Single Nucleotide Variations In Spectrin-1β Accentuate The Red Blood Cell Storage Lesion

Abstract Introduction During routine storage packed red blood cells (PRBC) undergo biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. Donor-to-donor variability in the severity of the storage lesion has been reported. The extent to which donor-associated differences in blood component storage affect blood product quality and post-transfusion outcome remains unknown. Murine models with single nucleotide variants (SNV) in gene encoding spectrin-1β were used to investigate the impact of mutations on the RBC storage lesion. Methods Two murine lineages with N-ethyl-N-nitrosourea (ENU) generated single SNV in Spnb1, encoding spectrin-1β (Table 1), were selected from the Australian Phenomics Facility library (http://databases.apf.edu.au/mutations). Using genetic selection, homozygous (HOM), heterozygous (HET) and unaffected (WT) mice from each strain were generated (C57BL/6 background strain). Murine blood was leucoreduced, prepared in SAGM (0.4 HCT) and stored at 4°C for time course assessment of RBC characteristics. At day (D), D2, D7, D14 and D21 of storage, RBC integrity and evidence of storage-related changes were investigated using RBC osmotic fragility and flow cytometric analysis of CD44, CD47, TER119 and phosphatidylserine (PS). Data were generated from analysis of blood from Spnb1 (pedigree spectrin-1β a) homozygous (HOM, n=3), heterozygous (HET, n=3) and unaffected (WT, n=2 ); Spnb1 (pedigree spectrin-1β b) HOM (n=4), HET( n=4); C57BL/6 (n=4). The Mann-Whitney Test and ANOVA were utilised for statistical analyses (95% CI). Results At D2 of storage SNV in Spnb1 did not alter RBC characteristics, with all mice studied demonstrating a similar resistance to osmotic lysis and levels of CD44, CD47, TER119 and PS. By D7 of storage, clear pedigree-related differences in RBC characteristics were evident. At D7, RBC from spectrin-1β(a) HOM mice had significantly increased osmotic fragility and exposure of PS as well as significantly reduced CD44 and TER119 expression compared to unaffected siblings and background strain. Of note, these changes were not evident in the spectrin-1β(b) HOM mice at D7. For both strains at D7, heterozygous SNV did not exhibit altered storage parameters. By D14 both HOM and HET spectrin-1β(a) mice demonstrated a phenotype consistent with an exacerbated RBC storage lesion, characterised by significantly increased osmotic fragility and exposure of PS, and reduced CD44 and CD47 compared to background strain. At D14 there was also evidence of exacerbation of the storage lesion in stored RBC from HOM spectrin-1β(b) mice (significantly increased PS), though this was not to the extent observed in the spectrin-1β(a) mice. By D21 all murine RBC were substantially degraded under these storage conditions. Conclusions SNV in Spnb1,encoding RBC structural protein spectrin-1β, resulted in both early onset and exacerbation of the RBC storage lesion. Further, the degree of storage lesion and the point at which RBC degradation was observed was not only dependent on the homozygous or heterozygous status, but the mutation itself. These data demonstrate that minor genetic variation in genes encoding important RBC proteins contribute to donor related differences in PRBC storage. Disclosures: No relevant conflicts of interest to declare.

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Prediction of genome-wide effects of single nucleotide variants on transcription factor binding

Scientific Reports ◽

10.1038/s41598-020-74793-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sebastian Carrasco Pro ◽

Katia Bulekova ◽

Brian Gregor ◽

Adam Labadorf ◽

Juan Ignacio Fuxman Bass

Keyword(s):

Binding Sites ◽

Cancer Type ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Regulatory Regions ◽

Genome Wide ◽

Transcriptional Regulatory ◽

Gene Regulatory ◽

The Impact ◽

The Relationship

Abstract Single nucleotide variants (SNVs) located in transcriptional regulatory regions can result in gene expression changes that lead to adaptive or detrimental phenotypic outcomes. Here, we predict gain or loss of binding sites for 741 transcription factors (TFs) across the human genome. We calculated ‘gainability’ and ‘disruptability’ scores for each TF that represent the likelihood of binding sites being created or disrupted, respectively. We found that functional cis-eQTL SNVs are more likely to alter TF binding sites than rare SNVs in the human population. In addition, we show that cancer somatic mutations have different effects on TF binding sites from different TF families on a cancer-type basis. Finally, we discuss the relationship between these results and cancer mutational signatures. Altogether, we provide a blueprint to study the impact of SNVs derived from genetic variation or disease association on TF binding to gene regulatory regions.

