scholarly journals Elevated Red Cell Reduced Glutathione Is a Novel Biomarker of Diamond-Blackfan Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1342-1342
Author(s):  
Taiju Utsugisawa ◽  
Toshitaka Uchiyama ◽  
Hiromi Ogura ◽  
Takako Aoki ◽  
Isao Hamaguchi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Reduced Glutathione ◽  
Recent Observation ◽  
Positive Family History ◽  
Gene Mutations ◽  
Support Vector ◽  
Red Cell ◽  
Erythroid Cells ◽  
Linear Discriminant ◽  
Diamond Blackfan Anemia

Abstract Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by congenital anomalies and predisposition to cancer. Recent observation disclosed that heterogeneous mutations in ribosomal protein (RP) genes are present in approximately 50% of patients, suggesting that diagnosis should be made by clinical phenotypes such as age, hematological findings or positive family history. Although elevated activity of red cell adenosine deaminase (eADA) has been utilized as a useful biomarker for differential diagnosis of DBA, approximately 20% of DBA patients are eADA-negative. Recent observations suggested that ribosomal haploinsufficiency increases oxidative stress, leading to p53 gene activation and premature death of erythroid cells. We hypothesized that reduced glutathione (GSH), an essential antioxidant of erythroid cells, might be upregulated in red cells of DBA subjects. In order to test this hypothesis, we examined red cell GSH as well as eADA of 22 patients in 18 DBA families, in whom we had identified gene mutations in RPS19, RPL5, RPL11, RPS10, RPS17 or RPS35a. All except one DBA patients showed elevated GSH (>88.6 mg/dl RBC, M+SD), whereas 17 out of 22 patients exhibited elevated eADA (>2.31 IU/g Hb, M+3SD). We also examined 14 unaffected members of the DBA families, with 1 out of 14 subjects showing elevated GSH and none showing elevated eADA. We performed linear discriminant analysis between DBA and non-DBA subjects with both eADA and GSH using the Support Vector Machine (SVM) from 36 subjects, and successfully obtained a formula to discriminate DBA from unaffected subjects: 0.937*eADA+0.0702*GSH-7.9044 >0. By using this formula, all DBA examined can be diagnosed and unaffected family members can be excluded. Since approximately 50% of clinically diagnosed DBA cases have no causative RP gene mutations, the combined assessment of eADA and GSH might be quite useful for biochemical diagnosis of DBA. Disclosures No relevant conflicts of interest to declare.

5q- Syndrome In a Child: Is This Acquired Diamond Blackfan Anemia (DBA)?

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4430-4430
Author(s):  
Adrianna Vlachos ◽  
Jason Farrar ◽  
Eva Atsidaftos ◽  
Ellen Muir ◽  
Thomas C. Markello ◽  
...  
Keyword(s):  
High Resolution ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Comparative Genomic ◽  
Red Cell ◽  
Significant Family History ◽  
Diamond Blackfan Anemia ◽  
Red Cell Aplasia ◽  
Large Deletions ◽  
Patient Database

