New Insights into Mechanisms of Erythropoietin Receptor Mutations in Primary Familial and Congenital Polycythemia

Abstract Primary Familial and Congenital Polycythemia (PFCP) is a non-malignant pathology of the erythroid lineage, characterized by an isolated increase of the red cell mass without evolution into myelofibrosis or acutisation. Around twenty constitutive non-sense and missense mutations located in the exon 8 of the erythropoietin receptor (EPOR) gene have been described so far. They all lead to the truncation of the C-terminal part of the protein and the loss of several cytoplasmic tyrosines. The erythropoietin (EPO) hypersensitivity of the PFCP erythroid progenitors is usually explained by the disappearance of these negative signaling regulation and internalization domains (Figure 1a). Nonetheless, relatively few functional studies have been carried out. We therefore investigated the mechanism of EPOR mutations in PFCP. We identified and extensively studied a new constitutive heterozygous frameshift EPOR mutation, p.Gln434Profs*11, which generates a new 11 amino acid (AA) C-terminal tail and a STOP codon at position 444, leading to the truncation of 63 AA of the wild-type receptor (Figure 1c). The primary progenitor cells displayed a major hypersensitivity to EPO, similar to Polycythemia Vera (PV) patients, as well as a spontaneous and persistent JAK2 and STAT5 phosphorylation, compared to the control cells. To study the mechanism of this new EPOR mutant, Ba/F3 cells were transduced with different retroviruses encoding either the HA-tagged wild-type EPOR (EPOR WT)or a truncated receptor at position 444, p.Gln444* (EPOR STOP) or the frameshift EPOR p.Gln434Profs*11mutation (EPOR FS), identical to the patient's mutation (Figure 2). As observed in primary cells, EPOR FS conferred a spontaneous STAT5 phosphorylation and a 4- to 5-fold EPO hypersensitivity to Ba/F3 cells (IC50 of 0.003 U/mL vs 0.01 U/mL) compared to both EPOR WT and EPOR STOP. As expected, the loss of negative regulatory domains in the C-terminal part of the receptor induced a persistent STAT5 activation in EPOR FS and EPOR STOP Ba/F3 cells. Moreover, EPOR FS was more stable (half-life of 120 minutes vs 60 minutes) and displayed a higher level of localization at the cell surface (more than 2-fold), compared to EPOR WT and EPOR STOP. However, no modification of the EPOR FS internalization pattern was observed during 125I-EPO labeling experiments and cytometry analysis. Furthermore, a dileucine motif, known to be a potential clathrin-dependent endocytosis site, is lost in the new C-terminal tail of EPOR FS mutant, yet its abrogation in EPOR WT and EPOR STOP did not modify the phenotype of Ba/F3 cells. Therefore, unlike previous reports, the major EPO hypersensitivity induced by EPOR p.Gln434Profs*11 cannot be explained by the receptor truncation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling. We wondered if this model could be extended to other EPOR mutations already described in PFCP (Figures 1a-b and 2). We therefore measured the impact on Ba/F3 cells proliferation of the frameshift EPOR p.Pro438Metfs*6 and its non-sense mutant counterpart, p.Pro443*, which retains the tyrosine at position 426, a binding site for the negative signaling regulators SOCS3 and CIS. EPO-hypersensitivity (4- to 5-fold) was only induced by EPOR p.Pro438Metfs*6, suggesting a common mechanism for the frameshift EPOR mutations. Interestingly, two proximal non-sense mutations, EPOR p.Glu399* and p.Glu425*, lacking 7 of the 8 cytoplasmic tyrosines that compose EPOR negative regulatory and internalization domains, were also able to confer a high EPO hypersensitivity to Ba/F3 cells. To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We highlighted that this pathology is much more complex than expected, since different mechanisms are involved in the EPO hypersensitivity phenotype, according to the type of EPOR mutation. Indeed, extensive truncations are sufficient by themselves to confer the EPO hypersensitivity phenotype due to the loss of all negative regulatory and internalization domains, whereas more distal truncations induced by frameshift mutants confer EPO hypersensitivity because of the appearance of a new C-terminal tail. The latter, by increasing EPOR stability at the cell surface, may cause pre-activation of both receptor and JAK2, constitutive signaling and hypersensitivity to EPO close to that of JAK2V617F-positive PVs. Disclosures No relevant conflicts of interest to declare.