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Re-evaluation of single nucleotide variants and identification of structural variants in a cohort of 45 sudden unexplained death cases

International Journal of Legal Medicine ◽

10.1007/s00414-021-02580-5 ◽

2021 ◽

Author(s):

Jacqueline Neubauer ◽

Shouyu Wang ◽

Giancarlo Russo ◽

Cordula Haas

Keyword(s):

Sudden Death ◽

Cardiac Diseases ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Sudden Unexplained Death ◽

Unexplained Death ◽

Pathogenic Variants ◽

The Impact ◽

Death Cases

AbstractSudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

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CSIG-22. CANCER-ASSOCIATED MISSENSE SINGLE NUCLEOTIDE VARIANTS REGULATE THE STABILITY AND SUBCELLULAR LOCALIZATION OF NF2/MERLIN

Neuro-Oncology ◽

10.1093/neuonc/noaa215.134 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii32-ii32

Author(s):

Charlotte Eaton ◽

Paola Bisignano ◽

David Raleigh

Keyword(s):

Tumor Suppressor ◽

Subcellular Localization ◽

Function Analysis ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Ferm Domain ◽

Binding Partners ◽

Subcellular Compartments ◽

The Stability ◽

The Impact

Abstract BACKGROUND Alterations in the NF2 tumor suppressor gene lead to meningiomas and schwannomas, but the tumor suppressor functions of the NF2 gene product, Merlin, are incompletely understood. To address this problem, we performed a structure-function analysis of Merlin by expressing cancer-associated missense single-nucleotide variants (mSNVs) in primary cancer cells for biochemical and cell biology experiments. METHODS All NF2 mSNVs were assembled from cBioPortal and COSMIC, and modelled on the FERM, a-helical, and C-terminal domains of Merlin (PDB 4ZRJ) using comparative structure prediction on the Robetta server and visually inspected using Pymol. mSNV hotspots were defined from sliding windows with at least 10 mutations within 5 residues in either direction. mSNVs from hotspots in meningiomas, schwannomas, or both, were selected for in vitro mechanistic analyses using immunofluorescence and immunoblotting of whole cell, plasma membrane, cytoskeletal, cytoplasmic, nuclear, and chromatin subcellular fractions from M10G meningioma cells and HEI-193 schwannoma cells. RESULTS We identified the following cancer-associated hotspot mSNVs in NF2, which were over-expressed for mechanistic studies: L46R, S156N, W191R, A211D, V219M, R418C and R462K. Endogenous Merlin was detected in all subcellular compartments, but was enriched in the nucleus. L46R and A211D mapped to hydrophobic pockets in the FERM domain, destabilized Merlin, and excluded Merlin from all subcellular compartments except the cytoskeleton. S156N, W191R and V219M also mapped to the FERM domain, but did not affect Merlin stability, and V219M attenuated chromatin localization, suggesting this motif may be involved in binding events that regulate subcellular localization. R418C and R463K mapped to the a-helical domain, but only R418C destabilized Merlin. CONCLUSION Our results suggest that cancer-associated mSNVs inactive the tumor suppressor functions of NF2 by altering the stability, subcellular localization, or binding partners of Merlin. Further work is required to identify and understand the impact of binding partners and subcellular localization on Merlin function.

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RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants

Genome Biology ◽

10.1186/s13059-019-1847-4 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 5

Author(s):

Hai Lin ◽

Katherine A. Hargreaves ◽

Rudong Li ◽

Jill L. Reiter ◽

Yue Wang ◽

...