Abstract Abstract 4430 Background: Diamond Blackfan anemia is a rare heritable red cell aplasia which usually presents in infancy but can also be diagnosed in childhood and even adulthood. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haplo-insufficiency have been reported in about 54% of the patients. The 5q- syndrome is an acquired myelodysplastic syndrome (MDS) characterized by a similar erythroid failure. Another RP gene included in the 5q deleted region, RPS14, has been identified as a causal gene in 5q- MDS but has not been reported in DBA. Purpose: Array Comparative Genomic Hybridization has been used to identify large deletions in patients with DBA. This report demonstrates the use of Single Nucleotide Polymorphism (SNP) genotyping array hybridization to identify a patient, previously thought to have DBA, as having a 5q- deletion consistent with 5q- syndrome. Method: Seventy-five patient samples from the Diamond Blackfan Anemia Registry of North America, a patient database of now 608 patients designed to better understand the biology and epidemiology of DBA, underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands from this cohort who did not have a mutation detected in either the resequencing project and/or the targeted sequencing efforts lead by Gazda and colleagues. Result: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one patient with presumed DBA. The patient presented at 5 years 10 months of age with anemia noted on a routine blood count. The hemoglobin was 8.4 grams/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The eADA was normal. The bone marrow showed decreased cellularity and megaloblastic changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. The cytogenetics performed at diagnosis in 1991 were reported as normal. The patient had no significant family history of anemia and was found to have no congenital physical anomalies. A diagnosis of non-classical DBA was presumed and the patient failed a trial of corticosteroids. At present the patient has marrow red cell aplasia and is on a chronic transfusion schedule. SNP array genotyping analysis identified mosaicism in two discrete regions covering ~17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33 though it excludes the 5q31 CDR associated with AML and more aggressive MDS as well as miR146a, a factor recently postulated to play a role in 5q- MDS. SNP array genotyping from purified peripheral blood populations indicated that lymphocytes were greater than 95% normal, while the myeloid cells were greater than 95% 5q-. CD34+ cells obtained from this patient showed a marked decrease in both myeloid and erythroid colony formation when compared with normal cells. Patient fibroblasts were normal and neither of the parents have any 5q anomalies by SNP array genotyping. Although the deletion was not identified in 1991 at the time of the diagnosis, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was able to be detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. Conclusion: Patients with non-classical DBA may have unique acquired 5q deletions with RPS14 haploinsufficiency. A search for other acquired somatic mutations or deletions in patients with DBA, in particular non-classical cases, is underway. SNP array genotyping is an essential diagnostic tool in this search. Disclosures: No relevant conflicts of interest to declare.

COL4A1 is a Novel Causative Gene Responsible for Congenital Hemolytic Anemia, Representing Characteristic Clinical Course in Infants

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 934-934
Author(s):  
Hiromi Ogura ◽  
Shouichi Ohga ◽  
Takako Aoki ◽  
Taiju Utsugisawa ◽  
Hidehiro Takahashi ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Small Vessel Disease ◽  
Gene Mutations ◽  
Basement Membranes ◽  
Red Cell ◽  
Missense Mutations ◽  
Causative Gene ◽  
Congenital Hemolytic Anemia

Abstract We have been working on the differential diagnosis of congenital hemolytic anemia, but even with extensive analysis of hemoglobin, red cell membrane and enzymes, approximately 40% of patients remained to be diagnosed. In this study, we analyzed 17 undiagnosed hemolytic anemia subjects under the age of 1 by whole-exome sequencing, and identified COL4A1 gene mutations in 5 cases (29.4%). All patients were de novo cases without family histories and exhibited moderate to severe neonatal hemolytic anemia: Hgb, 5.2-9.3 g/dl; MCV, 90.0-126.9; MCHC, 29.9-32.7; and reticulocyte count, 9.2-33.0%. Either schizocytes or poikilocytes were observed in peripheral blood smears of 3 cases, suggesting that the microangiopathy was attributable to hemolysis. Previous reports showed that mutation of COL4A1 accounts for brain small-vessel disease characterized by stroke and eye abnormalities and the most characteristic complications of the present cases were congenital anomaly in the central nervous system, such as porencephaly, schizencephaly, congenital hydrocephalus, cataracts or paraventricular calcification, as reported previously. Hemolytic anemia became less severe within 2 months after birth, and all cases no longer required red cell transfusion after Day 50. COL4A1 encodes subtype 1 of type IV collagen, which is most abundantly expressed in basement membranes, including the vasculature. The COL4A1 gene mutations identified in the cases were all novel missense mutations except one, located in exons 26, 27, 37, 38 and 51. Although the pathophysiological significance of the mutations remains unclear, COL4A1 is the first identified causative gene responsible for congenital hemolytic anemia without intrinsic defects of red blood cells, and mutation of COL4A1 is the most prevalent cause of neonatal hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 671-671
Author(s):  
Marc Gastou ◽  
Sarah Rio ◽  
Mickael Dussiot ◽  
Narjesse Karboul ◽  
Thierry Leblanc ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Expression Level ◽  
Hsp70 Expression ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
P53 Activation