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Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.

Blood ◽

10.1182/blood.v108.11.1297.1297 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1297-1297

Author(s):

Mariluz P. Mojica-Henshaw ◽

Caroline Laverdiere ◽

Jaroslav F. Prchal ◽

Josef T. Prchal

Keyword(s):

De Novo ◽

Stop Codon ◽

Cell Mass ◽

Hemoglobin Concentration ◽

Erythropoietin Receptor ◽

De Novo Mutations ◽

Erythroid Progenitors ◽

Increased Risk ◽

Genetic Mosaicism

Abstract Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband’s parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.

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Loss of the Wild-Type Allele Contributes to Myeloid Expansion and Disease Aggressiveness in FLT3/ITD Knock-in Mice

Blood ◽

10.1182/blood.v118.21.866.866 ◽

2011 ◽

Vol 118 (21) ◽

pp. 866-866

Author(s):

Li Li ◽

Emily Bailey ◽

Sarah M Greenblatt ◽

David Huso ◽

Donald Small

Keyword(s):

Median Survival ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Point Mutations ◽

Kinase Domain ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Lower Ratio ◽

The Impact

Abstract Abstract 866 Activating mutations of FLT3, either in the form of internal tandem duplication (ITD) mutations in the juxtamembrane domain or point mutations in the kinase domain, are one of the most frequent mutations in acute myeloid leukemia (AML). AML patients with FLT3/ITD mutations have poor prognosis. Loss of the wild-type FLT3 allele is associated with even worse prognosis when compared to those FLT3/ITD AML patients with the wild-type FLT3 allele still present. Also, FLT3/ITD patients with a high mutant-to-wild-type ratio have a significantly worse outcome than FLT3/ITD patients with a lower ratio. We have previously reported that heterozygous FLT3wt/ITD “knock-in” mice develop a slowly fatal MPN. In order to study the roles wild-type FLT3 play in the development of leukemia associated with FLT3/ITD mutations, we crossed FLT3wt/ITD mice with themselves or with FLT3 “knockout” (FLT3−/−) mice to obtain hemizygous (FLT3−/ITD) or homozygous (FLT3ITD/ITD) FLT3/ITD mice. Investigating phenotypic differences among them reveals the impact of wild-type FLT3 on the development of MPN resulting from FLT3/ITD mutations, and by extension, the effect on acute leukemia. FLT3−/ITD mice, with the loss of the wild-type allele, displayed a more severe MPN, as evidenced by even larger spleen, higher white blood counts and shorter survival, compared to FLT3wt/ITD mice. FLT3ITD/ITD mice had an even severe MPN compared to the FLT3−/ITD and FLT3wt/ITD mice. Fully transformed leukemia developed in some of the FLT3ITD/ITD (7%, 9/129), but not FLT3wt/ITD or FLT3−/ITD mice, with latency ranging from 139 to 304 days. Compared to FLT3wt/ITD mice, FLT3−/ITD and FLT3ITD/ITD mice displayed a further increase in the fraction of primitive hematopoietic cells, with notable increases in ST-HSCs and MPPs. Phosphorylation of STAT5, one of the key downstream targets for constitutively activated FLT3, was increased in FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD mice compared to the wild-type control. FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD BM also showed increased PU.1 expression and decreased GATA-1 expression, resulting in the subsequent expansion of granulocytic/monocytic/lymphocytic progenitors and a decrease in megakaryocytic/erythrocytic progenitors. It appears that the extent of myeloproliferation in FLT3/ITD mice correlates with loss of the wild-type allele (FLT3wt/ITD vs. FLT3−/ITD) and with the dose of mutant allele (FLT3−/ITD vs. FLT3ITD/ITD). In order to further explore the potential moderating effect of wild-type FLT3 expression on FLT3/ITD-associated MPN, we transduced wild-type FLT3 (wtFLT3, with the lentiviral vector co-expressing GFP) into lineage-depleted FLT3−/ITD CD45.2 BM cells and injected them into lethally irradiated CD45.1 recipients. When injected with sorted (GFP+) BM, vector alone-transduced GFP+FLT3−/ITD BM recipients died of MPN, with a median survival of 62 days. 100% of the recipients in the other three groups, i.e., those injected with vector alone-transduced GFP+ wild-type BM, wtFLT3-transduced GFP+ wild-type BM or wtFLT3-transduced GFP+ FLT3−/ITD BM, remained viable even after the point in time at which all of the recipients in the vector alone-transduced GFP+FLT3−/ITD group died. Similarly, recipients transplanted with unselected (including GFP+ and GFP− populations) vector alone-transduced FLT3−/ITD BM also died early, with a median survival of 73 days and overt signs of MPN. The percentages of GFP+ and GFP− cells in the BM of the dying recipients were comparable to those shortly after transplantation, indicative of the similar expansion ability of the GFP+ and GFP− populations in the BM. In contrast to the wtFLT3-transduced GFP+FLT3−/ITD BM recipients, which have a very prolonged survival, recipients injected with unselected wtFLT3-transduced FLT3−/ITD BM died of MPN, with a median survival of 91 days. Interestingly, 99% of the BM cells in the BM of the dying recipients were GFP−, demonstrating a proliferative/survival advantage for the FLT3−/ITD cells that had not been successfully transduced with wild-type FLT3. These results suggest that the presence of wild-type FLT3 delays and moderates the development of MPN caused by FLT3/ITD mutations. These results suggest that loss of the wild-type allele contributes to the development of a more severe phenotype. Thus, the wild-type FLT3 allele seemingly functions as a “tumor suppressor” in leukemia harboring FLT3/ITD mutations. Disclosures: No relevant conflicts of interest to declare.

