Journal articles: 'HBF 4'

1

Alsultan, Abdulrahman, Duyen A. Ngo, John J. Farrell, et al. "Co-Inheritance of Delta Thalassemia Might Contribute to the High Fetal Hemoglobin in Sickle Cell Anemia Patients with the Saudi-Indian Haplotype." Blood 118, no. 21 (2011): 1056. http://dx.doi.org/10.1182/blood.v118.21.1056.1056.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

2

Kesuma, Suryanata, and Elita Octavia. "Gambaran Fraksi Hemoglobin Penderita Talasemia Menggunakan Metode Elektroforesis Kapiler." Meditory : The Journal of Medical Laboratory 6, no. 2 (2019): 116–24. http://dx.doi.org/10.33992/m.v6i2.450.

Full text

Abstract:

Thalassemia is a hereditary blood disease that is found in Indonesia. One method of examining modern thalassemia is the examination of hemoglobin fraction using the Capillary Electrophoresis. This method has a high level of accuracy and precision for quantification of hemoglobin variants.The purpose of this study was to determine the hemoglobin fraction in thalassemia patients using the Capillary Electrophoresis method. This type of research is descriptive research. The sample of this study was 3 blood specimens from thalassemia sufferers.The results showed that the first specimen, age 4 years had HbA levels of 59.9%, HbA2 levels of 4.3%, HbF levels of 14.7% and HbE levels of 21.1%. In the second specimen, 8 years of age had 88.7% HbA, 2.5% HbA2, 3.2% HbF and 5.6% HbE. In the third specimen, the age of 13 years had HbA levels of 93.8%, HbA2 levels of 4.9% and HbF levels of 1.3%.The conclusion of this study was the first specimen, HbA levels decreased, HbA2 levels increased and HbF levels increased and hemoglobin variants were found, namely HbE. In the second specimen, HbA levels decreased from the normal range, HbA2 levels were in the normal range and HbF levels increased and hemoglobin variants were found, namely HbE. In the third specimen, HbA levels decreased from the normal range, HbA2 levels and HbF levels increased, but no hemoglobin variants were found in this specimen.

APA, Harvard, Vancouver, ISO, and other styles

3

Sverrisson, Kristinn, Josefin Axelsson, Anna Rippe та ін. "Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with α1-microglobulin and tempol". American Journal of Physiology-Renal Physiology 306, № 4 (2014): F442—F448. http://dx.doi.org/10.1152/ajprenal.00502.2013.

Full text

Abstract:

Extracellular fetal hemoglobin (HbF) and adult hemoglobin (HbA) are proinflammatory and generate ROS. Increased plasma levels of extracellular HbF have recently been reported to occur in early preeclampsia. α1-Microglobulin (A1M) is a physiological heme-binding protein and radical scavenger that has been shown to counteract vascular permeability increases induced by HbA in the perfused placenta. The present study was performed to investigate whether HbF and HbA will increase glomerular permeability in vivo and to test whether A1M and tempol, a ROS scavenger, can prevent their effects. Anesthetized Wistar rats were continuously infused intravenously with either HbA, HbF, or cyano-inactivated HbF together with FITC-Ficoll-70/400, inulin, and 51Cr-labeled EDTA for 2 h. Plasma samples and urine samples (left ureter) were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients for Ficoll of radius 10–80 Å. In separate experiments, A1M or tempol was given before and during Hb infusions. Extracellular HbF caused rapid, transient increases in glomerular permeability to large Ficoll molecules (50–80Å), contrary to the effects of HbA and cyano-inactivated HbF. For HbF, glomerular sieving coefficients for Ficoll of radius 60Å increased from 3.85 ± 0.85 × 10−5 to 2.60 ± 0.96 × 10−4 at 15 min, changes that were abrogated by tempol and reduced by A1M. In conclusion, our data demonstrate that extracellular HbF, infused systemically, can acutely increase glomerular permeability through inducing oxidative stress.

APA, Harvard, Vancouver, ISO, and other styles

4

Alsultan, Abdulrahman, Idowu Akinsheye, Nadia Solovieff, et al. "Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of Saudi Patients From the Eastern Province." Blood 116, no. 21 (2010): 1627. http://dx.doi.org/10.1182/blood.v116.21.1627.1627.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

5

Sebastiani, Paola, John J. Farrell, Shuai Wang, et al. "BCL11A enhancer Haplotypes Are Associated with the Distribution of HbF in Arab-Indian and African Haplotype Sickle Cell Anemia but Not the Different Population Levels of HbF." Blood 124, no. 21 (2014): 4066. http://dx.doi.org/10.1182/blood.v124.21.4066.4066.

Full text

Abstract:

Abstract Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia. In the Middle East and India the HbS gene is often on an Arab-Indian HBB haplotype that is associated with high HbF levels. HbF is “normally” distributed in this population with a mean ~20%. In African HbS haplotypes, HbF levels are much lower (mean value ~6%) with a highly skewed distribution. BCL11A is an important modulator of γ-globin gene (HBG2 and HBG1) expression and BCL11A is regulated by erythroid specific enhancers in its 2nd intron. The enhancers consist of 3 DNase hypersensitive sites (HS) +62, +58 and +55 kb from the transcription initiation site of this gene. Polymorphisms (SNPs) in these enhancers are associated with HbF. The strongest association with HbF levels in African Americans with sickle cell anemia was with rs1427407 in HS +62 and to a lesser extent, rs7606173 in HS+55. Using the results of whole genome sequencing of 14 AI haplotype patients—half with HbF <10%, half with HbF >20%—6 SNPs in the BCL11A enhancer region, rs1427407, rs7599488, rs6706648, rs6738440, rs7565301, rs7606173 and 2 indels rs3028027 and rs142027584 (CCT, CCTCT and AAAAC respectively), were detected as possibly associated with HbF level. There were no novel polymorphisms detected. We genotyped the 6 SNPs and studied their associated haplotypes in 137 Saudi (HbF18.0±7.0%) and 44 Indian patients (HbF23.0±4.8%) with the Arab-Indian HBB haplotype; 50 African Americans with diverse African haplotypes, including 4 Senegal haplotype heterozygotes, (20 with HbF 17.2±4.6% and 30 with HbF 5.0±2.5%) and imputed genotypes for these SNPs in 847 African Americans with sickle cell anemia and diverse haplotypes (HbF 6.6±5.5%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) in the HS sites showed consistent associations with HbF levels in all 4 cohorts. Haplotype analysis of these 4 SNPs showed that there were 4 common and 10 rare haplotypes. The most common, GCAG, was found in ~54% of Arab-Indian haplotype carriers (HbF, ~20%) and in ~33% of African origin haplotype carriers (HbF, ~5.5%). Two haplotypes, GTAC and GTGC, were carried by ~40% of African American patients and were associated with lower levels of HbF (3.6%-4%). These same haplotypes were carried by 18% of Arab-Indian haplotype carriers and their average HbF level was 17%. These differences were significant. Haplotype TCAG was present in 20% of Arab-Indian and 25% of African haplotype cases, and carriers had on average higher HbF levels (~22% in the Arab-Indian haplotype, ~8% in African Americans). The analysis shows that: BCL11A enhancer haplotypes are differentially distributed among patients with the HbS gene on Arab-Indian or African origin haplotypes; haplotype pairs TCAG/TCAG and GTAC/GTGC are associated with the highest and lowest HbF levels in all the studied groups; the population-specific prevalence of HbF BCL11A enhancer haplotypes are likely to explain the different distributions of HbF in African origin and Arab-Indian haplotypes but do not account for the differences in average population levels of HbF or the high HbF of the Arab-Indian haplotype. Novel SNPs in BCL11A do not explain the high HbF of the Arab-Indian haplotype. Other important loci must have a predominant role in the differential expression of HbF among HbS Arab-Indian haplotype carriers. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

6

Pongpaksupasin, Phitchapa, Tiwaporn Nualkaew, Suradej Hongeng, Suthat Fucharoen, Natee Jearawiriyapaisarn та Orapan Sripichai. "Lysine-Specific Histone Demethylase 1 Inhibition Enhances Robust Fetal Hemoglobin Induction in Human β0-Thalassemia/Hemoglobin E Rrythroid Cells". Hematology Reports 13, № 4 (2021): 9215. http://dx.doi.org/10.4081/hr.2021.9215.

Full text

Abstract:

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.

APA, Harvard, Vancouver, ISO, and other styles

7

Alsultan, Abdulrahman, Nadia Solovieff, Aamer Aleem, et al. "Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of Saudi Patients From the Southwestern Province." Blood 116, no. 21 (2010): 1621. http://dx.doi.org/10.1182/blood.v116.21.1621.1621.

Full text

Abstract:

Abstract Abstract 1621 Sickle cell disease (SCD) is common in the Eastern and Southwestern (SW) Provinces of Saudi Arabia. Patients from the SW Province have many complications of this disease but they have phenotypic differences from African Americans that include a high prevalence of splenomegaly, rare CNS disease and absence of leg ulcers. In contrast, Eastern Province patients have a milder phenotype that is related to a nearly uniformly high fetal hemoglobin (HbF) level that is associated with the Saudi-Indian haplotype of the HBB gene-like cluster. SW Province patients have variable HbF levels and do not have the Saudi-Indian haplotype. We studied patients from the SW Province to determine the associations of known HbF quantitative trait loci (QTL) with HbF concentration. Seventy-seven Saudi patients, aged ≥4 yrs, with SCD were studied, 53 HbS homozygotes and 24 with HbS-β0 thalassemia. Their age was 17.7±10 (range 4–46) yrs and 45% were on hydroxyurea. HBB gene cluster haplotypes were 58 (75%) Benin, 17 (22%) Bantu, 1(1.5%) Senegal/Bantu, and 1(1.5%) Senegal. HbF level was measured by capillary electrophoresis and we used the lowest HbF level after age 4 yrs, the age where HbF levels stabilized, for analysis. Genotyping was done by TaqMan SNP genotyping assays, and included SNPs in BCL11A (rs4671393, rs766432), HBS1L-MYB (rs28384513, rs9399137, rs4895441), and OR51B5/6 (rs5006884). Results are summarized in the Table. QTLs showed similar trend in their effects on HbF level when patients on hydroxyurea were excluded from analysis. BCL11A was the sole QTL associated with HbF level in Saudi patients from the SW Province whereas the HBS1L-MYB and OR51B5/6 loci had no effect. For comparison, we selected cases from 2 studies of African American with SCD. All were HbS homozygotes and none took hydroxyurea, and we compared them to Saudi patients who were HbS homozygotes and not on hydroxyurea (n=29). Compared with African Americans with similar HBB haplotypes, and after adjusting for the BCL11A genotype, Saudi cases from SW Province had HbF levels almost twice that of African Americans (p <0.0001). Given that Saudi and African American patients had a nearly identical distribution of HBB gene cluster haplotypes, we examined ancestral origin of Saudi and African American patients. Using a genome-wide set of SNPs, we performed a principal component analysis with the Saudi, the African Americans with SCD and African, Arab, Asian and European populations from the Human Genome Diversity Project. We estimated the Fst statistic between these populations which is defined as the proportion of genetic diversity due to allele frequency differences among populations and can be interpreted as the distance between populations. African American cases were in close proximity to Yoruban, Mandenka and Bantu populations while Saudi patients resembled Arab populations. African American patients were the farthest from Saudi patients as compared with Asian and European populations. The commonality of HBB haplotypes in these Saudi cases and African Americans, coupled with the genetic distance between these populations suggest that genetic modifiers remote from the HBB cluster or unknown environmental influences are likely to account for the higher HbF in these Saudi patients. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

8

Vathipadiekal, Vinod, Abdulrahman Alsultan, John Farrell, et al. "Polymorphisms Associated with the Arab-Indian Haplotype of Sickle Cell Anemia Are Candidate Fetal Hemoglobin Gene Modulators." Blood 126, no. 23 (2015): 3388. http://dx.doi.org/10.1182/blood.v126.23.3388.3388.

Full text

Abstract:

Abstract Fetal hemoglobin (HbF) inhibits HbS polymerization. Because of this, sufficient HbF in most sickle erythrocytes can lead to a milder disease phenotype. HbF levels differ amongst the β-globin gene (HBB) cluster haplotypes of sickle cell anemia. In the Arab-Indian (AI) haplotype, HbF was about 20% compared with 5-10% in the Bantu, Benin, and Senegal haplotypes. Functional elements linked to the HBB haplotype are likely to regulate the expression of HbF in addition to the effects of trans-acting modulators. To identify cis-acting SNPs in the HBB gene cluster that differentiate the AI haplotype from all others, including the Senegal haplotype-the Senegal haplotype shares some SNPs with the AI haplotype but its carriers have lower HbF-we studied patients with sickle cell anemia who were homozygous for HBB haplotypes by genome-wide SNP association analysis (GWAS; Table). First, we compared the results of GWAS of 42 Saudi AI haplotype homozygotes with GWAS in 71 Saudi Benin haplotype homozygotes. The only variants distinguishing these 2 populations with genome-wide significance (p-values between 9.6E-07 and 2.7E-45) were 223 SNPs in chromosome 11p15 from positions 3.5 to 6.5 mb. This region included the HBB gene cluster, its locus control region (LCR) and the upstream and downstream olfactory receptor gene clusters. The minor allele frequency of SNPs in MYB (chr 6q23), BCL11A (chr 2p16) and KLF1 (chr 19), trans-acting loci that affect expression of the HbF genes, were similar in these 2 cohorts. A novel candidate trans-acting locus was not found, however our power to detect such an association was low. We followed-up these observations by comparing allele frequencies in 303 African American cases homozygous for the haplotypes shown in the Table. Thirteen GWAS-significant SNPs, in addition to rs7482144 and rs10128556, were present in all AI haplotype cases but not in 83 Senegal haplotype chromosomes. The allele frequency of these SNPs was replicated in 62 independent AI haplotype cases. Rs2472530 is in the coding region of OR52A5; rs16912979, rs4910743 and rs4601817 are in the HBB gene cluster LCR; rs16912979 in DNase I hypersensitive site-4 altered motifs for POLR2A, GATA1, and GATA2 binding.The minor allele of rs10837771 causes a missense mutation in OR51B4 an upstream olfactory receptor gene. To see if any of these or other alleles might sometimes be associated with HbF in the Bantu and Benin haplotyes, we selected homozygotes and compound heterozygous for these haplotypes who had unexplained and uncharacteristically high HbF. Thirty-one African Americans, aged ≥5 yrs. who had a HbF of 21% were compared with 350 similar cases who had a mean HbF of 3%. Four additional SNPs on chromosome 11, from positions ranging from 5536415 to 5543705 in the UBQLNL/HBG2, region and present in 45-48% of AI haplotype and 3-13% of other haplotypes, were found at higher frequencies in the high HbF group compared with the low HbF group. These SNPs also altered transcription factor binding motifs. Loci marked by SNPs that distinguish the AI from the Senegal and other HBB haplotypes might contain functionally important polymorphisms and account in part for high HbF in AI haplotype sickle cell anemia, independent of, or in addition to, the effects associated with rs7482144 or rs10128556. They might also be rarely associated with high HbF found in other haplotypes. These observations provide a foundation for mechanistic studies focused on the role of these variants in the expression of their linked HbF genes.Table 1.non-codedallelegenomic locationSaudi AI(n=42)Saudi ben.ben(n=71)AA ben.ben(n=264)AA ban.ban(n=31)AA sen.sen(n=8)HbF (%)1711669rs10837771Gexon OR51B410.020.0200rs4601817GLCR10.020.0400rs4910743CLCR10.010.0100rs16912979CLCR00.960.920.111rs10488675Gintron HBE110.01000rs7482144*AHBG210001rs10128556#TIntron HBBP110001rs7935470COR51V110.020.0300rs10837582GOR51V1100.0200rs11036227TOR51V110000rs10734485COR51A1P00.990.9711rs10837461AOR52A110.01000rs2472522GOR52A110.01000rs2472530Gexon OR52A510.01000rs2499948TOR52A510.020.010.020Allele frequencies in haplotypes of sickle cell anemia. * Xmn1 5' HBG 2 restriction site. This SNP, not present on the SNP microarray, was genotyped independently; # LD with rs7482144; AA designates African Americans; ben-Benin; ban-Bantu; sen-Senegal. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

9

Rok, M., P. Szklarz, and G. Bator. "Structures and phase transitions in molecular complexes containing tetrafluoroboric acid and tetramethylpyrazine." CrystEngComm 20, no. 38 (2018): 5772–81. http://dx.doi.org/10.1039/c8ce01231h.