Abstract Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

Impact of SERPINC1, PROC and PROS1 Mutations on the Thrombotic Phenotype in Children with Venous Thromboembolism: An Observational Multicenter Cohort Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 583-583
Author(s):  
Verena Limperger ◽  
Gili Kenet ◽  
Christine Heller ◽  
Karin Kurnik ◽  
Ralf Knoefler ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Family History ◽  
Conflicts Of Interest ◽  
Positive Family History ◽  
Purpura Fulminans ◽  
Gene Mutations ◽  
High Risk Population ◽  
Cerebral Veins ◽  
Multicenter Cohort Study ◽  
Significant Difference

Abstract Background: Venous thromboembolism [TE] is a multifactorial disease and antithrombin [ATD] -, protein C [PCD] - or protein S [PSD] -deficiency constitutes a major risk factor. The objective of the present study was to i) evaluate the prevalence of ATD, PCD and PSD in a white Israeli-German cohort of children with TE, ii) the underlying gene mutations, and iii) the clinical presentation of ATD, PCD and PCS on symptomatic TE in children. Methods: In 367 unselected children (0.1-18 years) with TE recruited between July 1996 and December 2013 from Germany & Israel, a comprehensive thrombophilia screening was performed. Along with standardized plasma-based coagulation assays and the use of age-dependent reference values ATD, PCD and PSD were confirmed by family studies and/or molecular diagnosis [gene sequencing & multiplex ligation-dependent probe amplification]. Apart from descriptive analysis non-parametric statistics was performed. To compare the rates of deficiency phenotypes, locations of TE, spontaneous versus provoked TE, presence or absence of a positive family history of TE, Chi-square or Fisher’s exact test was applied. Results: 6.6% of children were ATD, 6.8% PCD including purpura fulminans (1.4%) and 8.2% patients carried PSD. Mean age at first TE was 13 years (range 0.1 to 18) with no statistically significant difference found between deficiency phenotypes (p=0.32). 38 children were male. 72 of 76 children (95%) showed type 1 deficiency, whereas in 4 cases [ATD] a type 2 deficiency was found (p=0.004). Underlying gene mutations were in the majority of cases missense mutations (SERPINC1: 75%; PROC: 93%; PROS1: 72%), with 24% presence of the Herleen polymorphism (CM951058) in children with PSD. Homozygous genotypes were found in 4 cases [ATD], 6 cases [PCD] and in one PSD carrier [p=0.07]. ATD co-occurred with the factor 5 mutation [F5] at rs6025 in one and the factor 2 susceptibility variant [F2] at rs1799963 in two children. PSD co-occurred with the F5 or the homozygous F2 in one case each. Apart from purpura fulminans which was seen only in neonates with homozygous PCD, thrombotic locations were similarly distributed (p=0.11): multiple veins (n=7), cerebral veins/stroke of venous origin (n=18), deep veins [DVT; n=38], DVT & pulmonary embolism (n=13). The rates of provoked TE were 57% [ATD], 48% [PCD] and 60% in PCD (p=0.06). A positive family history was present in 43% [ATD], 40% [PCD] and 57% [PSD; p=0.41]. After withdrawal of anticoagulation, - performed on an individual basis according to CHEST guidelines (updated according to year of publication) -, recurrence rates were 38% [ATD], 24% [PCD] and 6.6% in carriers of PSD (p=0.017). Conclusion: Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high risk population Disclosures No relevant conflicts of interest to declare.

The First Trimester Human Placenta Is An Embryonic Hematopoietic Organ with An Unexpected Function in Primitive Erythroid Maturation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1508-1508
Author(s):  
Ben Van Handel ◽  
Sacha Prashad ◽  
Andy Huang ◽  
Nargess Hassanzedeh-Kiabi ◽  
Mattias Magnusson ◽  
...  
Keyword(s):  
Human Placenta ◽  
De Novo ◽  
Conflicts Of Interest ◽  
First Trimester ◽  
Red Cell ◽  
Erythroid Cells ◽  
Cell Nuclei ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Placental Macrophages

Abstract Abstract 1508 Poster Board I-531 Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-18-SCI-18
Author(s):  
Janis L. Abkowitz ◽  
Raymond T Doty ◽  
Zhantao Yang ◽  
Xiaowei Yan ◽  
Christopher Lausted ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Macrocytic Anemia ◽  
Protein Translation ◽  
Cell Surface Expression ◽  
Red Cell ◽  
Erythroid Cells ◽  
P53 Expression ◽  
Diamond Blackfan Anemia ◽  
Heme Synthesis ◽  
Globin Synthesis