Keyword(s):

Rna Splicing ◽

Evolutionary Conservation ◽

Random Forest Classifier ◽

Training Data ◽

Reporter Assay ◽

Single Nucleotide Variants ◽

Excellent Performance ◽

Computational Framework ◽

Single Nucleotide ◽

The Impact

AbstractSingle nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.

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Fido-SNP: the first webserver for scoring the impact of single nucleotide variants in the dog genome

Nucleic Acids Research ◽

10.1093/nar/gkz420 ◽

2019 ◽

Vol 47 (W1) ◽

pp. W136-W141 ◽

Cited By ~ 1

Author(s):

Emidio Capriotti ◽

Ludovica Montanucci ◽

Giuseppe Profiti ◽

Ivan Rossi ◽

Diana Giannuzzi ◽

...

Keyword(s):

Matthews Correlation Coefficient ◽

Genomic Variation ◽

Gradient Boosting ◽

Binary Classifier ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Coding Regions ◽

Variation Data ◽

Boosting Algorithm ◽

The Impact

Abstract As the amount of genomic variation data increases, tools that are able to score the functional impact of single nucleotide variants become more and more necessary. While there are several prediction servers available for interpreting the effects of variants in the human genome, only few have been developed for other species, and none were specifically designed for species of veterinary interest such as the dog. Here, we present Fido-SNP the first predictor able to discriminate between Pathogenic and Benign single-nucleotide variants in the dog genome. Fido-SNP is a binary classifier based on the Gradient Boosting algorithm. It is able to classify and score the impact of variants in both coding and non-coding regions based on sequence features within seconds. When validated on a previously unseen set of annotated variants from the OMIA database, Fido-SNP reaches 88% overall accuracy, 0.77 Matthews correlation coefficient and 0.91 Area Under the ROC Curve.

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Release of Procoagulant and Proinflammatory Cell-Derived Microparticles (MP) During Blood Storage: Effects of Storage Time, Leukoreduction, and Residual Platelets,

Blood ◽

10.1182/blood.v118.21.3373.3373 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3373-3373

Author(s):

Wenche Jy ◽

Sherry Shariatmadar ◽

Marco Ricci ◽

Orlando Gomez-Marin ◽

Carlos Bidot ◽

...

Keyword(s):