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Cloning of oestrogen receptor beta from Old and New World primates: identification of splice variants and functional analysis

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0320703 ◽

2004 ◽

Vol 32 (3) ◽

pp. 703-718 ◽

Cited By ~ 17

Author(s):

JE Sierens ◽

GA Scobie ◽

J Wilson ◽

PT Saunders

Keyword(s):

Splice Variant ◽

Oestrogen Receptor ◽

New World ◽

Stop Codon ◽

Full Length ◽

Peptide Sequence ◽

Wild Type ◽

New World Primates ◽

World Primate ◽

The Impact

Oestrogens have a major impact on reproductive function in both males and females. Two oestrogen receptor genes known as ERalpha (ESR1NR3A1) and ERbeta (ESR2NR3A2) have been cloned. Splice variant isoforms of the ERbeta gene have been identified in human, bovine and rodents and it has been suggested that the existence of these forms can influence oestrogen responsiveness. In the human, splicing of an alternative eighth exon results in the formation of a C-terminal variant called hERbetacx, or hERbeta2, but this isoform has not been identified in other species. The aim of the present study was to clone ERbeta cDNAs from primates so as to determine how closely they resembled the ERbeta isoforms found in the human. The two species studied were the stump-tailed macaque (Macaca arctoides), an Old World primate, and the common marmoset (Callithrix jacchus jacchus), a New World primate. Full length ERbeta (wild type, ERbeta1) cDNAs were cloned from macaque and marmoset; they encoded proteins of similar size to those found in human (59 and 54 kDa, long and short forms respectively) and shared significant sequence homology (97.5% in macaque and 93.8% in marmoset) with the human peptide sequence. Full length cDNAs homologous to the hERbeta2 variant were identified in both primates. Marmoset ERbeta2 was slightly shorter than that of human ERbeta2 (54 kDa compared with 55 kDa) and did not contain the peptide sequence used to raise an anti-hERbeta2 antibody. All the macaque ERbeta2 cDNAs contained 56 bp of intronic sequence which included an in-frame stop codon resulting in translation of a truncated protein ( approximately 35 kDa). In all three species, truncated, alternatively spliced mRNAs lacking exon 5 were isolated on multiple occasions from all tissue extracts. In transient transfection assays, ERbeta2-containing constructs were unable to induce transcription of an oestrogen response element (ERE) reporter plasmid in the presence of oestradiol. ERbeta1 from human, macaque and marmoset exhibited minor differences in their ability to induce transcription of the ERE reporter when incubated with different ligands (oestradiol, PPT, DPN, 5-alpha-androstane-3-beta, 17beta-diol (3betaAdiol), genistein) and this may be due to amino acid substitutions within their ligand binding domains. In conclusion, we have identified and cloned wild type ERbeta (ERbeta1) from macaque and marmoset and demonstrated that splice variant mRNAs homologous to hERbeta2 are formed in both species. The marmoset monkey, therefore, provides a suitable animal model in which to investigate the impact of ERbeta variant expression on tissue responsiveness to oestrogens.

