimal dose for each patient. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of Saudi Patients From the Eastern Province

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1627-1627
Author(s):  
Abdulrahman Alsultan ◽  
Idowu Akinsheye ◽  
Nadia Solovieff ◽  
Hazem Ghabbour ◽  
Duyen A. Ngo ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Snp Array ◽  
Fetal Hemoglobin ◽  
Homozygosity Mapping ◽  
Eastern Province ◽  
Saudi Patients

Abstract Abstract 1627 In the Eastern Province of Saudi Arabia, sickle cell anemia (HbSS) is associated with the Saudi Indian (SI) HBB-gene cluster haplotype, high levels of fetal hemoglobin (HbF) and milder disease, when compared with Southwestern Province HbSS patients who have lower HbF levels and different HBB haplotypes. An association between HbF and the Xmn1 restriction site in the HBG2 promoter present in both the SI and African-derived Senegal haplotypes is well known, but the causal elements of this association are unknown. Moreover, among individuals with the SI haplotype, only HbSS patients have high HbF while individuals with sickle cell trait (HbAS) or normal hemoglobin (HbAA) do not. Furthermore, HbF levels are far higher in SI haplotype patients, as shown below, compared with Senegal haplotype homozygotes. For example African patients homozygous for the Senegal haplotype had 12.3±5.3% HbF. To better understand the genetic basis for high HbF in SI haplotype HbSS cases, we compared sequences in the HBB gene cluster in patients with SI and Senegal haplotypes. We hypothesized that the causal elements that modify HbF in Saudi patients are in linkage disequilibrium (LD) with the βS globin gene in this population. Accordingly, we studied 5 Saudi families from the Eastern Province. Seven SI haplotype patients with HbSS (median age 5 yrs, range 2.5–49 yrs) were homozygous for the Xmn I site and had Hb 9.7 ± 1.6 g/dL, MCV 76.5 ± 8.3 fl and median Hb F 30.3 (range 18–41). Seventeen SI haplotype individuals had HbAS (median HbF 1.2, range 0–4.2); and 2 were normal. We first determined the genotypes of 3 known HbF QTLs, BCL11A (rs766432); HBS1L-MYB (rs7775698 and rs9399137); and OR51B5/6 (rs5006884). There were no consistent genotypes among these 7 patients to explain their universal high HbF. Next, we performed homozygosity mapping using Illumina Human610-Quad SNP array and identified runs of homozygosity (RoH) of variable length (from 160 kb to nearly 2 mb) within and surrounding the HBB cluster only in HbSS patients. RoH were absent elsewhere in the genome in HbSS. The RoH that was shared by all HbSS patients was 126.6 kb in chr11:5153026-5279647 (NCBI36/hg18) and contains SNPs from rs11036090 to rs7118113 of the Illumina Human610-Quad SNP array. This region contains: OR51B4, the complete HBB cluster, and OR51V1. Homozygosity mapping in 6 Senegal haplotype homozygotes showed a slightly larger RoH from chr11:4909490-5314457 and SNPs rs840713-rs10837822. Both the Saudi patients and Senegal homozygotes had the same homozygous genotypes for the overlapping region of chr11:5205580-5235931 ranging from rs11036364 to rs5010981.To identify potential genetic modifiers of HbF level in the region detected in the Saudi cases, we sequenced areas within or near the Corfu deletion that is known to cause HPFH, the HBD-HBG1 intergenic region, and core regions of HS- 2, 3, and 4 in the LCR. Core regions of HS-3 and HS-4 were identical to the reference sequences. In the core of HS-2, the 10TA.2CA.2TA.CG.12TA motif was present. This motif is known to be associated with the SI haplotype but not with any other haplotypes. Within the region of the Corfu deletion, many polymorphisms were identified highlighting the complexity of SI haplotype and HBB haplotypes in general. Many of these polymorphisms lead to creation or abolition of transcription factor binding sites when this was examined in silico using the TFBS search program ConSite (consite.genereg.net). Some of these putative sites bind transcription factors presumed to have regulatory roles in globin gene expression. Complete sequencing of the 126.6 kb interval with comparison to other HBB haplotypes associated with high and low HbF might focus attention on areas of interest that can be examined in functional studies. Disclosures: No relevant conflicts of interest to declare.