Inflammatory Markers ◽

Storage Time ◽

Time Course ◽

Conflicts Of Interest ◽

Annexin V ◽

Bioactive Substances ◽

Platelet Counts ◽

The Impact ◽

Stored Blood ◽

Over Time

Abstract Abstract 3373 Introduction Several studies have indicated that transfusion with older blood carries more risk of adverse reactions than transfusion with younger blood, but this remains controversial. It is not clear why older blood may carry increased risks, or what the “safe age” of stored blood is. It is known that multiple bioactive substances are generated from blood during storage, and one or more of these substances may be involved in transfusion-related complications. Among them, MP are a recognized marker of the storage lesion, and their involvement in transfusion-related complications has been postulated. However, questions such as MP species, quantity, biological activity, and factors affecting their release are not well elucidated. The purpose of this study was to quantify MP species and their activity in stored RBC as a function of storage time, and to evaluate the impact of leukoreduction and residual platelets on MP release. Methods (I) MP generation and functional activity Thirty-four bags of packed RBC (16 non-leukoreduced, 18 leukoreduced) of known blood types (A+, B+, AB+, O+) were obtained from the blood bank within 2–4 days of drawing, and then stored at 4°C. Time of receipt was defined as day 0. At days 0, 10, 20, 30, and 40, 40 mL samples were centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for: (1) subtypes of MP by flow cytometry comprising (a) red cell MP (RMP) assessed by CD235a, (b) leukocyte MP (LMP) by CD45, (c) platelet MP (PMP) by CD41, (d) endothelial MP (EMP) by CD144, and (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV); (2) MP-mediated thrombin generation assay (TGA); (3) MP-mediated inflammatory activity by CD 11b expression in neutrophils following incubation with MP. (II) Reconstitution of increasing platelet counts in leukoreduced RBC. To investigate the effect of residual platelets on RMP generation, we mixed a constant amount of RBC with increasing amounts of type-matched platelets (0 to 250,000/μL f.c.) in standard storage bags and measured time-dependent MP release. Results (A) Time-course of MP generation (i) Non-leukoreduced. RMP, PMP and LMP all increased with time, but with different patterns. RMP increased little to day 10 but then rose exponentially, and by day 40 they were 4–6 fold higher than at day 0. PMP counts rose steadily from day 0 and peaked at day 20, being 2–3 fold higher than at day 0. LMP showed no significant change until day 20 when they started to increase, and then increased sharply after day 30, and by day 40 were 1.5–2 fold higher than at day 0. Levels of PMP (days 0 to 20) and RMP (days 20 to 40) correlated with increasing MP-mediated procoagulant and inflammatory markers. (ii) Leukoreduced. Pre-storage leukoreduction decreased RMP generation by 20–40%, completely suppressed PMP and LMP generation, and reduced total MP-mediated procoagulant and inflammatory markers by 40–60%. CBC showed that leukoreduction not only removed >99% WBC but also reduced residual platelets by >95% (from 90 ±30 ×103/μL to 3.5 ±1.3 ×103/μL). This suggests that residual leukocytes and platelets potentiate RMP generation. (B) Effects of residual platelets on RMP generation. To further study the effects of platelets on RMP generation, we mixed known counts of platelets with leukoreduced RBC, and then evaluated RMP generation over time. We found that RMP levels released were proportional to the platelet counts, as were the procoagulant and inflammatory markers. These results show that platelets in stored RBC play a key role in RMP generation. Conclusion Multiple MP types (PMP, LMP, RMP) are released during storage, and their levels increase over time but their patterns of change are different. Procoagulant and inflammatory markers increase in parallel with PMP and RMP. Our data support the hypothesis that age of stored blood could be important in transfusion-related complications, via MP production. Leukoreduction sharply reduces MP generation and procoagulant and inflammatory markers, suggesting that known benefits of leukoreduction may be attributable to reduced MP production. The finding that residual platelets in stored RBC can potentiate RMP generation suggests that minimizing platelets in non-leukoreduced packed cells could reduce the risk of transfusion-related complications. (Supported by NIH grant 1R01HL098031). Disclosures: No relevant conflicts of interest to declare.

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Association of Functional Single Nucleotide Variation in the NLRP3 Gene with Survival Outcomes After Unrelated Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v120.21.3140.3140 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3140-3140

Author(s):

Akiyoshi Takami ◽

J. Luis Espinoza ◽

Keitaro Matsuo ◽

Yasuo Morishima ◽

Makoto Onizuka ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Single Nucleotide ◽

Hematopoietic Stem ◽

Nlrp3 Gene ◽

Transplant Outcomes ◽

Functional Relevance ◽

The Impact

Abstract Abstract 3140 NLRP3 is an intracellular trigger of IL-1β production that plays important roles in the regulation of inflammation and apoptosis. A single nucleotide variation in the 3'-untranslated region of the NLRP3 gene, rs10754558 (+29940G>C), is linked to several immunological diseases. When we examined the impact of the NLRP3 genotype in a cohort consisting of 392 pairs of patients with hematologic malignancies and their unrelated HLA 12/12 matched bone marrow donors transplanted through the Japan Donor Marrow Program, the recipient NLRP3 GG genotype was found to be associated with a significantly worse 5-year overall survival (OS) rate (34% vs. 50%, P=0.006) (Fig. 1) and a trend toward a higher transplant-related mortality (TRM) rate (39% vs. 27%, P=0.09) than the recipient CC or CG genotype. The recipient GG genotype remained statistically significant in the multivariate analysis for OS (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.22 to 2.22; P=0.004) and TRM (HR, 2.28; 95% CI, 1.20 to 4.35; P=0.01). The donor NLRP3 genotype did not significantly influence the transplant outcomes. Next, we investigated the functional relevance of the NLRP3 +29940G>C variant. When leukocytes from healthy individuals were stimulated in vitro with NLRP3 ligand, the leukocytes with the NLRP3 GG genotype produced significantly more IL-1β than those with the NLRP3 CC or CG genotype (Fig. 2). These findings substantiate the functional relevance of the NLRP3 variant, and suggest that the higher IL-1β secretion in the peri-transplant period by recipients with the NLRP3 GG genotype likely accounts for their poor transplant outcomes. NLRP3 genotyping could therefore be useful in predicting prognoses and creating therapeutic strategies for improving the final outcomes of patients who undergo allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