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Alpha-Catenin Is Dispensable for Normal Hematopoietic Stem Cell Function.

Blood ◽

10.1182/blood.v114.22.1438.1438 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1438-1438

Author(s):

Natallia Mikhalkevich ◽

Michael W. Becker

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Conflicts Of Interest ◽

Fusion Product ◽

Wild Type ◽

Hematopoietic Stem ◽

Myeloid Neoplasms ◽

Increased Risk ◽

Significant Difference ◽

The Impact

Abstract Abstract 1438 Poster Board I-461 We previously demonstrated the loss of expression of alpha-E-Catenin, the product of the CTNNA1 gene, in primary leukemic stem cells isolated from patients with advanced Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) associated with loss of all or part of the long arm of chromosome 5. To formally assess the impact of loss of Ctnna1 expression on hematopoiesis, we employed a murine model for the hematopoietic specific conditional loss of Ctnna1 expression. We demonstrate that Ctnna1 deficiency is associated with normal hematopoietic maturation and proliferation as assessed by peripheral blood examination and methycellulose colony assays. We assessed stem cell and early progenitor frequencies using both flow cytometry and functional assays. Ctnna1 deficiency was associated with equivalent frequencies of Sca1+C-Kit+CD135-Lineage- HSCs in both experimental animals and controls. Short term HSC and MPP frequencies were likewise unaltered. We assessed HSC function using transplantation studies. In competitive repopulation experiments, HSCs deficient for Ctnna1 maintained stable engraftment of recipient mice for up to 1 year. Limiting dilution analyses detected no significant difference in HSC frequency between wild type and Ctnna1 deficient mice. We examined the potential role of Ctnna1 deficient hematopoietic stem cells in two murine models for myeloid neoplasms 1.) exposure to mutagen ENU and 2.) a model for murine AML driven by the HoxA9-Nup98 fusion product. Following exposure of HSCs to ENU, loss of Ctnna1 was not associated with an increased risk of development of a myeloid neoplasm. Expression of the HoxA9-Nup98 fusion product by retroviral infection of Ctnna1 deficient and wild type Sca1+C-Kit+Lineage- cells resulted in no difference in time to development of the previously characterized myeloproliferative disorder or acute leukemia. Taken together, these data demonstrate that in the absence of specific genetic abnormalities, loss of Ctnna1 expression in primary murine HSCs is not associated with aberrant HSC function or the development of myeloid neoplasms. Further studies are necessary to define a role for of loss of Ctnna1 expression in human myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

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Mutagenesis of Erythropoietin Receptor Cytoplasmic Lysines Uncouples Erythropoietin-Dependent Growth From Downstream Signal Transduction Cascades.

Blood ◽

10.1182/blood.v114.22.3611.3611 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3611-3611

Author(s):

Terri D Richmond ◽

Natasha Matthew ◽

Dwayne L. Barber

Keyword(s):

Cell Surface ◽

Cell Signaling ◽

Ubiquitin Ligase ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Erythropoietin Receptor ◽