Full text

Abstract:

Crystals of tetrafluoroboric acid with tetramethylpyrazine, TMP + HBF4 + H2O (1 : 1 : 2, A), TMP + HBF4 (1 : 1, B) and TMP + HBF4 (1 : 2, C) were synthesized and characterized by physicochemical experimental methods.

APA, Harvard, Vancouver, ISO, and other styles

10

Carol Illa, Ariadna, Jesper Petersen, Søren Skov, Andreas Glenthoej, and Carsten Dan Ley. "Is Epigenetic Modulation to Induce Fetal Hemoglobin Translatable in Sickle Cell Mice?" Blood 142, Supplement 1 (2023): 5261. http://dx.doi.org/10.1182/blood-2023-173922.

Full text

Abstract:

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the β-globin subunit of hemoglobin, resulting in the formation of HbS. Under deoxygenation, HbS polymerizes leading to red blood cell sickling, increased hemolysis and multi-organ damage. During human fetal development, due to expression of the γ-globin gene, the predominant type of hemoglobin produced is fetal hemoglobin (HbF). However, after birth, this shifts to adult globin (HbA or HbS in SCD). This is driven by epigenetic repression of γ-globin due to an increased methylation of its gene promoters. DNA methyltransferase 1 (DNMT1) is an important enzyme for DNA methylation and is known to be pivotal in regulation of HbF expression. Reactivation of HbF provides a functional hemoglobin, and therefore has the potential to treat SCD symptoms ( Steinberg et al. JAMA 2003;289:1645-51). HbF induction is an accepted treatment strategy in SCD; even small increases result in decreased vaso-occlusive crises and mortality ( Kato et al. Nat Rev Dis Primers 2018;4:18010). Decitabine inhibits DNMT1 activity, thus promoting γ-globin expression and thereby increasing HbF levels. While inhibition of DNA methylation is known to cause HbF induction in humans and non-human primates, the effects observed in SCD mouse models are more ambiguous. This study investigated to which extent decitabine treatment could induce HbF expression in the Townes SCD model. In Townes mice, the murine globin genes have been substituted for their sickle human counterparts, HBA, HBB sickle and HBG1, and mimic many features of human SCD. Mice aged 4-5 weeks were dosed subcutaneously 3 times a week for 12 weeks with research-grade decitabine (0.6 mg/kg or 0.4 mg/kg) or vehicle. Blood samples were collected for analysis of HbF protein levels by HPLC (represented as % of total Hb) and F-cells by flow cytometry (represented as % of the total RBC population positive for HbF), complete blood counts, histology, and other markers of disease and organ damage. Results are presented as mean ± SD. Groups were compared using an ANCOVA model, adjusting for multiple comparisons (Dunnett) with p&lt;0.05 considered statistically significant. For both treatment groups, HbF was significantly elevated compared to the control group, with the maximal response observed at 12 weeks (Figure 1). F-cells at the end of the study were increased in both 0.4 mg/kg (11.4±1.5%) and 0.6 mg/kg (11.8±1.4%) groups compared to vehicle (4.5±0.6%). The same was observed for protein levels: 0.4 mg/kg group (1.2±0.3%), 0.6 mg/kg (1.4±0.3%) compared to vehicle (0.4±0.1%). Interestingly, after 12 weeks RBC and reticulocyte counts were significantly decreased in treatment groups and mean corpuscular volume (MCV) was significantly increased. No changes on LDH, bilirubin and liver markers (ALP, ALT, AST) were observed at 6 or 12 weeks. The results showed that treatment groups had significantly higher HbF levels than the vehicle group. However, the level of induction was lower than observed for inhibition of DNA methylation in humans and primates. The decrease in RBCs and reticulocytes could be partially explained by a combination of HbF induction, reduction of sickle RBC, and inhibition of erythropoiesis. The lack of changes in disease markers suggests that the increase in HbF levels in Townes mice might not be sufficient to alleviate the disease. This observation is consistent with recently published data (Woodard et al. Dis Model Mech. 2022;15(6)) that indicates that Townes mice are less suitable for studying this mechanism of action since, even though they have all human globin genes and some proximal regulatory elements, they lack key cis-acting distant DNA elements that regulate the globin genes. However, in primates the globin structure and regulation mechanisms are similar to human (Hardison. Cold Spring Harb Perspect Med. 2012;2(12)). In conclusion, although the measured levels of HbF protein were no higher than 2%, we found that decitabine can induce HbF expression in the Townes mouse model of SCD. However, within the 12-weeks, there was no clear beneficial effect on the disease markers studied. The successful induction of HbF suggests that better outcomes might be achieved with further optimization of the dosing and duration of the study. This indicates that SCD mice may be useful for preclinical studies of HbF induction, but caution is needed when extrapolating results to humans.

APA, Harvard, Vancouver, ISO, and other styles

11

Hebert, Nicolas, Rakotoson Marie Georgine, Gwellaouen Bodivit, et al. "Fetal Hemoglobin Measurement per Red Blood Cell Provides Biological and Clinical Protective Thresholds." Blood 134, Supplement_1 (2019): 1008. http://dx.doi.org/10.1182/blood-2019-126727.

Full text

Abstract:

Introduction: Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity by decreasing the HbS polymerization. However, the distribution of HbF in red blood cells (RBC) is heterogeneous in SCD patients. In the hypothesis of an HbF "threshold" in RBCs for inhibiting the HbS polymerization, accurate measurement of the HbF content in each red blood cell (HbF/RBC) is mandatory. To this purpose we developed a new and accurate method allowing the direct measurement of HbF content per RBC. Thanks to it, as a proof of concept, we analyzed HbF distribution and content in RBC from SCD patients before and after 6 months of treatment by hydroxyurea (HU). To determine if a threshold of HbF/RBC could modulate SCD, we analyzed the associations between the %RBC reaching different thresholds of HbF (in picograms), and biological parameters and the incidence of severe VOC. Methods: 14 SCD (βS/βS or βS/β0) patients were included to study HbF distribution during HU for a period of 6 months. RBCs were collected during each outpatient visit (Week 0, Week 2, Week 4, Week 12, and ≥ Week 24). HbF content was measured in RBCs using an anti-Human-HbF antibody by flow cytometry. Normalized RBC fluorescence intensity was then converted in picograms of HbF/RBC by using the linear association between mean HbF content and mean RBC fluorescence obtained from subjects presenting homogeneous HbF distribution (patients with hereditary persistence of HbF (HPFH), or β-Thalassemia or δβ-Thalassemia). Quantitative analysis were performed before and during HU treatment to characterize the response by comparing percentages of RBC classes based on different ranges of HbF/RBC during HU treatment. We therefore analyzed the associations between HbF/RBC thresholds (%RBC containing at least 2, 4, 6, 8, 10 or 20 pg HbF) and biological parameters before and ≥ 6 months of HU treatment. Finally HbF/RBC thresholds at Week 0 were compared to the incidence of hospitalized VOC within 3 years before W0, and HbF/RBC thresholds at Week 24 were compared to the incidence of hospitalized VOC within 3 years after W24 at a stable dose. Results: After 6 months of HU, mean %HbF, assessed by HPLC, raised from 6.16% (±3.5) to 15.2% (±8.7) (mean ± standard deviation). Quantitative analysis of HbF/RBC revealed a statistically significant decrease between D0 and ≥M6 of 24% of RBCs containing less than 2 pg (p = 0.0015) and a 2-fold increase of RBCs containing between 2 and 4 pg (p = 0.0025) (Friedman test). For biological parameters we observed an increase in mean %HbF, MCV and MCH and a decrease in RBC count significantly associated (p<0.001 - Spearman test) with %RBC containing ≥ 2 pg of HbF. The incidence of VOC within 3 years after HU treatment was not statistically significant than during the 3 years before (p = 0.4414 - Wilcoxon test). VOC incidence under treatment decreased in 6/14 patients, did not change in 4/14 and increased in 4/14. The incidence of VOC over 3 years was not associated with the %HbF assessed by HPCL (r = -0.0358; p = 0.8564 - Spearman test). We observed a statistically significant correlation between the incidence of VOC over 3 years and the HbF threshold of 4 pg (r = -0.5068; p = 0.0059). We therefore determined the percentage of RBCs by thresholds of HbF, associated to ≤ 1 VOC over 3 years (Table 1). For example, if more than 20% RBCs have ≥ 4 pg of HbF, we calculated a sensitivity and a specificity of 58.3% and 100% respectively, and a positive and a negative predictive value of 100.0% and 76.2% respectively, to have ≤ 1 VOC over 3 years. Conclusion: Our results strengthen the hypothesis that the percentage of RBC above a threshold of HbF is the important parameter to measure. These results need to be replicated in a larger cohort but they open up interesting prospects for analysis of new therapeutic efficacy, including gene therapy and HbF inducers. Disclosures Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Agios: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

APA, Harvard, Vancouver, ISO, and other styles

12

Park, So Hyun, Ciaran M. Lee, Yankai Zhang, Alicia Chang, Vivien A. Sheehan та Gang Bao. "Engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP2) with Sickle or β-Thalassemia Mutation: An in-Vitro System for Testing Pharmacological Induction of Fetal Hemoglobin". Blood 132, Supplement 1 (2018): 3478. http://dx.doi.org/10.1182/blood-2018-99-114241.

Full text

Abstract:

Abstract Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia. Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A<T) or codon 6 [-G] β-thalassemia mutation into the HBB locus of HUDEP2 cells. Edited HUDEP2 cells were single-cell sorted into multiple 96-well plates and expanded. The genotype of the clones was determined using a probe-based droplet digital PCR assay and confirmed through Sanger sequencing. Native polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC) were used to confirm the hemoglobin phenotype. Normal parental cell line, sickle clone, and two individual β-thalassemia clones were used to test the pharmacological induction of HbF. We initiated drug treatment in the expansion phase with 30 µM hydroxyurea. Trypan Blue staining and CD71/CD233/CD235 staining determined the effect of the drugs on the viability, growth rate and erythroid development of HUDEP2 lines. After 10 days of drug treatment, differentiated HUDEP2 were analyzed for globin expression through RT-qPCR and HPLC, and HbF positive cells (F-cells) were quantified via flow cytometry. Cells were placed at 2% O2 for four hours, fixed in glutaraldehyde, stained, and viewed under magnification to assess sickling potential. Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A<T) and codon 6[-G] β-thalassemia HUDEP2 clones, respectively. No change was observed in CD71/CD235 positive HUDEP2 cells in the presence hydroxyurea. This finding demonstrated that hydroxyurea treatment induces HBG expression as well as HbF and F-cells in engineered sickle and β-thalassemia HUDEP2 clones. Future work will include screening other pharmacological compounds as well as studying the mechanism of HbF induction by using HUDEP2 clones. Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

13

Shang, Shengzhe, Sangam G. Goswami, Xia Li, Tin Truong, David C. Williams, and Gordon Ginder. "Association of LRF with MBD3-NuRD Versus MBD2 NuRD Mediates Opposing Effects on Developmental Globin Gene Regulation." Blood 144, Supplement 1 (2024): 413. https://doi.org/10.1182/blood-2024-203712.

Full text

Abstract:

Sufficiently high levels of HbF have been shown to ameliorate the pathophysiologic effects of HbS in sickle cell anemia patients bearing the homozygous codon 6 GAG to GTG mutation in the beta globin gene (HBB).Leukemia Related Factor (LRF, also known as ZBTB7A), BCL11A, and the MBD2-NuRD complex are major mediators of HbF silencing in adult erythroid cells, and combined depletion of both LRF and BCL11A was shown to result in nearly 100% HbF in adult erythroid cells (Masuda, etal, Science 2016). Both LRF and BCL11A are associated with a NuRD complex. In the case of BCL11A we have shown that this is specifically MBD2a-NuRD, and that silencing results from positioning of a nucleosome over the proximal gamma globin gene (HBG) promoter and is at least partially dependent on promoter CpG methylation. (Shang etal, Proc.Natl.Acad.Sci. USA, 2023). To define the specific NuRD complex that participates in LRF mediated HBG silencing we have investigated the roles of both MBD2-NuRD and MBD3-NuRD.NOME SEQ assays performed in MBD2 KO HUDEP-2 cells, showed the loss of a nucleosome positioned over the -189 GATA-1 site binding, which has been shown to be critical for HBG expression, and at which GATA-1 binds when the -198 HBG promoter binding site for LRF is mutated ( Doerfler, etal, Nature Genet., 2021). This suggests that MBD2-NuRD associates with LRF to silence HBG expression, consistent with the lack of effect of MBD3 depletion on HBG expression. When both MBD2 and MBD3 were knocked out in HUDEP-2 cells, over 90% HBG/ (HBG+HBB) RNA levels were observed, similar to the levels observed with knockout of both LRF and BCL11A. However, the increase in HBG/(HBG+HBB) RNA ratio and corresponding HbF/ (HbF+HbA) ratio were due primarily to a 4-5-fold decrease in HBB RNA and protein, as the level of HBG RNA was the same or slightly less than in MBD2 KO cells.Chromatin conformation capture (3C) assays showed that combined depletion of MBD2 and MBD3 resulted in significantly decreased interaction of the Locus Control Region (LCR) with the HBB gene, increased LCR interaction with the BGLT3 locus and a very large increase in interaction of the 3' beta globin locus enhancer with the HBB gene. These results demonstrate that MBD3-NuRD plays a critical role in the interaction of the LCR with the HBB gene to maintain its high level of expression in adult erythroid cells. Depletion of LRF in MBD2 KO HUDEP-2 cells resulted in the same &gt;90% HBG/(HBG+HBB) RNA level, as well as the same decrease in HBB RNA as depletion of MBD3. LRF depletion alone in WT HUDEP-2 cells also resulted in a significant decrease in HBB expression without a significant increase in HBG expression. This contrasts with the effect of mutation of the -198 LRF binding site in the HBG promoter, which results in high levels of HBG expression without a significant decrease in HBB expression ( Antoniou, etal, Nature Comm.,2022). Immunoprecipitation assays showed that LRF interacts with either MBD2-NuRD or MBD3-NuRD. These results are consistent with the fact that the repressive action of LRF, like that of BCL11A, requires association specifically with MBD2-NuRD to position a nucleosome and block the binding of a positive acting transcription factor, in this case GATA-1. Accordingly, disruption of MBD2-NuRD results in high level HBG expression while disruption of MBD3-NuRD does not, as we and others have shown. Conversely, LRF in association with MBD3-NuRD is required for maximum expression of the HBB gene in adult erythroid HUDEP-2 cells. Since combined disruption of the BCL11A-MBD2-NuRD complex and the LRF-MBD3-NuRD complex results in nearly 100% Hb F levels, these findings have implications for the treatment of sickle cell anemia, in which maximizing HbF levels while lowering HbS levels should provide the optimum protection from RBC sickling and its pathophysiologic consequences.