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Aberrant Expression of DOCK4 Leads to Disruption of the F-Actin Skeleton and Altered Membrane Stability in MDS Erythroblasts and Mature Erythrocytes

Blood ◽

10.1182/blood.v120.21.924.924 ◽

2012 ◽

Vol 120 (21) ◽

pp. 924-924 ◽

Cited By ~ 1

Author(s):

Sriram Sundaravel ◽

Tushar D. Bhagat ◽

Carolina Schinke ◽

David Ebenezer ◽

Hui Liu ◽

...

Keyword(s):

Actin Polymerization ◽

Conflicts Of Interest ◽

Osmotic Fragility ◽

Membrane Stability ◽

Refractory Anemia ◽

P Value ◽

Down Regulation ◽

Acute Leukemias ◽

Aberrant Expression ◽

The Impact

Abstract Abstract 924 Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders that are commonly characterized by anemia due to ineffective hematopoiesis. Even though a third of patients with MDS may transform to acute leukemias, cytopenias drive morbidity for most patients. Anemia remains a major cause of morbidity from fatigue. Most of the morbidity experienced by such patients is due to low red blood counts and therefore studies on the molecular pathogenesis of dysplastic erythropoiesis leading to anemia are critically needed. We have identified DOCK4, an important cofactor for various GTPases located on the chromosome 7q segment as a novel silenced gene in MDS and show that it's down regulation leads to disruption of normal erythropoiesis. In an attempt to uncover genes aberrantly expressed in MDS, we initially performed an integrative genomic analysis of primary hematopoietic cells from MDS patients. These studies revealed that DOCK4 is significantly under-expressed and hypermethylated in MDS stem and progenitor cells. Immunohistochemcial analysis revealed significantly reduced levels of DOCK4 in MDS erythroblasts. We then evaluated DOCK4 expression in a large published cohort of MDS gene expression datasets (N=183) and found that DOCK4 expression was strikingly reduced in the subset of 55 MDS patients with refractory anemia (RA; P value = 0.006). The RA subset of patients only has isolated anemia as the clinical presentation and has no apparent abnormalities in white cells or platelets. This association strongly alludes to a role of DOCK4 down-regulation in the erythroid dysplasia and anemia seen in this disease. Inorder to elucidate the functional implications of aberrant DOCK4 expression during erythropoiesis we used a dynamic model of human erythropoiesis to determine normal expression pattern during terminal differentiation and the impact of silencing DOCK4 expression on healthy primary erythroblasts. These studies revealed that DOCK4 is highly expressed during late stages of normal erythropoiesis and knockdown of DOCK4 in primary erythroblasts disrupted the F-actin skeleton. We then examined F-actin skeletal disruption in CD34+ derived erythroblasts from MDS patients. In order to quantify the extent of actin filament disruption directly in patient derived erythroblasts we first developed an assay based on multispectral flow cytometry (ImageStream™). This assay not only allowed us to visualize individually F-actin-stained cells but also allowed us to determine the percentages of cells in a given sample that contained shorter fragmented F-actin. These experiments revealed that approximately 85% of the cells in MDS patients with -7q deletion and/or hypermethylated promoter region in the DOCK4 gene contained shorter disrupted actin compared to the healthy controls that showed only 10% of the cells with disrupted F-actin. The level of F-actin disruption in healthy samples treated with cytochalasin D, an inhibitor of actin polymerization was 90%. We then examined the membrane stability of -7q MDS erythrocytes by performing osmotic fragility assays and found that these patients possessed erythrocytes that were more fragile compared to healthy erythrocytes. Based on these results we conclude that DOCK4 is an important signaling intermediate that is instrumental in maintaining erythroblast membrane homeostasis and silencing of DOCK4 in MDS contributes to anemia. Disclosures: No relevant conflicts of interest to declare.