Signaling Cascades ◽

Dependent Growth

Abstract Abstract 3611 Poster Board III-547 Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. Fundamental to this action is the ability of EPO to bind to its cognate receptor on the cell surface, the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Erythrocyte production relies heavily on precise control of EPO-mediated cell signaling cascades. The availability of EPO-R to EPO and the activation of subsequent signaling cascades are tightly regulated by the transit of mature EPO-R to the cell surface from the Golgi, and the mechanisms by which the receptor is internalized and down-regulated. Thus, small changes in plasma membrane EPO-R levels may be reflected in the intensity and duration of downstream signaling protein activation. EPO-R is rapidly ubiquitinated and down-regulated from the cell surface upon EPO stimulation. EPO-R ubiquitination relies on more than one E3 ubiquitin ligase, the U-box containing E3 ubiquitin ligase, p33Rul, and the RING finger E3 ubiquitin ligase SCFβTrcp. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome lead us to examine the role of EPO-R ubiquitination on cell signaling and proliferation. We hypothesized that EPO-R ubiquitination would result in down-regulation of EPO-mediated signaling cascades. This study characterized which lysines determined EPO-R ubiquitination, and assessed the ability of EPO-R lysine mutants to support EPO-mediated signaling and proliferation. Surprisingly, substitution of all EPO-R cytoplasmic lysines (EPO-R K5R) abolished the ability of EPO-R to support EPO-mediated proliferation in BaF3 cells. However, EPO-dependent phosphorylation of EPO-R, AKT, PKB and STAT5 was detected, albeit at reduced levels. While ubiquitination defects commonly prolong receptor signaling, the kinetics of EPO-R K5R-mediated signaling pathways paralleled Ba/F3-EPO-R cells. Mutation of membrane proximal lysines, K256 and K276, resulted in decreased JAK2 phosphorylation and EPO-R phosphorylation and inhibited proliferation at 0.5U/ml EPO. However, the interaction between JAK2 and EPO-R was not affected. In addition, any EPO-R cytoplasmic lysine could support signaling and proliferation at physiologically elevated EPO concentrations demonstrating significant redundancy. The requirement of EPO-R cytoplasmic lysines to promote CFU-E formation is currently being evaluated by introduction of EPO-R mutants into fetal liver cells derived from EPO-R null mice. These results suggest that the EPO-R cytoplasmic lysines play a critical role in transmitting EPO-dependent growth signals within the cell. Disclosures: No relevant conflicts of interest to declare.

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Association of A313g Glutathione S-Transferase P1 (GSTP1) Inborn Polymorphism with Susceptibility to De Novo MDS

Blood ◽

10.1182/blood.v120.21.1445.1445 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1445-1445

Author(s):

Sophia Zachaki ◽

Chryssa Stavropoulou ◽

Aggeliki Daraki ◽

Marina Kalomoiraki ◽

Panagoula Kollia ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Type A ◽

Common Mechanism ◽

Genotype Distribution ◽

Distribution Analysis ◽

Wild Type ◽

Increased Risk ◽

Variant Genotype

Abstract Abstract 1445 Models for the pathogenesis of myelodysplastic syndromes (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in detoxification of a variety of electrophilic compounds, such as benzo [a]-pyrene and other polycyclic aromatic hydrocarbons (PAHs), chemotherapy drugs and products of oxidative stress. GSTP1 acts through a common mechanism of conjugating reactive oxygen species (ROS) with glutathione, enabling their detoxification and elimination and thus defending tissues against DNA damage. The corresponding gene is subject to a single-nucleotide polymorphism (A313G) leading to abolished enzyme activity. Thus, individuals homozygous for the variant G allele (G/G) have a lower conjugating activity than individuals homozygous for the wild type A allele (A/A), while heterozygotes (A/G) display intermediate activity. The aim of the present study was to evaluate whether the GSTP1 polymorphism influences susceptibility to MDS and/or promote specific chromosomal aberrations. We conducted a case-control study in 310 de novo MDS patients and 370 unrelated healthy controls using both a conventional PCR-RFLP assay and a novel Real-Time PCR genotyping method using hybridization probe technology. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. Comparison of the genotype distribution between controls and MDS cases revealed a significantly higher frequency of the variant genotypes (heterozygotes A/A and homozygotes G/G) among MDS patients, as compared to controls (p<0.0001, χ2=31.167, df=2). The most marked statistical difference between MDS patients and controls was observed between the wild-type (A/A) and the homozygous variant genotype (G/G), since subjects carrying the G/G variant genotype showed a 4.1-fold increased risk of MDS prevalence than subjects carrying the wild-type A/A genotype (p=0.000, χ2=30.5, d.f.=1, OR=4.098, 95%CI=[2.433–6.897]). Allele frequencies distribution analysis between patients and controls, showed that MDS patients exhibited a 1.9-fold increased risk of carrying at least one variant G allele, as compared to the controls (p<0.0001, d.f.=1, OR =1.9, 95%CI=[1.48–2.34]). There was no association between the GSTP1 polymorphism and gender or any specific cytogenetic subgroup, while stratification of patients according to age showed a differential GSTP1 genotype distribution (p=0.007). Our results, derived from the larger series of primary MDS cases tested for the GSTP1 genetic background, reveal an increased incidence of the GSTP1 variant genotypes among MDS patients, providing evidence for a potential pathogenetic role of the GSTP1 polymorphism on de novo MDS risk. Disclosures: No relevant conflicts of interest to declare.