APA, Harvard, Vancouver, ISO, and other styles

14

Bohmer, Ralph M., Thomas A. Campbell, and Diana W. Bianchi. "Selectively increased growth of fetal hemoglobin-expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta." Blood 95, no. 9 (2000): 2967–74. http://dx.doi.org/10.1182/blood.v95.9.2967.009k21_2967_2974.

Full text

Abstract:

We have studied the effect of transforming growth factor beta (TGFβ) on erythropoiesis in cultures from adult peripheral blood, using flow cytometric enumeration of fetal hemoglobin (HbF)-containing cells. TGFβ caused a dramatic increase in the proportions of cells that accumulated HbF together with adult hemoglobin (HbA) (F+A+ cells). This highly significant (P &lt; .0001) increase in F+ cell proportion was achieved by TGFβ treatment during the first 4 days of culture and was sustained during further culture expansion in the absence of TGFβ. The increase in F+ cell proportions did not depend on the cytokine combination (EPO+SCF+IL3, EPO+SCF, EPO+IL3, SCF+IL3) used during the phase of TGFβ treatment. Increased F+ cell proportions were paralleled by an increased molecular ratio of HbF/ HbF+ HbA, measured by cation exchange high-performance liquid chromatography (HPLC). In addition to the effect on F+ cell proportions, TGFβ caused a dramatic increase in overall cell division potential. By the time cultures reached terminal growth arrest (12-14 days in controls and 18-26 days after TGFβ), the overall numbers of F+ cells produced per initially seeded clonogenic cell was approximately 10 times higher in the TGFβ-treated cultures than in the controls. We propose to investigate whether the TGFβ-induced increase in relative and absolute numbers of nucleated F+ cells, as demonstrated in vitro, can be translated into increased F+ erythrocytes in vivo, allowing therapeutic application for some beta-hemoglobinopathies.

APA, Harvard, Vancouver, ISO, and other styles

15

Walters, Mark C., David H. K. Chui, John J. Farrell та ін. "Response of Patients with Transfusion-Dependent β-Thalassemia (TDT) to Betibeglogene Autotemcel (beti-cel; LentiGlobin for β-Thalassemia) Gene Therapy Based on HBB Genotype and Disease Genetic Modifiers". Blood 136, Supplement 1 (2020): 1–3. http://dx.doi.org/10.1182/blood-2020-137642.

Full text

Abstract:

Introduction We investigated the impact of β-thalassemia genotypes and disease genetic modifiers including HBA and KLF1 genotype and sentinel single-nucleotide polymorphism (SNP) genotypes at 3 major HbF quantitative trait loci (QTL) on clinical outcomes of TDT patients treated with beti-cel gene therapy in two phase 3 studies, HGB-207 (NCT02906202) and HGB-212 (NCT03207009). Methods HBA deletions and triplications were determined by gap-polymerase chain reactions. HBG2 (including rs7482144, Xmn1 site) and HBG1 promoters, HBA2, HBA1, and KLF1 underwent individual nucleotide sequencing. Multiplex amplification refractory mutation system (ARMS) tests were used to identify HbF QTL SNPs (rs10128556 in HBBP1; rs766432, rs1427407, rs10189857 in BCL11A; rs9399137, rs66650371 in HMIP). Thalassemia severity score (TSS) was calculated as defined by Danjou et al, Haematologica, 2015, considering gender, HBB and HBA genotypes, and 4 SNPs in HbF QTL (HBG2, BCL11A, HMIP). Correlative analyses were performed to assess relationships between genotype, presence/absence of non-HBB mutations (HBA2, HBA1, KLF1), presence/absence of HbF QTL SNPs (HBG2, BCL11A, HMIP), and TSS with the achievement of transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without red blood cell [RBC] transfusions for ≥12 months). Correlation coefficients used percentage bend correlation. Statistical significance threshold was p ≤ 0.05. Results As of 3 March 2020, 38 patients were treated in HGB-207 and HGB-212 (β0/β0 genotype n=9; non-β0/β0 genotype n=29 [β+/β+ n=8; β0/β+ n=15; βE/β0 n=6]). All patients were heterozygous or homozygous for mutations or SNPs that may modulate disease severity; 20 patients were homozygous for ≥1 mutation or SNP. Patients had the following alleles associated with higher HbF synthesis: HBG2 rs7482144 C&gt;T (Xmn1 site), C/T n=9, T/T n=1; BCL11A rs1427407 G&gt;T, G/T n=8; BCL11A rs10189857 A&gt;G, A/G n=16, G/G n=18; HMIP rs9399137 T&gt;C, T/C n=9, C/C n=2. Three patients were heterozygous for single α-globin gene deletion (-α/αα) and 2 were heterozygous for α-globin gene triplication (αα/ααα). Median TSS was 3.65 (min - max 0.4 - 8.1). TI was achieved by 23/27 (85%) evaluable patients; 4 patients with ≥ 12 months follow-up have been transfusion free for &gt; 10 months but were not yet evaluable for TI (Figure). β-thalassemia genotype did not strongly correlate with TI (two-sided Fisher's Exact Test, p-value = 0.78). Month 12 median (min - max) peripheral blood vector copy number (PB VCN) was 1.5 (0.2 - 5.0) c/dg in TI or transfusion-free patients (n=27) and 0.2 (0.2 - 0.4) c/dg in patients who did not achieve TI (n=4). The transfusion-free patient (β0/β0) with the lowest month 12 PB VCN was homozygous for T/T at rs7482144 (HBG2Xmn1 site) and G/G at rs10189857 (BCL11A), and heterozygous for single α-globin gene deletion. Endogenous Hb (5.9 g/dL HbF + 0.2 g/dL HbA2) and gene therapy-derived HbAT87Q (4.4 g/dL) at month 12 enabled this patient to stop transfusions. Tests of association of SNPs and mutations with TI were not significant; no p-value &lt; 0.31 (chi-squared test). As only 4 patients did not achieve TI, the power to detect an association was limited. Larger sample sizes are needed to determine if individual SNPs and mutations may have an impact on TI. In TI or transfusion-free patients (n=27), TSS correlated strongly with month 12 endogenous unsupported Hb (HbA + HbA2 + HbF + HbE without RBC transfusions for 60 days) (correlation coeff. = -0.76, p &lt; 0.0001), but not with HbAT87Q (correlation coeff. = 0.26, p = 0.19) or unsupported total Hb (correlation coeff. = 0.32, p = 0.10). Beti-cel-related adverse events (AE) in &gt;1 patient were abdominal pain (n=2 non-β0/β0; n=1 β0/β0), thrombocytopenia (n=3 non-b0/b0). Serious AEs in ≥3 patients post-infusion were thrombocytopenia (n=2 non-β0/β0; n=1 β0/β0), pyrexia (n=1 non-b0/b0; n=2 β0/β0), veno-occlusive disease (n=3 non-β0/β0). Summary Genetic characterization of TDT patients treated with beti-cel revealed diverse HBB and non-HBB mutations and polymorphisms that may influence disease severity. Higher PB VCN and HbAT87Q levels were associated with increased likelihood of TI. In instances of lower HbAT87Q, higher endogenous HbF might be a determinant in whether TI is achieved. Despite genetic heterogeneity, beti-cel enabled patients to achieve TI regardless of β-thalassemia genotype, TSS, disease genetic modifiers including HBA, and HbF QTL SNP genotypes. Disclosures Walters: Veevo Biomedicine: Consultancy; AllCells, Inc: Consultancy; Editas: Consultancy. Chui:bluebird bio, Inc.: Other: Payment for lab use for the sequencing analyses done for the studies. Lal:bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Celgene, BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; La Jolla Pharmaceutical Company: Research Funding; Novartis: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Terumo Corporation: Research Funding; Chiesi USA: Consultancy; Insight Magnetics: Research Funding. Locatelli:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Apopharma: Research Funding; Bristol Myers Squibb: Consultancy; Imara: Consultancy; Celgene: Consultancy; Sangamo: Research Funding; Terumo Corp: Research Funding; Novartis: Research Funding. Porter:bluebird bio, Inc.: Consultancy, Honoraria; Vifor Pharmaceuticals: Honoraria; La Jolla Pharmaceuticals: Honoraria; Protagonist Therapeutics: Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Silence Therapeutics: Honoraria. Thuret:Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis pharma: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. Thrasher:4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Yannaki:bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau; SANDOZ: Speakers Bureau; Gilead: Speakers Bureau. Yang:bluebird bio,Inc.: Current Employment, Current equity holder in publicly-traded company. Whitney:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Petrusich:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Colvin:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson:BMS: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; CRISPR/Vertex: Research Funding; Biomarin: Research Funding; Baxalta: Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

16

Akinsheye, Idowu, Nadia Solovieff, Anita Malek, Duyen A. Ngo, Martin H. Steinberg, and David H. K. Chui. "Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of the High Fetal Hemoglobin Phenotype In African American Patients." Blood 116, no. 21 (2010): 2068. http://dx.doi.org/10.1182/blood.v116.21.2068.2068.

Full text

Abstract:

Abstract Abstract 2068 HbF interferes with deoxygenated HbS polymerization, and is a major genetic modifier of sickle cell anemia severity. In this study, we attempted to identify genetic factors responsible for HbF production in a small group of African American sickle cell anemia patients who have markedly elevated HbF levels. Initially, patients with HbF of more than 11% as determined by HPLC were selected. The following were excluded: age less than 5; MCV greater than 100; presence of HPFH 1 and 2 based on specific gap-PCR tests; other β-globin gene deletions as determined by multiplex ligation-dependent probe amplification (MLPA). We further excluded rare HBG1 and 2 promoter HPFH mutations by nucleotide sequencing. In the end, a unique group of 20 patients were identified for further studies. Their mean age was 16.3 ± 8.3 years; Hb 9.0 ± 1.3 g/dL; MCV 87.9 ± 7.3 fL; and Hb F 17.2 ± 4.8% (range 11–29%). A control group of 30 African American patients were chosen. They had similar age, Hb, and MCV, but their HbF was 5.0 ± 2.5% (range 0.5–8.8%). These patients were examined for the 3 known major HbF quantitative trait loci: the Xmn1 restriction site C/T polymorphism at NT -158 upstream of HBG2; the BCL11A polymorphism on Chr2p16; the HBS1L-MYB intergenic polymorphism on Chr6q23. These 3 HbF quantitative trait loci collectively account for 20–50% of HbF variance in different populations. We found a significant association between high HbF and BCL11A and HBS1L-MYB intergenic region QTLs in these patients, but these account for only 20% of HbF variance (Table). These results were further validated in 590 patients of similar age from the Cooperative Study of Sickle Cell Disease, 57 patients with HbF 20.6 ± 8.2% and 533 patients with HbF 3.1 ± 1.5% (Table). To further explore other possible causes of elevated HbF, we sequenced 8.6 kb DNA fragment between HBG1 and HBD in 15 high HbF and 15 control patients. This DNA fragment includes the 7.2 kb Corfu deletion that is associated with elevated HbF levels and also binding sites for BCL11A. Twenty SNPs were found. The minor allele frequencies were consistently higher in the high HbF group, but the difference was found to be statistically significant only in 4 SNPs, 3 SNPs between positions 49213 and 49994 and 1 SNP at position 54541 (P = 0.001 to 0.04), suggesting that polymorphisms in this region might contribute to HbF expression in African American sickle cell anemia patients. The G→A polymorphism at position 49876 creates a C/EBP binding site which is not present in the major allele. The G→A polymorphism at position 49994 eliminates an AP-1 and NF-E2 binding sites, which are present in the major alleles. All 3 factors are erythroid transcription factors. The possible functional roles of these minor alleles found in significantly higher frequencies in the high HbF patients need to be further investigated. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

17

Hahn, Cynthia K., and Christopher H. Lowrey. "Salubrinal Increases Human Fetal Hemoglobin Production Through a Post-Transcriptional Mechanism." Blood 120, no. 21 (2012): 250. http://dx.doi.org/10.1182/blood.v120.21.250.250.

Full text

Abstract:

Abstract Abstract 250 Sickle cell disease and β-thalassemia continue to cause significant morbidity and mortality. Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of patients with these diseases. While most previous studies have focused on induction of γ-globin gene expression as an approach to induce HbF, there is evidence that HbF may be post-transcriptionally regulated. For example, butyrate was shown to increase the translational efficiency of γ-globin mRNA and 5-azacytidine (5-Aza) induces HbF to a much greater degree than γ-globin mRNA steady state levels. These findings suggest that translational regulation may play an underappreciated yet important role in controlling HbF levels and that investigating the molecular mechanisms involved in this control may provide new therapeutic targets for HbF induction. We hypothesized that the Integrated Stress Response (ISR) pathway is involved in differentially regulating fetal and adult hemoglobin production. The ISR pathway has been shown to modulate globin protein synthesis in response to heme availability and other stresses. In the absence of heme, the heme-regulated inhibitor kinase phosphorylates eIF2α, downregulates general protein synthesis, but enables translation of a limited number of transcripts that are critical for coordinating the stress response. To test our hypothesis, we first evaluated the effects of salubrinal (Sal), a small molecule that activates ISR signaling by selectively inhibiting p-eIF2α dephosphorylation, in K562 cells. 3μM and 6μM Sal increased p-eIF2α and activated ISR signaling as evidenced by increased ATF4 and GADD34 protein levels and increased gene expression of ATF3 and CHOP, two transcriptional targets of ATF4. Once we verified that Sal increased p-eIF2α and ISR signaling, we extended testing to primary human erythroid cells to evaluate its effect on hemoglobin production. We first determined a dose range of Sal that increased p-eIF2α in primary cells without reducing cell viability. Both 3μM and 6μM Sal increased p-eIF2α and only reduced cell number by 15% when applied on days 15 and 18 of differentiation, the period of maximal hemoglobin synthesis. Next, we determined that 3μM and 6μM Sal slightly reduced γ-globin and β-globin steady state mRNA levels but did not change the γ/(γ+β) ratio relative to control. In contrast, Sal significantly induced HbF when evaluated by HPLC at the end of differentiation on day 20. Compared to untreated cells, 3μM Sal increased the percent HbF from 2.7% to 5.0% (1.8 fold) and 6μM Sal resulted in 12.9% HbF (4.7 fold) (n=4, p<0.05). The enhanced %HbF was due to increased HbF but also reduced HbA, providing evidence that HbF and HbA may be differentially or reciprocally regulated at the translational level. Importantly, Sal treatment did not significantly reduce the total hemoglobin content relative to the untreated control and did not alter cellular differentiation when assessed by flow cytometry for CD71 and CD235a. These results suggest that Sal increases HbF by a post-transcriptional mechanism potentially through ISR activation. Sal treatments earlier in the differentiation process (days 9 and 12) before considerable amounts of hemoglobin are synthesized failed to significantly increase HbF, further supporting this conclusion. We then evaluated whether Sal treatment could enhance HbF induction by known activators of γ-globin transcription, such as 5-Aza and hydroxyurea (HU). 200nM 5-Aza alone increased %HbF from 2.7% to 12.4%. When 200nM 5-Aza was combined with 3μM and 6μM Sal, the %HbF increased to 18.0% and 22.8%, respectively. Similarly, 10μM HU alone increased HbF from 2.9% to 4.9%, but co-treatment with 3μM and 6μM Sal increased HbF to 7.7% and 15.0%, respectively. For both HU and 5-Aza, combined treatment with Sal did not alter the γ/(γ+β) ratio from what was seen with HU or 5-Aza alone. Taken together, these results indicate that the novel method of HbF induction by Sal enhances the effect of transcriptional activators of γ-globin. In the future, utilization of transcriptional and translational mechanisms of HbF induction may provide an opportunity for combination therapy to achieve therapeutic HbF levels at reduced doses, thereby reducing toxicity. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

18

Jiang, Zhihua, Shengwen Huang, Hong Yuan Luo та ін. "Assessment of HbF QTLs Affecting Disease Severity and Genetic Analysis in Patients Homozygous for Codon 8 (–AA) β0-Thalassemia Mutation". Blood 124, № 21 (2014): 2690. http://dx.doi.org/10.1182/blood.v124.21.2690.2690.