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Hoxblinc Is Aberrantly Expressed in Acute Myeloid Leukemia and Functions As a Potent Oncogenic Long Non-Coding RNA in Leukemogenesis

Blood ◽

10.1182/blood-2018-99-112232 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 886-886

Author(s):

Ganqian Zhu ◽

Huacheng Luo ◽

Shi Chen ◽

Qian Lai ◽

Ying Guo ◽

...

Keyword(s):

Mononuclear Cells ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Three Dimensional ◽

Donor Cell ◽

Lower Percentage ◽

Leukemic Transformation ◽

Aberrant Expression ◽

The Impact

Abstract Aberrant expression of long non-coding RNAs (lncRNAs) might contribute to the development and progression of leukemia. However, functional studies on the actual role of lncRNAs during the development of leukemia remain scarce, and very few lncRNAs have been shown to be involved in leukemogenesis. HoxBlinc is an anterior HoxB gene-associated intergenic lncRNA. It is a cis-acting lncRNA and functions as an epigenetic regulator to coordinate anterior HoxB gene expression. Giving the dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation, HoxBlinc might be an oncogenic lncRNA of leukemia. To determine whether HOXBLINC lncRNA is aberrantly expressed in human AML samples, we performed RT-qPCR on bone marrow mononuclear cells (BMMNCs) from a cohort of 73 AML patients. A dramatic up-regulation of HOXBLINC was observed in over 60% of the patients. When TCGA-AML datasets of a cohort of 179 AML patients were analyzed for their HOXBLINC expression, a significant portion of these AML patients had high levels of HOXBLINC expression. Interestingly, AML patients with high HOXBLINC expression (the top thirty percentile of patients) had a significantly shortened survival as compared to patients with low HOXBLINC expression (the bottom thirty percentile). To investigate the impact of HoxBlinc overexpression on normal hematopoiesis and the pathogenesis of hematological malignancies in vivo, we generated a HoxBlinc transgenic(Tg) mouse model. Within 1 year of age, 67% of the HoxBlincTg mice (10 of 15) died or were sacrificed because of a moribund condition due to AML. We then assessed whether overexpression of HoxBlinc affects the pools of HSC/HPCs by flow cytometric analysis on the BM cells of young WT and HoxBlincTg mice (8-10 weeks of age). HoxBlincTg BM had a dramatically greater number of LT-HSC, ST-HSC, MPP cells, and a significantly higher percentage of GMP, but a lower percentage of MEP/CMP cell populations as compared to WT group. To determine the effect of HoxBlinc overexpression on the function of HSC/HPCs, we performed paired-daughter cell assay, replating assay and liquid culture on sorted LT-HSC, LSK or LK cells from young WT and HoxBlincTg mice, the results indicate that transgenic expression of HoxBlinc enhances HSC self-renewal and impairs HSC/HPC differentiation. To assess whether HoxBlinc overexpression-mediated changes in HSC/HPC function are cell-autonomous, we performed competitive transplantation assays to examine the repopulating capacity of HoxBlincTg BM cells. When the donor cell chimerism was analyzed kinetically in the PB of recipient mice, the CD45.2 cell population remained ~50% in mice receiving WT BM cells, whereas the CD45.2 chimerism in the recipients transplanted with HoxBlincTg BM cells steadily increased. Interestingly, mice receiving HoxBlincTg BM cells developed AML at 2-6 months after transplantation. Previous data reported that HoxBlinc can recruit the Setd1a/Mll1 histone H3K4 methyltransferase complex to mediate formation of the active topologically associated domain (TAD) in the anterior HoxB locus for transcription of the anterior HoxB genes. In this study, LSK or LK cells sorted from young WT and HoxBlincTg mice were analyzed by RNA-seq, ATAC-seq, H3K4me3 CHIP-seq and 4C analysis. Mechanistically, HoxBlinc overexpression alters HoxB locus chromatin three-dimensional organization to enhance enhancer/promoter chromatin accessibility and coordinate the expression of not only HoxB1-5 but also HoxA9, Runx1, Meis1 and so on, which are critical genes for HSC regulation and/or leukemogenesis. Our study provides novel insights into the HSC regulation by lncRNAs and identifies HOXBLINC, which coordinates to maintain an oncogenic transcription program for leukemic transformation, as a potent oncogenic lncRNA in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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Age of Blood Does Not Affect Quality of Life and Hemoglobin: N– of −1 Trials in Transfusion-Dependent Patients,