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The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

Blood ◽

10.1182/blood.v122.21.5159.5159 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5159-5159

Author(s):

Junichiro Yuda ◽

Toshihiro Miyamoto ◽

Yoshikane Kikushige ◽

Jun Odawara ◽

Yasuyuki Ohkawa ◽

...

Keyword(s):

Tyrosine Kinase ◽

Conflicts Of Interest ◽

Kinase Domain ◽

Molecular Response ◽

Healthy Individuals ◽

Wild Type ◽

Abl Kinase ◽

Tki Treatment ◽

The Impact ◽

Splicing Patterns

Abstract Background Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. Aims The aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. Methods We analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). Results We successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / Conclusion We have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures: No relevant conflicts of interest to declare.

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Frequent BRAF V600E Mutations Are Identified in CD207+ Cells in LCH Lesions, but BRAF Status does not Correlate with Clinical Presentation of Patients or Transcriptional Profiles of CD207+ Cells

Blood ◽

10.1182/blood.v118.21.1372.1372 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1372-1372 ◽

Cited By ~ 1

Author(s):

Tricia L Peters ◽

Tsz-Kwong Chris Man ◽

Jeremy Price ◽

Renelle George ◽

Phaik Har Lim ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Biological Significance ◽

Braf V600e ◽

Cell Of Origin ◽

Wild Type ◽

Braf Mutations ◽

Clinical Difference ◽

The Impact

Abstract Abstract 1372 Background: Very little is known about the cell of origin or the pathogenesis of LCH. There remains debate regarding LCH as a malignant disorder or the result of immune dysregulation. While multiple studies in the past failed to identify significant genetic lesions, an activating mutation (V600E) in the serine/threonine kinase BRAF was recently described in LCH biopsy samples (Badalian-Very et al., 2010). Objective: This study was designed to evaluate the frequency of BRAF mutations in LCH lesions, to identify the cells within the lesions carrying the mutation, and to evaluate the clinical and biological significance of the mutation. Design/Methods: Fresh LCH biopsy samples were collected, cells were sorted into CD3+ and CD207+ fractions, and RNA was purified then amplified into cDNA. Sanger sequencing as well as BRAF allele-specific PCR were performed for each sample. Categorical clinical data was compared to BRAF genotype to evaluate clinical significance of the mutation. Transcriptomes of CD207+ cells were also compared (wild-type BRAF vs V600E) with the Affymetrix U133Plus2.0 platform to determine the impact of the BRAF mutation on global gene expression. Results: The BRAF V600E mutation was consistently identified in cDNA generated from CD207+ cells in 17 of 32 (52%) LCH biopsy samples. Only the wild-type allele was detected in purified T (CD3+) cells from LCH lesions, control epidermal (CD207+) Langerhans cells, and control tonsil T (CD3+) cells. In two cases of recurrent disease, BRAF status was consistent in the presenting and the relapse CD207+ cells: wild-type BRAF in one case and V600E BRAF in another. However, mutation status did not correlate significantly with age (p=0.6), single lesion vs multifocal/systemic (p=1.0), or future recurrent/refractory disease (p=0.2) in this series. Furthermore, unsupervised clustering gene expression profiles CD207+ cells (wild-type BRAF vs V600E) did not segregate datasets based on BRAF status. Using standard statistical analysis, there were no genes identified as significantly up- or down-regulated as a result of the V600E mutation. Conclusion: The BRAF V600E point mutation is the first reproducible molecular abnormality identified in LCH. In this study, we validate the observation that it occurs with high frequency, and definitively localize the pathologic CD207+ cell as the source of the mutation in LCH lesions. Interestingly, while the frequency of the mutation implies some functional significance, in this series there is no statistically significant clinical difference between patients with wild-type or mutated BRAF lesions, and the transcriptomes of LCH CD207+ cells with wild-type and V600E BRAF are indistinguishable. It is possible that the mutation affects LCH pathogenesis at earlier stages in tumorigenesis, or there may be other routes of Ras pathway activation in LCH lesions with wild-type BRAF. While the role for BRAF in LCH pathogenesis remains to be defined, this is an important molecular foothold from which to investigate the biology of LCH. Disclosures: No relevant conflicts of interest to declare.