Full text

Abstract:

Abstract In β-thalassemia major, an inherited disorder caused by homozygosity or compound heterozygosity for β-thalassemia mutations, most patients are severely anemic and require chronic RBC transfusions. Elevated levels of HbF can ameliorate the disease severity and 3 major HbF quantitative trait loci (QTL) on chr 11p, 6q (the HBS1L-MYB intergenic polymorphism) and 2p (BCL11A) are associated with HbF levels in all populations studied. We studied Iraqi-American non-identical twins with markedly elevated HbF who are homozygous for codon 8 (–AA) β0-thalassemia mutation, but are clinically well and transfusion independent. MLPA analysis of the β-globin gene cluster did not show any hereditary persistence of fetal hemoglobin deletions or γ-globin gene quadruplication. Sequencing HBG2 and HBG1 promoters excluded other γ-globin gene promoter non-deletional HPFH mutations. We characterized 12 Turkish and Iraqi patients who are homozygous for codon 8 (–AA) frame-shift β-thalassemia mutation. Among this group, three patients from two Turkish families have a mild disease phenotype, six patients from four Turkish families have a severe phenotype, and three Iraqi patients from three families have a severe phenotype. To assess the contribution of 3 major HbF QTLs to disease severity, we genotyped polymorphisms in these QTL in all 14 patients (Table). Homozygosity for rs7482144 minor alleles (the -158 5' HBG2 Xmn1 C-T SNP) in the HBB gene cluster is present in 13 of 14 patients with the exception being one patient with a severe phenotype. No distinct pattern for the BCL11A sentinel SNP rs766432 minor allele is found between mild and severely affected patients. Homozygosity for rs9399137 minor alleles marking the HBS1L-MYB intergenic region is present in two patients with a mild phenotype, while heterozygosity for minor alleles and homozygosity for major alleles are present in both mild and severe patients. Although the Iraqi-American twins first studied have the most minor alleles (5 of 6 possible minor alleles), the other three patients with mild phenotype have 4, 3 and 2 minor alleles, respectively. Strikingly, one patient with mild phenotype is only homozygous for the rs7482144 minor alleles. Patients with severe phenotypes have 2 to 4 QTL minor alleles. Neither individual QTL genotypes nor the number of QTL minor alleles can distinguish the mild phenotype and severe phenotype in patients with β-thalassemia major of Iraqi and Turkish origin suggesting that other factors contribute to the mild phenotype and high HbF levels in these patients. The entire 14.7 kb intergenic region of between HBG1 and HBD of one twin was sequenced and revealed 19 known SNPs and 4 novel SNPs. The potential roles of these SNPs in regulating γ-globin gene expression are under investigation. We are also investigating whether there are any other potential cis- or trans-acting factors that contribute to mild disease phenotype in these patients using genome-wide SNP arrays, whole genome sequencing and whole exome sequencing. By studying these unusual cases we hope to provide new understanding and insights for elevated HbF production in β-hemoglobinopathies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

19

Sheehan, Vivien A., Thad A. Howard, Aniko Sabo, et al. "Genetic Predictors of Hemoglobin F Response to Hydroxyurea in Sickle Cell Anemia." Blood 120, no. 21 (2012): 241. http://dx.doi.org/10.1182/blood.v120.21.241.241.

Full text

Abstract:

Abstract Abstract 241 Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia exhibit wide variability in their laboratory and clinical phenotype, suggesting additional genetic modifiers exist beyond the sickle mutation. One of the most powerful and reproducible disease modifiers is fetal hemoglobin (HbF) level. The most widely used and safest method for increasing innate fetal hemoglobin levels in patients with sickle cell anemia is hydroxyurea. While hydroxyurea has disease modulating effects outside of HbF induction, the majority of its benefit is directly related to the %HbF produced in response to the drug. Unfortunately, the amount of the HbF produced in response to hydroxyurea is highly variable between individuals, with induced HbF levels ranging from 5 to >30% even for compliant patients on similar dosing regimens at the maximum tolerated dose (MTD). Hydroxyurea is an ideal target for pharmacogenomics investigation, since there is strong concordance of HbF response to hydroxyurea within sibling pairs, and amount of HbF produced at MTD is a quantifiable and objective phenotype. To address the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated pediatric patients treated prospectively with hydroxyurea. We analyzed patients enrolled in the HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) studies (n=183); all patients received an identical dose escalation regimen to MTD and had the most reliable HbF phenotypes available. To best identify genetic modifiers of hydroxyurea induction of HbF, we categorized study subjects according to their HbF response. Of a total cohort of 183 treated subjects, we identified 55 pediatric patients who represented the extreme ends of HbF response to hydroxyurea: 30 high responders (final HbF >30% and >25% change from %HbF at baseline) and 25 low responders (final HbF <20%, and <15% change from %HbF at baseline). There was no significant difference between high and low responders by age. Baseline fetal hemoglobin was similar between the two groups, suggesting that genetic predictors of drug response will differ from genetic regulators of endogenous HbF production. Absolute neutrophil count and hydroxyurea dose at MTD did not differ between high and low responders, evidence of uniform treatment. We performed whole exome sequencing on these 55 subjects and achieved 20X coverage in 95% of exonic sequences. In our statistical analysis, we compared nucleotide polymorphisms between low responders and high responders. Univariate testing identified ten nonsynonymous polymorphisms with p-values below 1×10−3, six of which are shown below. Additional candidate mutations with higher p-values were selected for further analysis based on known function in hematopoiesis or cell cycle arrest (Table). Whole exome sequencing genotyping was verified by TaqMan PCR or Sanger sequencing. Together, these variants represent excellent candidate genetic mutations to explain differences in HbF responses. These data represent the first examples of genetic predictors of HbF response to hydroxyurea using whole exome sequencing. Gene db SNP ID Amino acid change Function Direction of HbF response P-value PPP1R15A rs11541192 Gly312Ser Cell stress recovery Higher 1.87 × 10−4 HSD17B4 rs28943594 Met710Val Fatty acid oxidation Lower 4.16 × 10−4 HSD17B4 rs28943589 Lys122Asn Fatty acid oxidation Lower 4.18 × 10−4 FLVCR1 rs11120047 Ala52Pro Erythroid maturation Higher 4.21 × 10−4 LAMA5 rs6143021 His2036Arg Glomerular filtration Higher 4.21 × 10−4 ATP4A rs2733743 Val265Ala Iron absorption Higher 4.23 × 10−4 PPP1R15A rs611251 Val199Ala Cell stress recovery Higher 1.05 × 10−3 AKAP12 rs10872670 Lys19Glu Cell senescence Lower 1.28 × 10−3 SLC17A4 rs11754288 Ala372Thr Anion transporter Lower 1.86 × 10−3 RREB1 rs115093903 Leu983Ser Erythroid maturation Lower 2.31 × 10−3 DCHS2 rs61746132 Pro2676Leu Unknown Higher 2.90 × 10−3 SALL2 rs61743453 Pro840Arg Transcription factor Higher 9.17 × 10− Disclosures: Off Label Use: Hydroxyurea is FDA approved for adults but not children with sickle cell anemia.

APA, Harvard, Vancouver, ISO, and other styles

20

Steinberg, Martin H. "Fetal Hemoglobin in Sickle Hemoglobinopathies: High HbF Genotypes and Phenotypes." Journal of Clinical Medicine 9, no. 11 (2020): 3782. http://dx.doi.org/10.3390/jcm9113782.

Full text

Abstract:

Fetal hemoglobin (HbF) usually consists of 4 to 10% of total hemoglobin in adults of African descent with sickle cell anemia. Rarely, their HbF levels reach more than 30%. High HbF levels are sometimes a result of β-globin gene deletions or point mutations in the promoters of the HbF genes. Collectively, the phenotype caused by these mutations is called hereditary persistence of fetal hemoglobin, or HPFH. The pancellularity of HbF associated with these mutations inhibits sickle hemoglobin polymerization in most sickle erythrocytes so that these patients usually have inconsequential hemolysis and few, if any, vasoocclusive complications. Unusually high HbF can also be associated with variants of the major repressors of the HbF genes, BCL11A and MYB. Perhaps most often, we lack an explanation for very high HbF levels in sickle cell anemia.

APA, Harvard, Vancouver, ISO, and other styles

21

Paredes-Agurto, Mónica Santa María, Armando Fortunato Ugaz Cherre, José Manuel Marchena Dioses, and Robert Barrionuevo Garcia. "Aquatic Macroinvertebrate Diversity and Water Quality, La Gallega-Morropón Creek, Piura, Peru." Nature Environment and Pollution Technology 23, no. 4 (2024): 2397–402. https://doi.org/10.46488/nept.2024.v23i04.043.

Full text

Abstract:

Freshwater systems are one of the most important natural resources for life. Despite their value, these ecosystems have suffered great impacts caused by human activities, which directly affect the aquatic biota and the quality of water sources. Considering the value of aquatic macroinvertebrates as bioindicators of water quality, the richness, composition, and water quality of La Gallega-Morropón stream, Piura-Peru, were compared. Two field trips were conducted between November 2018 and May 2019 (contemplated wet and dry periods, respectively), performing 4 sampling stations. A total of 1772 individuals of macroinvertebrates were recorded, distributed in 22 families. Psychodidae had an abundance of 670 individuals, followed by morphospecies (Gasteropoda) with 379 individuals, Chironomidae with 275 individuals, and Elmidae with 136 individuals (all indicators of water quality). Finally, the water quality index method: 1) BMWP/Col, presented one station with good (HB1), acceptable (HB2), and critical (HB3 and HB4) quality, while 2) EPT exhibited two stations with good quality (HB3 and HB4), HB1 regular quality and HB2 poor (HB3 and HB4), HB1 regular quality and HB2 poor quality.

APA, Harvard, Vancouver, ISO, and other styles

22

Bartolucci, Pablo, Laura Bencheikh, Meghan Perkins, et al. "New Insights into VOC Incidence By Analyzing the Single Cell Quantification of HbF per RBC and Mchbs." Blood 142, Supplement 1 (2023): 5272. http://dx.doi.org/10.1182/blood-2023-184758.

Full text

Abstract:

Vaso-occlusive crisis (VOC), the main complication of sickle cell disease (SCD), is responsible for more than 50% of the associated deaths. The effect of hydroxyurea, the cornerstone treatment for SCD, on the occurrence of VOC is mainly mediated by increasing HbF levels, which is a protective factor by inhibiting hemoglobin S (HbS) polymerization. 1,2 However, HbF level is variable between patients, and its expression is heterogeneous among the red blood cell (RBC) population. 3 As a result, while an increase in HbF levels is associated with better survival, %HbF only partially explains patients' clinical manifestations. The protective effect of HbF is also related to the HbS content; the more HbS, the more HbF is needed to inhibit its polymerization. In this study, we wondered if the single-cell quantification of HbF that we developed could better explain the VOC incidence of SCD compared to %HbF. Patients and method We analyzed data from the FCDREP1 protocol, aiming to obtain a predictive score for VOC at 1 year. Inclusion criteria were SS and S- S-b 0 Thal SCD patients, more than 1 month after a vaso-occlusive crisis, 3 months after a transfusion, and on a stable dose of hydroxyurea (HU) for at least 3 months. After giving their consent (IRB no. 00003835) during a routine blood sampling visit, an additional sample was taken for HbF single cell quantification, providing HbF/cell distribution. VOC were collected prospectively for 1 year. HbF single cell quantification was measured by flow cytometry using a previously published protocol, 4 data are expressed as histograms representing % RBC in each HbF range in pg for the whole cluster. K-means clustering was performed on different combination of variables containing ranges of HbF/cell distribution versus %HbF, with MCHbS (MCH x %HbS) using UMAP for dimensionality reduction (into 2), after we applied Min-Max scaling to preprocess the features, with python 3 on sklearn and umap library. This clustering approach can group data points based on their similarity, providing insights into distinct patterns or subgroups within the dataset. Results One hundred two patients were included with a mean age of 38 ± 11 years, a sex ratio F/M of 0.56, 63% of patients were on HU. Patients had a total of 74 VOC with a mean of 0.71 ± 1.4 VOC per patient in the year. Analyses were performed finding 4 different clusters in different 2 groups. Group 1: clusters identified by the association of HbF/cell distribution and MCHbS (HbF/Cell) and Group 2: clusters identified by %HbF and MCHbS (%HbF). Clusters were numbered according to increasing HbF levels (Figure 1A and B). There was no significant difference in age or sex ratio between the groups whereas the percentage of patients on HU differed between groups (Figure 1F). The number of patients by cluster was also similar. Cluster analysis of Group 1 showed a decrease in VOC incidence (Figure 1E, blue bars) associated with an increase in HbF level per RBC (Figure 1A). If clusters 1 and 2 are similar regarding the % of HU-treated patients, cluster 1 shows a lower HbF/cell content (Figure 1A) but lower MCHbS and MCH (Figure 1C and 1D), possibly explaining a similar VOC incidence in the 2 clusters. With a higher % of HU-treated (Figure 1F), Cluster 3 and 4 stand out from each other by different HbF expression patterns and VOC incidence (Figure 1A). The clusters of group 2 does not show the same increasing incidence of VOC as a function of median %HbF (Figure 1B and E). If cluster 4, mostly treated with HU, corresponds to patients with high %HbF and low VOC; cluster 1, with little HU treatment and very low %HbF, has few VOCs, not explained by MCHbS. The difference in severity between clusters 2 and 3, which have the same percentage of HU, could be explained by the greater difference in %HbF distribution, with median %HbF values of 11.8 [5-14] and 13.8 [11-15] (p=0.06) respectively, as well as by the low MCHbS of cluster 3. Conclusion : Single cell quantification of HbF in RBC in association with MCHbS could help improve to better understand VOC incidence.

APA, Harvard, Vancouver, ISO, and other styles

23

Blau, CA, P. Constantoulakis, CM Shaw, and G. Stamatoyannopoulos. "Fetal hemoglobin induction with butyric acid: efficacy and toxicity." Blood 81, no. 2 (1993): 529–37. http://dx.doi.org/10.1182/blood.v81.2.529.529.

Full text

Abstract:

Abstract Butyric acid induces fetal hemoglobin (HbF), a property of potential therapeutic advantage in patients with disorders of globin chain synthesis. We performed dose escalation studies of this compound in baboons to assess whether clinically significant increases in HbF are achievable, and to define the associated toxicities. Additionally, the effect of butyrate in combination with erythropoietin on HbF induction was assessed. HbF induction in response to butyrate was dependent on the dose and duration of treatment. Doses of butyrate less than 4 g/kg/d were associated with minimal toxicity (hypokalemia) and significant HbF induction in these nonanemic animals, with 1 g/kg/d producing an increase in HbF-containing reticulocytes (F reticulocytes) from 0.9% to 8.7% and an increase in HbF from 0.8% to 1.4%. A dose of 2 g/kg/d resulted in an increase in F reticulocytes from 2.1% to 27.8% and an increase in HbF from 0.7% to 2.2%. Doses of 4 g/kg/d in another animal produced an increase in F reticulocytes from 1% to 21.6% and in HbF from 1.9% to 5.3%. Infusions in excess of 4 g/kg/d were complicated (after a variable amount of time) by a decreased level of alertness (caused by hyperosmolality or butyrate itself) and hematologic toxicity (with declines in reticulocyte, white blood cell, and platelet counts). Prolonged infusions of high doses of butyrate (8 to 10 g/kg/d) were associated with peak F reticulocyte percentages reaching 38% to 64.5% and HbF reaching levels in excess of 20%. These high doses (8 to 10 g/kg/d) were complicated in two animals with a striking and unique neuropathologic picture and, in one animal, multiorgan system failure. Erythropoietin in combination with butyrate, induced F reticulocytosis in an additive manner. We conclude that butyric acid is a strong inducer of HbF, particularly when administered in combination with erythropoietin. As chronic toxicities remain undefined, patients in future clinical trials of this and similar compounds should be monitored closely for evidence of neurologic toxicity.