Abstract Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1214-1214
Author(s):  
Husam Qanash ◽  
Kaari Linask ◽  
Jeanette Beers ◽  
Keyvan Keyvanfar ◽  
Sara Young-Baird ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Protein Translation ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Diamond Blackfan Anemia ◽  
Heme Synthesis ◽  
Free Heme ◽  
Erythroid Maturation

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

Application of Machine Learning Approaches for the Design and Study of Anticancer Drugs

2019 ◽  
Vol 20 (5) ◽  
pp. 488-500 ◽  
Author(s):  
Yan Hu ◽  
Yi Lu ◽  
Shuo Wang ◽  
Mengying Zhang ◽  
Xiaosheng Qu ◽  
...  
Keyword(s):  
Machine Learning ◽  
Drug Design ◽  
Anticancer Drugs ◽  
Nearest Neighbor ◽  
Cost Effective ◽  
Support Vector ◽  
Learning Approaches ◽  
K Nearest Neighbor ◽  
Activity Prediction ◽  
Linear Discriminant

Background: Globally the number of cancer patients and deaths are continuing to increase yearly, and cancer has, therefore, become one of the world&#039;s highest causes of morbidity and mortality. In recent years, the study of anticancer drugs has become one of the most popular medical topics. </P><P> Objective: In this review, in order to study the application of machine learning in predicting anticancer drugs activity, some machine learning approaches such as Linear Discriminant Analysis (LDA), Principal components analysis (PCA), Support Vector Machine (SVM), Random forest (RF), k-Nearest Neighbor (kNN), and Naïve Bayes (NB) were selected, and the examples of their applications in anticancer drugs design are listed. </P><P> Results: Machine learning contributes a lot to anticancer drugs design and helps researchers by saving time and is cost effective. However, it can only be an assisting tool for drug design. </P><P> Conclusion: This paper introduces the application of machine learning approaches in anticancer drug design. Many examples of success in identification and prediction in the area of anticancer drugs activity prediction are discussed, and the anticancer drugs research is still in active progress. Moreover, the merits of some web servers related to anticancer drugs are mentioned.

Colorectal Cancer Classification and Survival Analysis Based on an Integrated RNA and DNA molecular signature

2020 ◽  
Vol 15 ◽  
Author(s):  
Mohanad Mohammed ◽  
Henry Mwambi ◽  
Bernard Omolo
Keyword(s):  
Colorectal Cancer ◽  
Survival Analysis ◽  
Discriminant Analysis ◽  
Linear Discriminant Analysis ◽  
Negative Binomial ◽  
Sub Saharan Africa ◽  
Support Vector ◽  
Mutation Status ◽  
Linear Discriminant ◽  
Rnaseq Data

Background: Colorectal cancer (CRC) is the third most common cancer among women and men in the USA, and recent studies have shown an increasing incidence in less developed regions, including Sub-Saharan Africa (SSA). We developed a hybrid (DNA mutation and RNA expression) signature and assessed its predictive properties for the mutation status and survival of CRC patients. Methods: Publicly-available microarray and RNASeq data from 54 matched formalin-fixed paraffin-embedded (FFPE) samples from the Affymetrix GeneChip and RNASeq platforms, were used to obtain differentially expressed genes between mutant and wild-type samples. We applied the support-vector machines, artificial neural networks, random forests, k-nearest neighbor, naïve Bayes, negative binomial linear discriminant analysis, and the Poisson linear discriminant analysis algorithms for classification. Cox proportional hazards model was used for survival analysis. Results: Compared to the genelist from each of the individual platforms, the hybrid genelist had the highest accuracy, sensitivity, specificity, and AUC for mutation status, across all the classifiers and is prognostic for survival in patients with CRC. NBLDA method was the best performer on the RNASeq data while the SVM method was the most suitable classifier for CRC across the two data types. Nine genes were found to be predictive of survival. Conclusion: This signature could be useful in clinical practice, especially for colorectal cancer diagnosis and therapy. Future studies should determine the effectiveness of integration in cancer survival analysis and the application on unbalanced data, where the classes are of different sizes, as well as on data with multiple classes.

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