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Disparate Phenotypes Of SEC23B Deficiency In Humans and Mice

Blood ◽

10.1182/blood.v122.21.312.312 ◽

2013 ◽

Vol 122 (21) ◽

pp. 312-312

Author(s):

Rami Khoriaty ◽

Matthew Vasievich ◽

Morgan Jones ◽

Bin Zhang ◽

Lesley Everett ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Stop Codon ◽

Fetal Liver ◽

Cre Recombinase ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Cell Counts ◽

Frame Shift ◽

The Impact

Abstract Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs. SEC23 is a core component of COPII coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus. Bone marrow transplantation cures CDAII, suggesting that the pathologic defect in this disease is restricted to the hematopoietic compartment. However, the mechanism by which SEC23B-deficiency results in CDAII remains unknown. We previously reported that mice homozygous deficient for SEC23B (Sec23bgt/gt) exhibit massive pancreatic degeneration. The latter resulted in perinatal mortality precluding evaluation of the adult hematopoietic compartment. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, fetal liver cells (FLC) were harvested from Sec23bgt/gt or wildtype (WT) control E17.5 embryos and transplanted into lethally irradiated C57BL/6J mice. Recipients of Sec23bgt/gt FLC had normal peripheral blood counts and were indistinguishable from recipients of WT FLC. To test for a more subtle hematopoietic defect, Sec23bgt/gt FLCs were tested directly against WT FLC for their ability to reconstitute hematopoiesis in a competitive repopulation assay. SEC23B deficient FLC exhibited no competitive disadvantage at reconstituting erythropoiesis relative to WT FLC over 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. We also generated a second, conditional Sec23b allele, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 with Cre-recombinase results in a frame shift leading to a stop codon in exon 7. Mice with erythroid-specific SEC23B deficiency were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice maintained normal erythropoiesis indistinguishable from their WT littermates. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to p48-Cre or Pdx1-Cre transgenes generated phenotypes indistinguishable from complete SEC23B deficiency, demonstrating that loss of pancreatic Sec23b expression alone is sufficient to explain the perinatal lethality observed in Sec23bgt/gt and Sec23b-/- mice. Our results conclusively demonstrate that in contrast to humans, SEC23B-deficiency results in massive pancreatic degeneration in mice, but no CDAII in these animals. To investigate the cause of the disparate human and mouse SEC23B-deficient phenotypes, SEC23B/SEC23A expression ratios were examined in endogenous tissues from both species. This ratio (normalized to the ratio in liver mRNA as 1.0) was higher in mouse pancreas (12.7) compared to BM (2.6), with the reverse pattern observed in human BM (7.8) relative to pancreas (5.5). These data, taken together with the high degree of identity between SEC23A and SEC23B (∼ 85% amino acid identity), suggest that the tissue-specific functions of SEC23A and SEC23B may have shifted during evolution between humans and mice. To test the role of SEC23A, we generated a mouse with a conditional Sec23a allele, in which exon 3 is flanked by loxP sites (Sec23afl). Cre-recombinase mediated deletion of exon 3 results in a frame shift leading to a stop codon in exon 7 (Sec23a-). Mice with erythroid-specific SEC23A-deficiency (Sec23afl/-/EpoR-CreTg+) maintained normal red blood cell counts indistinguishable from their WT littermates. In summary, we have shown that SEC23B-deficient humans and mice exhibit disparate phenotypes. We have also demonstrated variations in the gene expression programs for SEC23A and SEC23B potentially explaining the pancreatic phenotype of SEC23B-deficiency in mice and the erythroid phenotype in humans. These results suggest that the two SEC23 paralogs have overlapping functions and that therapeutic strategies that increase the expression of either SEC23A or SEC23B in erythroid cells might be effective in CDAII. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII. Disclosures: No relevant conflicts of interest to declare.

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Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

Blood ◽

10.1182/blood.v128.22.1398.1398 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1398-1398

Author(s):

Azza Abdelaal ◽

David Jakab ◽

Pamela A Christopherson ◽

Robert R Montgomery ◽

Sandra L Haberichter

Keyword(s):

Conflicts Of Interest ◽

Stop Codon ◽

Premature Stop Codon ◽

Von Willebrand Disease ◽

Sequence Variants ◽

Cell Lysate ◽

Expression Studies ◽

Von Willebrand ◽

The Impact

Abstract von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar. We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype. Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure. 47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed. In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. Disclosures No relevant conflicts of interest to declare.

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