APA, Harvard, Vancouver, ISO, and other styles

24

Blau, CA, P. Constantoulakis, CM Shaw, and G. Stamatoyannopoulos. "Fetal hemoglobin induction with butyric acid: efficacy and toxicity." Blood 81, no. 2 (1993): 529–37. http://dx.doi.org/10.1182/blood.v81.2.529.bloodjournal812529.

Full text

Abstract:

Butyric acid induces fetal hemoglobin (HbF), a property of potential therapeutic advantage in patients with disorders of globin chain synthesis. We performed dose escalation studies of this compound in baboons to assess whether clinically significant increases in HbF are achievable, and to define the associated toxicities. Additionally, the effect of butyrate in combination with erythropoietin on HbF induction was assessed. HbF induction in response to butyrate was dependent on the dose and duration of treatment. Doses of butyrate less than 4 g/kg/d were associated with minimal toxicity (hypokalemia) and significant HbF induction in these nonanemic animals, with 1 g/kg/d producing an increase in HbF-containing reticulocytes (F reticulocytes) from 0.9% to 8.7% and an increase in HbF from 0.8% to 1.4%. A dose of 2 g/kg/d resulted in an increase in F reticulocytes from 2.1% to 27.8% and an increase in HbF from 0.7% to 2.2%. Doses of 4 g/kg/d in another animal produced an increase in F reticulocytes from 1% to 21.6% and in HbF from 1.9% to 5.3%. Infusions in excess of 4 g/kg/d were complicated (after a variable amount of time) by a decreased level of alertness (caused by hyperosmolality or butyrate itself) and hematologic toxicity (with declines in reticulocyte, white blood cell, and platelet counts). Prolonged infusions of high doses of butyrate (8 to 10 g/kg/d) were associated with peak F reticulocyte percentages reaching 38% to 64.5% and HbF reaching levels in excess of 20%. These high doses (8 to 10 g/kg/d) were complicated in two animals with a striking and unique neuropathologic picture and, in one animal, multiorgan system failure. Erythropoietin in combination with butyrate, induced F reticulocytosis in an additive manner. We conclude that butyric acid is a strong inducer of HbF, particularly when administered in combination with erythropoietin. As chronic toxicities remain undefined, patients in future clinical trials of this and similar compounds should be monitored closely for evidence of neurologic toxicity.

APA, Harvard, Vancouver, ISO, and other styles

25

Ware, Russell E., Barry Eggleston, Tatiana Abramova, Sherri A. Zimmerman, Alice Lail, and Thad A. Howard. "Influence of Globin Gene Modifiers on Baseline and Hydroxyurea-Induced Fetal Hemoglobin Levels in Children with Sickle Cell Anemia." Blood 106, no. 11 (2005): 3171. http://dx.doi.org/10.1182/blood.v106.11.3171.3171.

Full text

Abstract:

Abstract Fetal hemoglobin (HbF) is recognized as a major determinant of clinical disease severity in children and adults with sickle cell anemia (SCA). Patients with elevated HbF levels have a milder disease course, and many current therapeutic protocols for SCA include pharmacological induction of HbF. However, baseline and treatment HbF levels vary widely due to presumed genetic and environmental factors. Recognized globin gene modifiers of HbF include the beta globin haplotype and a potential contribution from concomitant alpha thalassemia. To characterize more fully the influence of globin gene modifiers on both baseline and treatment HbF levels, we retrospectively determined the beta globin haplotype (Benin, CAR, Senegal, Cameroon, or Arab-Indian) by selective gamma globin gene nucleotide sequencing and the alpha globin gene number (2, 3, or 4) by PCR for 67 African-American children with SCA receiving hydroxyurea therapy at stable maximal tolerated dose (MTD). The four beta globin haplotypes and frequencies identified in our cohort of children include Benin (0.61), CAR (0.17), Senegal (0.12), and Cameroon (0.10). The number of alpha globin genes and frequencies identified were 4 genes (0.72), 3 genes (0.25) and 2 genes (0.03). Baseline and MTD HbF levels were analyzed according to each variable. The average baseline HbF value for the entire cohort of children was 7.7 ± 4.4% (median 7.6%, range 1.3 – 19.3%), while the average treatment HbF value was 23.9 ± 7.2 % (median 22.9%, range 10.2 – 40.7%). All 67 children increased their HbF in response to hydroxyurea therapy (median 16.7%, range 5.0 – 28.8%). There was a modest but statistically significant correlation between the baseline and treatment HbF (r=0.66, p&lt;.0001). The estimated effect of one unit change in baseline HbF on treatment HbF was 1.11 (95% CI of 0.78, 1.43). When baseline %HbF was analyzed according to the beta globin haplotype, the overall ANOVA had a p-value of 0.02, indicating a statistically significant influence. Further analysis confirmed associations previously identified in adults with SCA, i.e. children with at least one copy of the CAR haplotype had a lower baseline HbF (5.9% vs 8.4%, p=.05), while those with at least one copy of the Senegal haplotype had a higher baseline HbF (11.1% vs 6.7%, p&lt;.001). When hydroxyurea MTD (treatment) HbF values were analyzed according to beta globin haplotype while adjusting for baseline HbF, however, the effect of beta globin haplotype was not statistically significant (p=.13). Analyses of HbF according to alpha globin gene number revealed no statistically significant effects on either baseline or treatment HbF values. Taken together, these data support the hypothesis that beta globin haplotype significant influences baseline HbF values for children with SCA, but has no significant effects on hydroxyurea MTD HbF values. Accordingly, children with SCA should be offered hydroxyurea based solely on clinical indications, without consideration of baseline HbF or beta globin haplotype. Even children with low baseline HbF values or the CAR beta globin haplotype can respond to hydroxyurea therapy with an elevated %HbF. Future studies designed to identify genetic modifiers of treatment HbF values should focus on sequence polymorphisms in non-globin genes that have trans-acting effects on gamma globin gene expression.

APA, Harvard, Vancouver, ISO, and other styles

26

Steinberg, Martin H. "Fetal hemoglobin in sickle cell anemia." Blood 136, no. 21 (2020): 2392–400. http://dx.doi.org/10.1182/blood.2020007645.

Full text

Abstract:

Abstract Fetal hemoglobin (HbF) can blunt the pathophysiology, temper the clinical course, and offer prospects for curative therapy of sickle cell disease. This review focuses on (1) HbF quantitative trait loci and the geography of β-globin gene haplotypes, especially those found in the Middle East; (2) how HbF might differentially impact the pathophysiology and many subphenotypes of sickle cell disease; (3) clinical implications of person-to-person variation in the distribution of HbF among HbF-containing erythrocytes; and (4) reactivation of HbF gene expression using both pharmacologic and cell-based therapeutic approaches. A confluence of detailed understanding of the molecular basis of HbF gene expression, coupled with the ability to precisely target by genomic editing most areas of the genome, is producing important preliminary therapeutic results that could provide new options for cell-based therapeutics with curative intent.

APA, Harvard, Vancouver, ISO, and other styles

27

Bruun-Rasmussen, Peter, Elena Dudukina, Lars Peter Korsholm, Julie Derving Karsbøl, Inga Hegemann, and Maarten Jan Wensink. "Large Scale Analysis of the Real-World Association between Fetal Hemoglobin and Vaso-Occlusive Crises in Sickle Cell Disease." Blood 144, Supplement 1 (2024): 1124. https://doi.org/10.1182/blood-2024-200196.

Full text

Abstract:

Background Fetal hemoglobin (HbF) attenuates the rate of vaso-occlusive crises (VOCs) in sickle cell disease (SCD), but exact quantification is lacking. Methods We analyzed two large historic datasets with contemporary statistical methods. Data, acquired through BioLINCC, were the Cooperative Study of SCD (CSSCD; USA, 1979-1988) and the Multicenter Study on Hydroxyurea (MSH; USA and Canada, 1992-1995). The CSSCD had 3 planned HbF assessments: baseline, 1st annual visit, 2nd annual visit. We included those aged ≥12 years with at least one HbF measurement at baseline, 1st, or 2nd annual visit (1395 unique individuals, median age 22 years). Follow-up was up to 8 years post baseline. We analyzed the data using one of three approaches: baseline HbF and all follow-up; three HbF readings and one year of follow-up after each visit; three HbF readings and all follow-up up to next visit or follow-up end. For the MSH (HbSS genotype, age ≥18 years, ≥3 VOCs within the pre-trial year, 299 unique individuals, median age 29 years), we used HbF at randomization in the placebo arm and after titration in the hydroxyurea (HU) arm. Placebo participants were offered HU after early trial termination and their follow-up was censored then. HbF was analyzed as a percentage of total hemoglobin. In the MSH data, F-cells (as a percentage of red blood cells [RBC]) were additionally analyzed. F-cells were averaged over follow-up. We used directed acyclic graphs for confounder identification and negative binomial generalized additive models to allow the data to suggest the functional relationship between HbF and the VOC rate. A straight line suggests a linear effect of HbF% on log VOC rate and implies that each percentage point (%pt) increase in HbF or F-cells reduces the VOC rate by a fixed percentage. We regressed VOC rate during follow-up on HbF and adjusted models for age at baseline, sex, ethnicity, and when applicable for β-globin haplotype, assigned treatment (HU/placebo), and received HU dose. Results HbF showed a linear protective effect on log VOC rate in two of the CSSCD analyses and a non-linear progressive effect for the remaining analysis: each additional %pt increase in HbF reduced the VOC rate by a slightly larger percentage. When fitting linear models, each %pt increase in HbF was associated with a 4% (95% CI: 2-6%) to 6% (95% CI: 3-8%) reduction in VOC rate. For MSH data, each %pt increase in HbF was associated with an 8% (95% CI: 4-11%) reduction in VOC rate. The model suggested that the reduction per %pt increase in HbF depended on the level of HbF%, with a larger reduction in VOC rate the higher the HbF%. We found no evidence of change in VOC rate up to 35% F-cells. Above 35% F-cells, there was increasing protection against VOCs. Individuals with ≥70% F-cells had less than half the VOC rate of those with 35% F-cells. Results did not change when the models were adjusted for total hemoglobin. Interpretation The results show that each %pt increase in HbF has a protective effect on VOC rate, which was larger for higher HbF% in two analyses. Since HbF intercalates in HbS polymerization, VOC rate may be driven by HbF/HbS ratio. Hence, increasing HbF% would be progressively more effective through the numerator effect in HbS (like odds). These data further highlight the importance of HbF distribution across RBC, as VOCs occurred at a progressively lower rate for F-cells &gt;35%. HbF content varies also among RBC that fail the F-cell threshold (6pg), and a small number of sickle RBC may suffice to cause a VOC. Translation of these data from its geographic reach to a current, global setting may be limited. However, data from a recent small study on HU in India suggested an upper limit of 9% VOC reduction for every %pt increase in HbF, if all protective effect of HU comes from HbF induction. This corresponds well with the 4-8% reduction in VOC rate found here. The CSSCD remains the largest real-world study on SCD, which contributes pre-treatment data, and the MSH yielded comparable results. Conclusion This large real-world study on prospectively collected data confirms the direct relationship between HbF and VOC occurrence. Further, our results suggest that one %pt increase in HbF% may be more beneficial for patients with a higher starting HbF%. These results highlight the importance of HbF distribution across the RBC population with a focus on HbF as an important SCD therapy target. Repeating the study on data of greater geographic reach would strengthen the interpretation.

APA, Harvard, Vancouver, ISO, and other styles

28

Zheng, Biao, Rongrong Liu, Xinhua Zhang та ін. "Efficacy and Safety of Brl-101, CRISPR-Cas9-Mediated Gene Editing of the BCL11A Enhancer in Transfusion-Dependent β-Thalassemia". Blood 142, Supplement 1 (2023): 4995. http://dx.doi.org/10.1182/blood-2023-186031.

Full text

Abstract:

BACKGROUND: β-Thalassemia is an inherited hemolytic disease that is prevalent worldwide. Over 200 mutations in the HBB gene, which encodes adult hemoglobin (HbA), result in β-thalassemia. Hereditary persistence of fetal hemoglobin (HbF) can alleviate the symptoms of anemia. CRISPR-Cas9-mediated disruption of the BCL11A erythroid enhancer results in the reduction of BCL11A expression and the induction of fetal γ-globin, which is a practicable therapeutic strategy for treating transfusion-dependent β-thalassemia (TDT). METHODS: We obtained mobilized autologous CD34+ cells from 4 TDT patients sponsor initiated in phase I/II clinical trial (NCT05577312) and 6 TDT patients in investigator initiated clinical study（NCT04211480, NCT04205435）respectively. These cells were edited with CRISPR-Cas9 RNP at the +58 erythroid specific enhancer region of the BCL11A gene and then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, neutrophil and platelet engraftment. Efficacy assessments included proportion of transfusion-independent (TI) subjects, levels of total hemoglobin and HbF, and proportion of red blood cells expressing HbF in peripheral blood. TI was evaluated for elimination of transfusions starting 42 days after the last transfusion and Hb above 90 g/L. RESULTS: Between October 15, 2019 and November 20, 2022, 10 patients with TDT were enrolled and received BRL-101 with a median age of 12.4 years (6-26). Among all the treated patients, 5 patients were β 0/β 0 phenotype, 4 patients were β0/β+ phenotype and 1 patient were β +/β + phenotype. As of July 20, 2023, the median follow- up was 24.6 months (4.3-39.2 m) (Tab.1). All of 10 patients achieved TI. The median duration of TI was 22.1 months (1.3 - 37.2 m). The longest duration of TI was 37.2 months (Fig.1). The levels of HbF ranged from 2.3 to 140.7g/L and the levels of total hemoglobin ranged from 105.7 to 149 g/L (Fig.2). At 6 months after BRL-101 infusion, the proportion of HbF-expressing red blood cells in peripheral blood had reached 96.5%, and then continued to rise and remained around 98-99% (Fig.3). Treatment-related adverse events were typical of those associated with myeloablation and autologous stem-cell transplantation, mainly manifested as hematological toxicities (Tab.2, Tab.3). 3 patients experienced adverse events (AEs) grade ≥ 3 which related to BRL-101. 4 patients experienced serious AEs (SAEs), including decreased platelet count, shock, febrile infection, soft tissue infection, and veno-occlusive liver disease. Only decreased platelet count may related to BRL-101, the others were due to busulfan treatment. All the SAEs were resolved. No study drug-related withdrawals or deaths occurred during treatment. CONCLUSIONS: Whether genotype was β 0/β 0 or non-β 0/β 0, BRL-101 demonstrated clinically meaningful increases in total Hb and HbF which occurred early and have been maintained over time, the safety profile of BRL-101 is generally consistent with that of myeloablative conditioning and autologous hematopoietic stem cell transplant. The updated data with 10 patients reported here are consistent with previous reports and support continued investigation of BRL-101 as a potential functional cure for patients with TDT.

APA, Harvard, Vancouver, ISO, and other styles

29

Gabbianelli, Marco, Ugo Testa, Adriana Massa, et al. "Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate." Blood 95, no. 11 (2000): 3555–61. http://dx.doi.org/10.1182/blood.v95.11.3555.

Full text

Abstract:

Abstract Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and γ-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% γ-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the β-globin gene cluster; KL may activate the γ-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and β-thalassemia therapy.