Imaging Flow Cytometry for Fully Automated Quantification of Percentage of Sickled Cells in Sickle Cell Anemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2105-2105 ◽  
Author(s):  
Eduard J. van Beers ◽  
Leigh Samsel ◽  
Laurel G. Mendelsohn ◽  
Rehan Saiyed ◽  
James S Nichols ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
New Technique ◽  
Outcome Variable ◽  
Imaging Flow Cytometry ◽  
Automated Method ◽  
Shape Ratio

Abstract Abstract 2105 Introduction In preclinical and early phase pharmacologic trials in sickle cell anemia (SCA) the percentage of sickled cells after deoxygenation, in a so called sickling assay, has been used as an outcome variable. Although this sickling assay is a highly useful test, this method has the disadvantage of being subjective, operator-dependent and with low sensitivity and high variability due to the lack of automated method to quantitate the percentage of sickled cells from a large sample. Imaging flow cytometry is an emerging technology with potential to improve this assay. Therefore, we have explored the capability of this new technique to discriminate sickled cells from unsickledcells in this assay. Methods This study had regional ethics committee approval, and all patients gave written informed consent. To perform the sickling assay peripheral blood was drawn, diluted 1:100 with HemOx buffer (TCS Scientific Corp., Southampton, PA, USA) and aliquoted onto a 96 well plate and placed in a glovebox in hypoxic conditions (2% oxygen) for 2 hours (or as otherwise specified). After incubation, samples were fixed with glutaraldehyde, washed and placed on ice before analyzing the samples with imaging flow cytometry. Cells were analyzed on the ImageStreamx imaging flow cytometer (Amnis Corporation, Seattle, Washington, USA). Data were acquired using the INSPIRE acquisition software and using the 60X objective. Data from a minimum of 5000 cells were collected for each sample and analyzed using IDEAS 5.0 software. Single in-focus cells were identified using data from the brightfield images using various masks. As a learning population for the IDEAS software we hand tagged populations of sickled and unsickledcells. Results Using various combinations of pre-defined and self-defined masks we found that shape-ratio (the ratio between the shortest width of the mask divided by the longest part of the mask) identified our hand-tagged populations the best. We customized the algorithm with tight masks and a spot count feature to eliminate doublets and other artifacts. We were able to identify sickled or unsickled cells and a continuum between the two morphological extremes (figure 1a,1b). We selected three different shape ratio cut-off values for further analysis (table 1). To test the classifying algorithm, we spiked normal control blood with different amounts of SCA blood before incubation under variable hypoxia. At 3% oxygen the relation between percentage of sickled cells and percentage of SCA blood in the sample was strong (a Spearman rho for all cut-off values higher than 0.925) and significant (P≤0.001). At 4% oxygen the relation was less strong (Spearman rho higher than 0.725 for all cut-off values) but still significant (P≤0.05). As an additional validation, we found (as expected) lower percentages of hypoxia-induced sickling in blood from patients with SCA according to the level of fetal hemoglobin (HbF) expression (figure 1c). At all shape ratio cut-off values HbF percentage seems to suppress the amount of cells identified as sickled. While additional experiments are underway in an attempt to validate this finding, we preliminarily observe that fetal hemoglobin has a large effect on this flow cytometry sickling assay. Experiments assessing how sensitive this new technique is in detecting the effect of the anti-sickling agent 5-hydroxymethyl-2-furfural (Aes-103) are ongoing. Conclusion This study shows that imaging flow cytometryhas potential as a fully automated, operator independent method to quantify sickled cells in a sickling assay. While additional experiments are ongoing, our early data suggest that the presented technique seems discriminative enough to identify patient dependent and independent differences in sickling capacity of SCA red cells. Disclosures: No relevant conflicts of interest to declare.

Nitrite Levels Are Associated To Fetal Hemoglobin Concentration In Patients With Sickle Cell Anemia: Implications In Inflammatory Process

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4666-4666
Author(s):  
Thassila Nogueira Pitanga ◽  
Bruno A. V. Cerqueira ◽  
Wendell Vilas Boas ◽  
Sanzio S. Santana ◽  
João O. Reis ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
High Performance ◽  
Conflicts Of Interest ◽  
Density Lipoprotein ◽  
Fetal Hemoglobin ◽  
Hemoglobin Concentration ◽  
Serum Levels ◽  
Chronic Inflammatory Disease ◽  
Control Group

Introduction Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises. Aims The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil. Methods Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients. Results Our results showed a significant increase in NO-2 (p<0.0001) in adult patients with SCA compared to the control group (healthy volunteers). Data demonstrated a negative correlation between nitrite and fetal hemoglobin (p=0.004, r=-0.627). No correlation was found between fetal hemoglobin or nitrite with LDL-Cl and triglycerides. Conclusion The results reinforce that the serum nitrite can predict the clinical disease course. Disclosures: No relevant conflicts of interest to declare.

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