Blood ◽

10.1182/blood.v118.21.3369.3369 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3369-3369 ◽

Cited By ~ 2

Author(s):

Maayan Seitelbach ◽

Ian H. Chin-Yee ◽

Jeff Kinney ◽

Justin Chia ◽

Katrina Ormond ◽

...

Keyword(s):

Quality Of Life ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Group Analysis ◽

Adverse Outcomes ◽

Fresh Blood ◽

Storage Lesion ◽

The Impact

Abstract Abstract 3369 Introduction Red blood cell concentrates (RBCs) are stored for up to 42 days. Prolonged storage of RBCs results in “the storage lesion” which has been associated with adverse outcomes in patients with critical illness, sepsis, and post-cardiac surgery. No studies have assessed the impact of the storage lesion on the quality of life (QoL) of patients who require chronic transfusion support. This study assessed the effects of fresh versus standard issue blood on QoL and follow-up hemoglobin (Hb) in transfusion-dependent patients. Methods Transfusion-dependent patients age 18 years or older, were invited to participate in an N-of-1 trial. Transfusion dependence required at least one unit of RBCs every four weeks. Each participant was randomly assigned four fresh (less than seven days of storage) and four standard issue (seven to 42 days of storage) blood transfusions. Investigators, study participants, treating physicians, and nurses were blinded to product allocation. A questionnaire containing a visual analog scale (VAS) and the Functional Assessment of Cancer Therapy-Anemia questionnaire (FACT-An) was completed prior to and 24 hours after each transfusion. Changes were calculated as post-transfusion scores minus pre-transfusion scores. Hemoglobin levels were measured at each subsequent transfusion. Within-subject (each participant) between-treatment comparisons of QoL scores and hemoglobin levels were assessed by unpaired t-tests among participants who completed at least six transfusions. Between-group changes in QoL scores for all participants were assessed by paired t-tests and a mixed model approach. Results Twenty patients were enrolled (mean age 66.8 years, 12 females). Underlying diagnoses included myelodysplastic syndromes (12), β-thalassemia (3), myeloproliferative neoplasms (3), Diamond-Blackfan anemia (1), and chronic anemia of undetermined etiology (1). Mean ages of fresh and standard blood were 4.0 days and 23.2 days, respectively. Nine participants completed at least six transfusions. Among remaining participants, nine were non-compliant, and two died. All data were analysed in the between-group comparisons. There were no statistically significant differences in the effect of standard blood and fresh blood on the eight QoL parameters assessed in all analyses. This was seen in the within subject between-group analysis as well as the between-group analysis. Similarly, there were no statistically significant differences in the effect of standard blood and fresh blood on follow-up Hb levels. Conclusions No significant differences in QoL parameters or follow-up Hb levels were observed in patients receiving fresh versus standard issue blood. These results suggest that local blood transfusion laboratory practices do not need to be altered for transfusion-dependent patient populations. To our knowledge, this is the first study to assess the QoL effects of age of blood on transfusion-dependent patients using N-of-1 trials. While larger studies are needed to confirm these findings, our results demonstrate that N-of-1 studies are feasible and informative in the management of individual patients. The randomized controlled multiple crossover design of N-of-1 studies may be also useful in addressing questions in transfusion medicine regarding differences in the quality of blood products. Disclosures: No relevant conflicts of interest to declare.

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