APA, Harvard, Vancouver, ISO, and other styles

30

Gabbianelli, Marco, Ugo Testa, Adriana Massa, et al. "Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate." Blood 95, no. 11 (2000): 3555–61. http://dx.doi.org/10.1182/blood.v95.11.3555.011k16_3555_3561.

Full text

Abstract:

Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and γ-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% γ-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the β-globin gene cluster; KL may activate the γ-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and β-thalassemia therapy.

APA, Harvard, Vancouver, ISO, and other styles

31

Fibach, E., P. Prasanna, GP Rodgers, and D. Samid. "Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia." Blood 82, no. 7 (1993): 2203–9. http://dx.doi.org/10.1182/blood.v82.7.2203.2203.

Full text

Abstract:

Abstract In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

APA, Harvard, Vancouver, ISO, and other styles

32

Fibach, E., P. Prasanna, GP Rodgers, and D. Samid. "Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia." Blood 82, no. 7 (1993): 2203–9. http://dx.doi.org/10.1182/blood.v82.7.2203.bloodjournal8272203.

Full text

Abstract:

In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

APA, Harvard, Vancouver, ISO, and other styles

33

Vieira, Benjamin, Vu P. Hong, Kunal Desai, Martin K. Safo, and David R. Light. "LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences." Blood 132, Supplement 1 (2018): 1074. http://dx.doi.org/10.1182/blood-2018-99-116319.

Full text

Abstract:

Abstract Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs. HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016). Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. Disclosures Vieira: Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

APA, Harvard, Vancouver, ISO, and other styles

34

Kuo, Kevin H. M., Sylvia Titi Singer, Gershwin Theophilus Blyden, et al. "Initial Results in a Phase 1b Trial of PB-04 in Sickle Cell Disease Demonstrate Fetal Hemoglobin Induction, Additive Activity with Hydroxyurea, and Improved Red Blood Cell Sickling Parameters." Blood 142, Supplement 1 (2023): 1148. http://dx.doi.org/10.1182/blood-2023-184627.

Full text

Abstract:

Extensive evidence has shown fetal hemoglobin (HbF) is a major modulator of sickle cell disease (SCD) phenotype. Clinical benefit with any increment of HbF was shown in the NIH-sponsored Natural History Study and the Multicenter Study of Hydroxyurea (HU), with mean HbF increases of 3.6% above baseline (BL), to improve survival, reduce painful vaso-occlusive crises, and recently stroke prevention and improved cardiac and neurocognitive function with HU are reported. As some patients do not tolerate optimal HU doses due to cytopenias, additional inducers suitable for combined use are desirable. Proportions of F-cells and HbF-quantity/cell have been cited as important. PB-04, a repurposed drug with a well-known safety profile, is an oral fetal globin stimulant identified in high throughput screening, which suppresses 4 repressors of the HMG gene promoter, ( BCL-11A, LSD-1, HDAC-3, KLF-1). PB-04 induces fetal globin in nonhuman primates, transgenic mice, with additive effect when combined with HU in SCD erythroid progenitors, and was shown to reduce infarcts in multiple organs with preserved splenic function in transgenic sickle mice. An ongoing Phase 1b trial in beta thalassemia intermedia (BTI) ( NCT04432623) of 3 doses (1,3, or 5 mg/kg QD) given 3 (TIW) or 5 times/week (Wk) for 12 or 24 Wks in 18 courses in 10 subjects demonstrates a favorable safety profile, dose-proportional PK with 5 &gt;3 mg/kg doses and 5 &gt;3 doses/Wk, Median fold-increases from baseline (BL) in F-cells of 4.4x BL (range 2.3-7x), 2.8x F-reticulocytes (1.6-14.8x), and 6.5x (range 3-11x) increases in mean fluorescence intensity (MFI, representing amount of HbF/RBC). Mean increases in % HbF were 6.5%. Evaluation of PB-04 has now begun in 7 subjects with SCD, 19-51 yrs; 3 were receiving HU with HbF responses (12-22%). Adverse events, F-cells, F-reticulocytes, MFI (amount of HbF/RBC or HbF/reticulocyte) by FACS and ImageStream Analysis and % HbF are assessed Q 4 wks; flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) and sickling kinetics are assessed q 12 wks. In 4 subjects on 3 mg/kg QD TIW for 24 wks, changes observed from BL values included mean increases of 6% F-cells and 27% F-reticulocytes (2-fold) . In 3 ongoing subjects receiving 5 mg/kg QD TIW for 12 wks mean changes above BL are currently 13% F-cells, 15% F-reticulocytes; HbF/cell or HbF/reticulocyte have increased up to 10-fold. Mean % HbF has increased above HU effects in 2/3 pts on the second dose by 5-12.6% (mean 8.8%), producing 81-95% F-cells, and 35-38% HbF. Decline in FA-WB-VCAM) was observed ± HU and with both doses in 3 subjects. Correlation of HbF quantity/cell with RBC survival and sickling dynamics is being explored. These early results with not-yet-optimized doses of PB-04 suggest additive activity with HU, producing significant HbF and F-cell levels, and support further PK-optimized studies in patients with SCD.

APA, Harvard, Vancouver, ISO, and other styles

35

Peslak, Scott A., Talla Abbas, Eugene Khandros, et al. "Protein Phosphatase 6 Complex: Novel Regulator of Fetal Hemoglobin and Potential Therapeutic Target in Sickle Cell Disease." Blood 142, Supplement 1 (2023): 13. http://dx.doi.org/10.1182/blood-2023-181989.

Full text

Abstract:

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen in HUDEP2 cells targeting all 218 serine/threonine protein phosphatases with 6 independent sgRNAs each. A single phosphatase, PPP6C, emerged as a potential HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6 (PP6), a serine/threonine phosphatase complex that has been implicated in cell cycle regulation, autophagy, and innate immunity mechanisms in other cell types, but its role in erythropoiesis and HbF regulation has not previously been described. CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells elevated mRNA levels of HBG (the fetal form of β-globin) in a dose-dependent manner up to 3-4 fold, increased HbF levels 3-fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis, and did not measurably impair erythroid maturation. In addition, PPP6C loss in primary SCD patient-derived cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by up to 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation proceeds at least in part via loss of BCL11A. Importantly, xenotransplantation data (NBSGW) showed ~4-fold induction of HBG at 16 weeks post-transplant, suggesting that PPP6C deficiency leads to effective, sustained HbF induction in vivo. We next set out to determine whether any PP6 subunits have erythroid specificity that could be exploited therapeutically. In contrast to PPP6C, which is highly expressed in all hematopoietic lineages, the PP6 facultative subunit PPP6R1 appears to be enriched in erythroid precursors, suggesting that depletion of PPP6R1 may function as an erythroid-selective target to increase HbF levels. CRISPR-Cas9-based depletion of PPP6R1 in primary human erythroid cells showed a 2-3-fold increase in HBG and near-doubling of HbF-expressing cells, recapitulating the majority of PPP6C-mediated HbF induction. Functional studies are currently in progress to specifically determine whether additional PP6 subunits play a key role in HbF regulation. Furthermore, ongoing work utilizing dTAG-based acute depletion of PPP6C and PPP6R1 paired with phospho-proteomic studies will narrow down direct targets of the PP6 complex and provide insights into erythropoietic and HbF regulatory pathways impacted by PP6. Taken together, our data indicate that PP6, including its catalytic subunit PPP6C and erythroid-enriched regulatory subunit PPP6R1, inhibit HbF production in part via modulating BCL11A levels, and may serve as a red-cell specific therapeutic target in the treatment of SCD.

APA, Harvard, Vancouver, ISO, and other styles

36

Gardner, Kate, Tony Fulford, Helen Rooks, et al. "A Genetic Predictive Model for HbF in Sickle Cell Disease." Blood 128, no. 22 (2016): 319. http://dx.doi.org/10.1182/blood.v128.22.319.319.

Full text

Abstract:

Abstract Fetal hemoglobin (HbF) is strongly associated with clinical severity in the β-hemoglobinopathies, including sickle cell disease (SCD). In recent years, the three major HbF genetic loci (at BCL11A on chromosome 2p, HMIP-2 on chromosome 6q and Xmn1-HBG on chromosome 11p) have been more clearly characterized and mechanisms of the likely causal variants better defined. In this study, we have combined this new biological understanding with statistical methods to create a genetic "predictor" for HbF in SCD. We chose 7 variants to represent the 3 HbF quantitative trait loci (QTL) to investigate their utility in predicting HbF levels, and, in turn, clinical severity of SCD. For BCL11A, we used 2 markers: rs1427407 (62kb downstream of BCL11A) localizes to an erythroid-specific enhancer (Bauer et al. Science 2013) and rs6545816 tags a second signal 58kb downstream of BCL11A. The genetic architecture of HMIP-2 as a QTL comprises two elements, A and B (Menzel et al. Ann Hum Genet 2014). We have represented HMIP-2A withthe 3bp deletion rs66650371, shown as a causal variant (Stadhouders et al. JCI 2014) plus the ethnicity marker rs9376090. HMIP-2B is less well-characterized, we selected: rs9494142 (near the MYB enhancer) and rs9494145. For the β-globin locus, we used the long-established Xmn1 marker (rs7482144) in the proximal promoter 158kb upstream of HBG2. This is likely not the variant itself, but in tight linkage disequilibrium with the causal element. Of 892 initial patients (516 females, 376 males), we excluded 17 children aged under 5 because of the non-linear relationship between age and HbF at a young age (we confirmed this finding in our cohort). This left: 658 with HbSS, 206 with HbSC, 8 with HbSβ0 thalassemia, and 20 with HbSβ+ thalassemia. We then genotyped 666 patients with HbSS/HbSβ0 thalassemia for the 7 genetic variants. For each patient, we selected 'validated' HbF levels i.e. HbF not influenced by transfusion, drugs (especially hydroxyurea) or pregnancy. HbF levels were log-transformed (Ln). We then used multiple linear regression models to identify variants which were independently associated with Ln-HbF levels. Using only age and sex as covariates revealed predictive power r2~10% which was orthogonal to (i.e. additive) the predictive power of the variants, and so we did not include them in subsequent analysis. Also, by adding α-globin status to the model where known (N=272), the r2 remained unchanged and is not significant for α-globin status. We then normalized the 7 variants to take account of the mean allele count (a strongly predictive but rare variant may not explain much of the total population variance). We performed multiple linear regression to rationalize the 7 variants, and found 4 markers (rs6545816, rs1427407, rs66650371 and rs7482144) independently contributing HbF-boosting alleles (see table). Combining these 4 variants into a genetic risk model, as per the table, allows us to predict 21.8% of variability (r2) of HbF in our HbSS / HbSβ0 thalassemia patients. We validated the 4-variant risk score first with a 5-fold cross-validation within the cohort which demonstrated a mean r2=22% for the 5 folds. We then replicated the findings in the cohort of HbSC patients (N=206) and found the 4-variant model to predict HbF with variability r2=27.5% (i.e. towards r2=44% seen in non-anemic individuals). Thus, our 4-variant model provides a robust approach to genetic prediction of HbF in SCD. The predictive power appears to be larger for HbSC compared to HbSS (r2=27.5% vs 21.8%) which may be related to stress erythropoiesis in HbSS patients releasing immature erythrocytes as a non-genetic factor modifying HbF levels. This process is a first step towards creating a global genetic predictive score in SCD: stratifying patients with SCD early in life would enable us to offer curative therapy (i.e. hematopoietic stem cell transplant) to those identified as genetically severe. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

37

Gaudreau, Pierre-Olivier, Xiaoduan Weng, Marilyn Brillant, et al. "Prospective Evaluation of Fetal Haemoglobin Induction in Maternal Erythrocytes: A Preliminary Analysis of a Cohort of 345 Parturients." Blood 126, no. 23 (2015): 3370. http://dx.doi.org/10.1182/blood.v126.23.3370.3370.

Full text

Abstract:

Abstract Introduction. The mechanisms of haemoglobin (Hb) subtype switch remain largely unknown even though it has a therapeutic potential (Bauer et al., 2012). In fact, reactivation of fetal haemoglobin (HbF) production in adult erythrocytes (F-cells) is protective in many Hb disorders. Pregnancy may represent the only physiological condition where HbF production increases transiently (Ibrahim et al, 2009; Yamada et al., 2012). Moreover, flow cytometry (FC), by intracellular staining using anti-HbF antibodies, has improved the evaluation of maternal F-cells (Corcoran et al., 2014; Chen et al., 2000). In this study, the objectives are: 1) to document HbF expression prospectively peri- and post-partum and confirm its maternal origin; 2) to assess potential causes of HbF modulation throughout pregnancy (i.e. β-hCG, EPO) and; 3) to identify gene differences between HbF-positive and HbF-negative pregnant women. Methods. In this observational, single institution, prospective study, 879 pregnant women were screened. A total of 345 participants were included in the study: the first consecutive 176 women with negative total HbF expression (<1%), and the first consecutive 169 women with positive total HbF expression (≥1%). Hb studies were conducted with high performance liquid chromatography (HPLC), and FC was performed if total HbF expression was ≥1%. Other assessments included DNA sampling, complete blood counts, β-hCG and EPO levels, and qualitative data (i.e. to account for other factors such as tobacco, progesterone use, etc.). Analyses were conducted at each trimester except DNA sampling, which was done during the first visit only. HbF-positive women had an additional post-partum visit to assess changes of HbF expression and account for hereditary persistence of HbF. Descriptive statistics and Pearson correlations were planned for this analysis. Results. At the time of analysis, 93 HbF-positive and 147 HbF-negative women had completed follow-up. Results revealed that, among all pregnant women screened, 22.07% (n = 194) had positive HbF expression. Of this percentage, 90.72% (n = 176) filled the inclusion criteria. Of all women with positive HbF expression during 1 or more of the first 3 visits, 72.04% showed a post-partum decrease of HbF below 1%. Furthermore, FC analyses in HbF-positive women confirmed the maternal origin of HbF in all cases and revealed an average of 13.28% (sd = 5.38%) F-cells when total HbF was ≥ 1%. Only modest correlations could be found so far between HbF levels and β-hCG (ρ = 0.25) or EPO (ρ = 0.07). Comparative DNA analyses of HbF-positive and HbF-negative women are currently underway and will be presented at the meeting. Conclusions. To our knowledge, this represents the largest prospective study evaluating HbF expression throughout pregnancy, as well as the first to evaluate post-partum HbF dynamics and the genetic background of HbF-negative and HbF-positive pregnant women. Preliminary data shows that: 1) a significant percentage of pregnant women have a transient elevation of HbF which cannot be explained by fetomaternal hemorrhage nor hereditary persistence of HbF; 2) the proportion of F-cells in women remains constant peri- and post-partum when HbF ≥ 1%; 3) a large proportion of maternal erythrocytes express HbF at low levels and; 4) HbF modulation during pregnancy is only modestly correlated to β-hCG and EPO. These data may lead to the study of a new paradigm of Hb subtype switch as well as HbF-induction therapeutics for the benefit of Hb disorders. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

38

Koshy, Mabel, Louise Dorn, Linda Bressler, et al. "2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia." Blood 96, no. 7 (2000): 2379–84. http://dx.doi.org/10.1182/blood.v96.7.2379.

Full text

Abstract:

Abstract Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.

APA, Harvard, Vancouver, ISO, and other styles

39

Koshy, Mabel, Louise Dorn, Linda Bressler, et al. "2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia." Blood 96, no. 7 (2000): 2379–84. http://dx.doi.org/10.1182/blood.v96.7.2379.h8002379_2379_2384.

Full text

Abstract:

Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.

APA, Harvard, Vancouver, ISO, and other styles

40

Fevereiro-Martins, Mariza, Laura Aguiar, Ângela Inácio, et al. "Fetal Hemoglobin as a Predictive Biomarker for Retinopathy of Prematurity: A Prospective Multicenter Cohort Study in Portugal." Biomedicines 13, no. 1 (2025): 110. https://doi.org/10.3390/biomedicines13010110.

Full text

Abstract:

Background/Objectives: Retinopathy of prematurity (ROP) is a leading cause of vision impairment in preterm infants, with its pathogenesis linked to oxygen exposure. Red blood cell (RBC) transfusions, commonly performed in neonatal intensive care units (NICUs), reduce fetal hemoglobin (HbF) fraction, altering oxygen dynamics and potentially contributing to ROP. We aimed to investigate the relationship between RBC transfusions, HbF percentage, and ROP, evaluating HbF as a potential predictive biomarker. Methods: A multicenter, prospective study was conducted across eight Portuguese NICUs, involving infants born at <32 weeks gestational age (GA) or <1500 g. ROP staging followed the International Classification of ROP (ICROP2). Clinical data were collected during hospitalization, and HbF fractions were measured from blood samples in the first four weeks of life using standardized methods. Infants were stratified by ROP presence and treatment requirement. Statistical analysis was performed using SPSS 28.0, with p < 0.05. Results: Eighty-two infants (mean GA: 28.1 ± 2.1 weeks, birth weight: 1055.8 ± 258.3 g) were included. Among them, 29 (35.4%) presented ROP and 4 (4.9%) required treatment. Infants with ROP had more RBC transfusions and lower HbF percentages than those without ROP (p < 0.05). Lower HbF was associated with more RBC transfusions (p < 0.001). Kaplan–Meier survival curves showed a higher ROP risk in infants with reduced HbF (p < 0.05). Conclusions: Low HbF percentage in the first four weeks of life may increase ROP risk in preterm infants. HbF could serve as a biomarker for ROP prediction. Interventions preserving HbF may reduce ROP risk. Further studies are needed to validate HbF as a biomarker and refine prevention strategies.

APA, Harvard, Vancouver, ISO, and other styles

41

Press, Katharine Rose, Jeffrey Keefer, Steven D. Gore, Hetty E. Carraway, Sarah Sakoian, and Thomas Prebet. "Clinical Evaluation of Combined Epigenetic Therapies on the Induction of Fetal Hemoglobin in Patients with Hematologic Malignancies." Blood 126, no. 23 (2015): 960. http://dx.doi.org/10.1182/blood.v126.23.960.960.

Full text

Abstract:

Fetal hemoglobin induction with hydroxyurea (HU) is a mainstay of therapy for β-hemoglobinopathies, especially sickle cell disease (SCD). A high level of fetal hemoglobin (HbF) has a direct relationship with acute clinical status in SCD patients including pain crises, acute chest syndrome, and death. However, not all patients benefit from HU, and more effective HbF induction strategies are needed. DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors have been shown in vitro to induce HbF production through epigenetic modification of the β-globin gene cluster. Azacitidine (AZA) is a DNMT already used in some SCD patients resistant to HbF modulation with HU. Entinostat (MS-275) is an orally available histone deacetylase inhibitor with a long half-life and established antitumor activity in preclinical models. Recent studies suggest that drugs, which act with different molecular and epigenetic mechanisms, have synergistic effects on induction of fetal hemoglobin (Fard et al. IJHOSCR 2013). In this study, we evaluated the effects of a combination of AZA and MS-275 on HbF levels. This was preformed as a correlative study of a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We sequentially measured the level of HbF the peripheral blood in 33 patients receiving different doses of AZA (range: 30mg/m2 to 50mg/m2 per day for 10 doses) and MS-275 (range: 2 to 8 mg/m2 orally on days 3 and 10). Patients completed a minimum of four 28-day cycles of combined therapy. HbF levels were measured in peripheral blood at baseline, at day 15 or 16 and day 29 or 30 of cycle 1, and after cycles 2, 4, and if applicable 6. Azacitidine dose positively correlated with HbF fold increase (mean of 1.1, 2.3, and 2.1 for doses of 30, 40, and 50 mg respectively, p=0.07) while MS-275 dose had a slightly negative correlation with HbF level (mean of 3.0, 1.8, and 1.3 for doses of 2, 4, and 6mg respectively, p=0.13). There was no correlation between baseline HbF and HbF fold increase after exposure to treatment (p=NS) and no correlation between baseline HbF levels and clinical disease response (p=0.19). Interestingly, we demonstrated a correlation between HbF fold increase and clinical disease response: median fold increase of 3.5 for patients achieving hematologic normalization (complete response, partial response, or trilineage hematological improvement) versus 1.4 in non-responders (p=0.006). The positive correlation between AZA dose and HbF increase is consistent with prior work showing that this drug induces HbF production. The correlation between clinical response and HbF induction could reflect a greater susceptibility to AZA potentially related to differing methylomes. Alternatively, it may also represent a known increase in HbF in the setting of stress erythropoiesis. The slight inverse correlation between MS-275 and HbF level was surprising, as other HDAC inhibitors are known to induce HbF in vitro. However, these results are in line with the methylation data found in the more recent randomized phase 2 trial of AZA +/- MS-275 (E1905 trial) that showed a potential pharmacodynamic antagonism of the combination (Prebet et al. J Clin Oncol. 2014). Overall, this work supports the use of AZA as a clinical inducer of HbF. It also shows the importance of trialing various combinations of HbF inducers, as not all drugs work synergistically and some may even be antagonistic in combination. Disclosures Off Label Use: Azacitidine (AZA) is a DNA methyl transferase (DNMT) inhibitor. Entinostat (MS-275) is an orally available histone deacetylase inhibitor. Both drugs were used in a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). . Keefer:MAST therapeutics: Employment. Gore:Celgene: Consultancy, Honoraria, Research Funding. Prebet:CELGENE: Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

42

Pavlenko, Anastasiia, Sławomir Lasota, Dawid Wnuk, et al. "Bronchial Fibroblasts from Asthmatic Patients Display Impaired Responsiveness to Direct Current Electric Fields (dcEFs)." Biomedicines 11, no. 8 (2023): 2138. http://dx.doi.org/10.3390/biomedicines11082138.

Full text

Abstract:

Accumulating evidence suggests that an important role is played by electric signals in modifying cell behaviour during developmental, regenerative and pathological processes. However, their role in asthma has not yet been addressed. Bronchial fibroblasts have recently been identified having important roles in asthma development. Therefore, we adapted an experimental approach based on the lineages of human bronchial fibroblasts (HBF) derived from non-asthmatic (NA) donors and asthmatic (AS) patients to elucidate whether their reactivity to direct current electric fields (dcEF) could participate in the asthmatic process. The efficient responsiveness of NA HBF to an electric field in the range of 2–4 V/cm was illustrated based on the perpendicular orientation of long axes of the cells to the field lines and their directional movement towards the anode. These responses were related to the activity of TGF-β signalling, as the electrotaxis and re-orientation of NA HBF polarity was impaired by the inhibitors of canonical and non-canonical TGF-β-dependent pathways. A similar tendency towards perpendicular cell-dcEF orientation was observed for AS HBF. However, their motility remained insensitive to the electric field applied at 2–4 V/cm. Collectively, these observations demonstrate the sensitivity of NA HBF to dcEF, as well as the inter-relations between this parameter and the canonical and non-canonical TGF-β pathways, and the differences between the electrotactic responses of NA and AS HBF point to the possible role of their dcEFs in desensitisation in the asthmatic process. This process may impair the physiologic behaviour of AS HBF functions, including cell motility, ECM deposition, and contractility, thus promoting bronchial wall remodelling, which is a characteristic of bronchial asthma.

APA, Harvard, Vancouver, ISO, and other styles

43

Hernandez, Britney, Ashwin P. Patel, Erica N. Evans, et al. "Rheological Comparison of Red Cells from Individuals with SCD Treated with Gene Therapy, Haploidentical Transplantation or Hydroxyurea." Blood 144, Supplement 1 (2024): 4956. https://doi.org/10.1182/blood-2024-210578.

Full text

Abstract:

Background: Sickle cell disease is caused by point mutation in the beta-globin gene, (HBB) resulting in rigid, dense red blood cells (RBC) that sickle under hypoxic conditions. Outcomes from matched sibling donors (MSD) are excellent, but donor availability is limiting. Patients without a MSD may consider hydroxyurea (HU), haploidentical hematopoietic cell transplant (HaploHCT), or gene therapy (GT). Two GT strategies are FDA approved; a lentiviral gene addition approach that introduces a normal copy of the HBB gene, and a CRISPR/Cas9 strategy that induces fetal hemoglobin (HbF) through disruption of the erythroid enhancer element of the globin gene transcriptional regulator BCL11A(CTX001). Our goal is to assess the red blood cell (RBC) quality of different gene therapy strategies (FDA approved and in clinical trials) and compare to rheology of HaploHCT and to optimal medical management in a cohort of individuals with an excellent HbF response to HU. Methods: Individuals treated on NCT05329649 (CTX001), NCT04293185 (BB305), and HU treated individuals provided samples under an Emory IRB approved protocol in EDTA tubes. NCT04443907 (OTQ923) and NCT04819841 clinical trial samples were provided by the sponsor. HU samples were obtained from individuals at maximum tolerated dose exhibiting a final %HbF of 20% or more, without hereditary persistence of fetal hemoglobin. HaploHCT samples were collected under a St Jude Children's Research Hospital sponsored clinical trial (NCT04362293). All samples were collected without exogenous HbA present, 6-12 months after graft infusion; 18 months for NCT04819841. Complete blood counts (CBC) were obtained on an ADVIA hematology analyzer which provides percent dense red blood cells (%DRBC). %HbF was measured using high performance liquid chromatography (HPLC). Oxygen gradient ektacytometry (LoRRca) was used to assess red cell deformability; readouts are elongation index minimum (EIMin) deformability under hypoxia, elongation index maximum (EIMax) deformability under normoxia, and point of sickling (PoS), the oxygen tension at which HbS polymerization begins. Kruskal-Wallis or Mann Whitney tests were used for comparisons, and interquartile range (IQR) calculated to assess spread of values within each group. Results: We compared the GT (CTX001, n=2, BB305, n=2, and sponsored clinical trial OTQ923, n=4), HaploHCT (n=4), and HU (n=15) groups. EImin, EImax, DRBC, and RBC were significantly different in HU compared to HaploHCT (p: 0.001, 0.021, 0.01, and 0.03, respectively). The median and IQR were as follows: EImax (HU: 0.50(0.47-0.56),GT: 0.57(0.49-0.59), HaploHCT: 0.59(0.59-0.60)), EImin (HU: 0.15 (0.13-0.19),GT:0.29 (0.23-0.37), HaploHCT: 0.59(0.57-0.59)), DRBC (HU:4.9 (3.4-6.3), GT:2.1(1.6-6.6), HaploHCT:0.1(0.1-1.1)), and RBC (HU: 2.8(2.5-3.4), GT:3.4 (3.0-4.0),HaploHCT: 3.9 (3.4-4.1)). The range of red cell function values was narrow for HaploHCT, wide for GT and HU. Significantly lower EImin and EImax in HU compared to HaploHCT indicates worse RBC deformability under normoxia and hypoxia. %HbF levels were comparable between HU and GT. There was no statistically significant difference in PoS between GT and HU; HaploHCT treated individuals' RBC did not sickle and had no PoS. Conclusions: RBC function testing provides an opportunity to compare optimal HU response and curative strategies for SCD, including GT and HaploHCT. We observed a wide range of red cell function values in the GT group, suggesting that with appropriate clinical correlations, these tests can help compare different GT strategies by assessing the degree of red cell correction achieved by each one. We found that the RBC quality and function of HU was comparable to GT, when samples are selected from individuals having a HbF/functional hemoglobin response comparable to that that of individuals post GT. Post HaploHCT, individuals exhibited nearly normalized RBC rheology, with very similar inter-individual results. Further studies are needed to confirm our findings in a larger SCD patient cohort, including samples pre- and post-transplant; longer follow-up of both strategies are also needed.

APA, Harvard, Vancouver, ISO, and other styles

44

Hebert, Nicolas, Erica B. Esrick, Myriam Armant, et al. "Effects of BCL11A Shmir-Induced Post-Transcriptional Silencing on Distributions of HbF in Single-RBCs and Reticulocytes." Blood 138, Supplement 1 (2021): 967. http://dx.doi.org/10.1182/blood-2021-150186.

Full text

Abstract:

Abstract NH and EE equally contributed. ADW and PB co-signed. The expression of fetal hemoglobin (HbF) is one of the main targets of sickle cell disease treatment, as it inhibits the polymerization of hemoglobin S. The hypothesis of an inhibitory threshold of HbF per red blood cell (RBC) has been suggested, 1 although not well defined, as the overall percentage of HbF does not reflect the heterogeneous distribution of HbF per cell. Likewise, the qualitative analysis of RBCs containing HbF, called F cells, is neither reproducible nor clinically interpretable, due to low expression. 2 We have developed a technique for measuring the amount of HbF per cell, to determine thresholds of HbF expression per RBC correlated with clinical and biological effects. 2 Among genes controlling its expression, BCL11A has a major repressive effect on the expression of gamma globin/HbF during the fetal to adult hemoglobin switch. Post-transcriptional silencing of BCL11A, using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression, is safe and demonstrates high levels of %HbF in a pilot clinical study (NCT 03282656). 3 Here, we show the quantitative measurement of HbF per RBC and reticulocyte. Methods: During patient follow-up, HbF quantification per single cell RBC was performed using a fluorescent HbF antibody. 2 Addition of an anti-CD71 fluorescent antibody allowed selection of reticulocyte sub-populations for determining their HbF content. Fold-increase in percentage of RBC versus percentage of reticulocyte were calculated. Kinetics of HbF/RBC and HbF/Reticulocyte were modeled using mixed effects polynomial linear regression to account for the correlation between repeated data over time. Results: With a median follow-up of 15 months [12-20] after gene transfer, figure 1 shows the mathematical modeling of single-RBC HbF measurement representing RBC percentage containing at least 2, 4, 6, 8 and 10 pg of HbF. Percentage of RBC above each threshold was higher compared to 14 hydroxyurea treated patients for 6 months. Figure 2 shows fold increase between reticulocytes and RBCs with same thresholds of HbF/cell. For low thresholds, RBCs were found in same percentage as reticulocytes whereas RBCs containing increasing levels of HbF were found in higher percentage than reticulocytes, until 6pg/cell showing a clear selective advantage for red cells with a threshold ≥ 6pg/cell of HbF. Figure 3 shows different kinetics of HbF increase according to two different transduction strategies with 2 enhancers in patients 2-4 compared to one enhancer in patients 6-8. Conclusion: BCL11A down-regulation in six clinical trial subjects was associated with an in vivo selection process RBCs with ≥ 6pg HbF per cell attained with different engraftment kinetics, depending on transduction processes, and ultimately stable high level and broadly distributed HbF. 1 Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014 Jan 23;123(4):481-5. 2 Hebert N, Rakotoson MG, Bodivit G, et al. Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease. Am. J. Hematol. 3 Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215. Figure 1 Figure 1. Disclosures Esrick: bluebird bio: Consultancy. Audureau: GBT: Honoraria. Higgins: Sebia, Inc.: Honoraria; Danaher Diagnostics: Consultancy. Williams: BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Bartolucci: AGIOS: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; Jazz Pharma: Other: Lecture fees; Emmaus: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; INNOVHEM: Other: Co-founder; Hemanext: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Fabre Foundation: Research Funding.

APA, Harvard, Vancouver, ISO, and other styles

45

Adorno, Elisangela V., Jose P. Moura Neto, Isa Lyra, et al. "A New Sequence Change in the Hs2-Lcr (G→A − 10.677) and Gg-Globin Gene Promoter Region (T→C − 157 and a 4 Bp Deletion − 222 to − 225) in Sickle Cell Anemia Patients with Fetal Hemoglobin Level and bS-Globin Gene Haplotypes Diversity." Blood 108, no. 11 (2006): 3803. http://dx.doi.org/10.1182/blood.v108.11.3803.3803.

Full text

Abstract:

Abstract The sickle cell anemia has a very high prevalence in Bahia, a state of Brazil. The HbF levels and bS-globin gene haplotypes of 125 sickle cell anemia patients were investigated and the Gγ and Aγ gene promoter and HS2-LCR regions of ten patients were selected for DNA sequence. The study was approved by the Oswaldo Cruz Research Foundation’s human research ethics committee. The bS- globin gene haplotypes were investigated using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques; the HbF levels were estimated by high performance liquid chromatography (HPLC). The Gg and Ag-globin gene promoter and HS2-LCR regions were sequenced in an automatic sequencer. The distribution of bS- globin gene haplotypes showed 64 (51.2%) patients with genotype CAR/Ben; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; two (1.6%) CAR/Aty; two (1.6%) Ben/Cam; one (0.8%) Car/Cam; one (0.8%) CAR/Arab-India and one (0.8%) Sen/Aty. Among these patients, four CAR/CAR had HbF ≥10.0%; three Ben/Ben had HbF ≤ 5.0%; 11 CAR/Ben had HbF ≥15.0% and 18 had HbF ≤ 5.0%; two Cam/Ben patients had HbF levels ≥15.0%. Ten individuals presenting HbF and bS-globin gene haplotypes diversity were selected to sequence the Gg and Ag-globin gene promoter and HS2-LCR regions. The HS2-LCR sequence analyses demonstrated a G→A change (−10.677) (GeneBank DQ873522) in five sickle cell anemia patients with Ben haplotype and high HbF levels (Table 1); the CAR/Ben and Ben/Ben patients with low HbF levels did not have this sequence variation; among the small patient group investigated, there were two CAR/CAR patients with high HbF levels without this substitution. The Gg gene promoter sequence analyses showed a T→C substitution (−157) (GeneBank DQ873521) among all patients and a 4 bp deletion (−222 to −225) (GeneBank DQ873519) related to Cam haplotype (Figure 1). The HS2-LCR sequence change reported here is located in the proximity to a binding site of the GATA-1 and a ubiquitous trans-acting factor and can constitute a motif associated to the Ben chromosome and may play an important role in g-globin gene transcription regulation. The polymorphic site on Gg-globin gene promoter region can be a common sequence characteristic among the Brazilian sickle cell anemia patients. The results described here suggest new polymorphisms in the HS2-LCR and Gg gene promoter and confirms the genotypic heterogeneity among Brazilian sickle cell anemia patients, justifying further studies to investigate its correlation with HbF synthesis and clinical profile in sickle cell anemia patients. Figure Figure

APA, Harvard, Vancouver, ISO, and other styles

46

Stevenson, F. Craig, and Adrian M. Johnston. "Annual broadleaf crop frequency and residual weed populations in Saskatchewan Parkland." Weed Science 47, no. 2 (1999): 208–14. http://dx.doi.org/10.1017/s0043174500091633.

Full text

Abstract:

The development of problematic weed populations is a concern in western Canadian fields where canola and pea are grown in a 4-yr sequence with spring cereal grains. Weed densities were examined at a site near Melfort, Saskatchewan, Canada, from 1994 to 1997 in seven zero-till managed crop rotations. Four rotations that included canola, pea, or flax in at least 3 of 4 yr (HBF: high broadleaf–crop frequency) were compared with three rotations that included broadleaf crops grown in 2 of 4 yr (LBF: low broadleaf–crop frequency). Spring wheat and barley were the cereal crops in rotation. Residual (postherbicide application) weed density for each weed species in a given year was summed across all phases for each rotation to reflect the overall weed infestation. Four annual broadleaf weed species were most abundant in 1996 and a second group of three species, having a variety of reproductive strategies, became progressively less abundant as the study progressed. The difference between the HBF and LBF rotations for the density of these species varied and was most prominent in years when environmental conditions were conducive for their growth. More frequent applications of ethafluralin, with its residual weed control, best explained why wild oat and catchweed bedstraw generally were less abundant in the HBF rotations. Of particular interest was the 8 plants m−-2greater density of dandelion and perennial sowthistle in the HBF vs. LBF rotations in the last year of the study. It is thought that the limited herbicide options for the control of these species could present a future problem if they continued to develop in the HBF rotations. Differences in herbicide use between the HBF and LBF rotations were considered the primary factor controlling the rotation effects on weed density.

APA, Harvard, Vancouver, ISO, and other styles

47

Vichinsky, EP, and BH Lubin. "A cautionary note regarding hydroxyurea in sickle cell disease." Blood 83, no. 4 (1994): 1124–28. http://dx.doi.org/10.1182/blood.v83.4.1124.1124.

Full text

Abstract:

Abstract Hydroxyurea can increase fetal hemoglobin (HbF) and improve the clinical course of sickle cell disease (SCD) patients. However, several issues of hydroxyurea therapy remain unresolved, including differences in patients' drug clearance, predictability of drug response, reversibility of sickle cell disease-related organ damage by hydroxyurea, and the efficacy of elevated HbF. We treated two patients with hydroxyurea for periods of 1 to 4 years, monitoring clinical course and laboratory parameters at regular intervals. The first patient (patient A) had a history of chronic pain and extensive hospitalizations. The second patient (patient B) had a history of stroke and refused to continue with chronic transfusion therapy and chelation. Both patients showed a fivefold to tenfold increase in HbF (5% to 25%, 3% to 31%). However, patient A developed an acute chest syndrome, despite an HbF level of 20%. After red blood cell transfusions for hypoxia, the HbF level decreased to 5%. When hydroxyurea dosage was increased, pancytopenia developed and was not resolved until 2 months after hydroxyurea was discontinued; Patient B developed a cerebral hemorrhage on hydroxyurea; he died shortly thereafter. His HbF level was 21% before death. We noted an increase in HbF and a general improvement in the two patients. However, both experienced major SCD-related complications despite HbF levels over 20%. Our findings also suggest that the progressive vascular changes associated with SCD are unlikely to be dramatically affected by increased HbF levels. Because neither the efficacy nor the toxicity of hydroxyurea have been thoroughly investigated, physicians should be cautious in prescribing hydroxyurea for patients with SCD before completion of the National Clinical Trial.

APA, Harvard, Vancouver, ISO, and other styles

48

Vichinsky, EP, and BH Lubin. "A cautionary note regarding hydroxyurea in sickle cell disease." Blood 83, no. 4 (1994): 1124–28. http://dx.doi.org/10.1182/blood.v83.4.1124.bloodjournal8341124.

Full text

Abstract:

Hydroxyurea can increase fetal hemoglobin (HbF) and improve the clinical course of sickle cell disease (SCD) patients. However, several issues of hydroxyurea therapy remain unresolved, including differences in patients' drug clearance, predictability of drug response, reversibility of sickle cell disease-related organ damage by hydroxyurea, and the efficacy of elevated HbF. We treated two patients with hydroxyurea for periods of 1 to 4 years, monitoring clinical course and laboratory parameters at regular intervals. The first patient (patient A) had a history of chronic pain and extensive hospitalizations. The second patient (patient B) had a history of stroke and refused to continue with chronic transfusion therapy and chelation. Both patients showed a fivefold to tenfold increase in HbF (5% to 25%, 3% to 31%). However, patient A developed an acute chest syndrome, despite an HbF level of 20%. After red blood cell transfusions for hypoxia, the HbF level decreased to 5%. When hydroxyurea dosage was increased, pancytopenia developed and was not resolved until 2 months after hydroxyurea was discontinued; Patient B developed a cerebral hemorrhage on hydroxyurea; he died shortly thereafter. His HbF level was 21% before death. We noted an increase in HbF and a general improvement in the two patients. However, both experienced major SCD-related complications despite HbF levels over 20%. Our findings also suggest that the progressive vascular changes associated with SCD are unlikely to be dramatically affected by increased HbF levels. Because neither the efficacy nor the toxicity of hydroxyurea have been thoroughly investigated, physicians should be cautious in prescribing hydroxyurea for patients with SCD before completion of the National Clinical Trial.

APA, Harvard, Vancouver, ISO, and other styles

49

Meier, Emily R., Colleen Byrnes, Maxine Weissman, et al. "Stressed Erythropoiesis In Children with Sickle Cell Disease Is An Indicator of Low Fetal Hemoglobin Production and Increased Disease Severity." Blood 116, no. 21 (2010): 2666. http://dx.doi.org/10.1182/blood.v116.21.2666.2666.

Full text

Abstract:

Abstract Abstract 2666 Acute erythroid stress causes elevated levels of fetal hemoglobin (HbF) in primates. However, relationships between the chronic erythroid stress in children with Sickle Cell Disease (HbSS) and HbF production are less well understood. For this study, peripheral blood from children with HbSS was analyzed within 72 hours of collection and storage at 4°C. The level of erythroid stress was determined by absolute reticulocyte counts (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (HiCD71, LoCD71 and NegCD71) and CD36. Immature reticulocytes were identified by HiCD71 expression and included the CD36+ population. Sorted populations from at least three subjects were utilized to assess fetal globin expression during reticulocyte maturation according to quantitative polymerase chain reaction (qPCR), and high pressure liquid chromatography (HPLC). While the number of gamma-globin mRNA copies/cell decreased with reticulocyte maturation, the ratio of gamma/gamma+beta mRNA remained stable. HbF protein levels in the HiCD71 and LoCD71 populations were equivalent for each patient with average values of 4.3±4.2% HiCD71 vs. 4.4±4.0% LoCD71, p=0.96. By comparison, HbF levels in the NegCD71 were increased in all subjects (14.2±9.5% NegCD71, p<0.05) consistent with prolonged survival of the HbF expressing cells. Unexpectedly, the percentage of immature reticulocytes neither correlated with the total ARC nor total HbF expression in the peripheral blood. Further analysis of 82 HbSS patients 4–21 years of age was performed to determine if ARC, by itself, might provide a simple clinical indicator of HbF expression or disease severity in this population. Thirteen patients had been treated with HU, 17 had received regular RBC transfusions (Tx), and the remainder (n=52) received neither HU nor monthly Tx within 4 months of analysis (Ctrl). A negative correlation between ARC (K/uL) and HbF levels was present in both the HU and Ctrl groups (HU r=-0.76, p=0.0016; Ctrl r=-0.34, p=0.013), but the correlation was lost in the Tx group (r=-0.21, p=0.43). Clinical events were defined as acute chest syndrome, splenic sequestration, conditional and abnormal transcranial Doppler (TCD),silent cerebral infarct, overt stroke, laparascopic cholecystectomy and bacteremia. There was no significant correlation between ARC and clinical events in the HU and Tx groups. Within the Ctrl group, those with an ARC below 300 (21 of 52, 40.4%) had a statistically lower event rate (ARC<300 mean event rate=0.23±0.18 events/yr vs. ARC>300 mean event rate=0.59±0.60 events/year, p=0.0028). These data suggest that increased levels of erythroid stress as measured by reticulocyte production and maturation in pediatric HbSS subjects is not associated with elevated gamma-globin mRNA or HbF levels. Instead, higher levels of reticulocytosis were associated with lower HbF levels and increased disease severity. Disclosures: Noel: NIH: Patents & Royalties.

APA, Harvard, Vancouver, ISO, and other styles

50

Azzouzi, Imane, Jeannine Winkler, Jean-Claude Fauchère, et al. "MicroRNA-96 Inhibits Fetal Hemoglobin Expression During Erythropoiesis." Blood 116, no. 21 (2010): 3220. http://dx.doi.org/10.1182/blood.v116.21.3220.3220.

Full text

Abstract:

Abstract Abstract 3220 Background: Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized until birth; it subsequently declines and switches to adult hemoglobin, HbA (α2β2). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in hemoglobinopathies. Analysis from e.g. Hb Corfu and δβ-thalassemia indicates that γ-globin gene expression is post-transcriptionally regulated (Efremov DG., Br J Haematol, Oct 1994; Chakalova L., Blood, 2005). The post-transcriptional regulation of HbF might involve regulatory non-coding RNAs. MicroRNAs (miRNAs) are a small non-coding family of 21 nucleotide RNAs that regulate gene expression post-transcriptionally by targeting mRNAs. After transcription and maturation the miRNA is incorporated into the miRNA-induced silencing complex (miRISC). The miRISC, which contains argonaute proteins (AGO), binds and silences the target mRNAs. Previous studies have reported a role of miRNAs in the developmental switch from HbF to HbA synthesis (Noh SJ, J Transl Med, 2009; Bianchi N., BMB Rep, 2009), however so far no direct interaction between miRNAs and the γ-globin mRNA has been reported. Moreover a function of miRISC in mature red blood cells (RBC) has not been shown yet. Therefore, we aimed (I) to characterize the miRISC composition in RBCs, (II) to identify miRNAs that are directly involved in the regulation of HbF and (III) to study their function during erythropoiesis. Methods: To characterize miRISC in RBCs, we performed co-immunoprecipitations of the miRISC using antibodies directed against human AGO. The immunoprecipitated complexes were analyzed by Western-blot, mass spectrometry and qPCR. To identify miRNAs potentially involved in the regulation of HbF miRNA expression patterns in RBCs from adult and umbilical cord blood were analyzed by qPCR. To confirm the role of selected miRNAs, γ-globin protein levels were measured by ELISA in HEL cell cultures transfected with miRNA precursors or inhibitors as well as in primary erythroid cultures overexpressing miRNAs after lentiviral transduction. The direct interaction between miRNAs and the γ-globin mRNA was analyzed by luciferase reporter assay. Results: AGO1, 2, 3 and 4 and Importin 8 were detected in RBCs both from adult and cord blood. In adult RBC samples (n=3), expressing <1% HbF of total Hb, γ-globin mRNA was bound to AGO2 but not to AGO1 containing miRISC. However in cord blood (n=3), containing 90 % HbF and 30 times more γ-globin mRNA molecules than adult RBCs, the amount of γ-globin mRNA bound to AGO2 containing miRISC was 200 times less (p<0.05). With these indications, we started to analyze miRNA patterns in adult and cord blood RBCs. miR-96, miR-146a, miR-330-3p, let-7a and miR-888 were significantly downregulated in cord blood RBCs compared to adult RBCs. However, luciferase reporter assays demonstrated that only miR-96 was able to bind to the predicted target site within the γ-globin mRNA. Transfection of HEL cells with miR-96 inhibitor showed a twofold induction (p<0.01; n=3) of the γ-globin protein level compared to control cells transfected with scramble RNA. Consistently, transfection with miR-96 precursor in HEL cells showed a 15% reduction (p<0.05, n=3) of the γ-globin protein level. To study the miR-96 mediated inhibition of HbF during erythropoiesis, cord blood derived primary erythroid cultures were transduced with miR-96 lentiviruses. A 40% decrease (p<0.05, n=3) of γ-globin protein expression was observed in erythroid cells overexpressing miR-96 compared to cells transduced with the negative control virus. This γ-globin reduction was observed on 10 and 14 days old cultures, which contained 80% and 65% CD235+ CD71+ erythroblasts, respectively. Conclusions: This study identifies miR-96 as direct inhibitor of the HbF expression during erythropoiesis. Specifically we observed that in cells with low HbF content, γ-globin mRNAs are bound to miRISC and are therefore directly inhibited by miRNAs. We demonstrated that miR-96 is binding to a seedless target site within the γ-globin open reading frame. Finally we showed that miR-96 overexpression reduces the expression of HbF in erythroleukemia cells and during late stages of cord blood derived erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'HBF 4'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles