ALBERT

Description: Abstract 2826 Polycythemia vera (PV) is a clonal blood disorder arising from a multipotent hematopoietic stem cell (HSC) associated in ∼95% of patients with the acquired somatic mutation, JAK2 V617F. Although important for the PV phenotype, we and others demonstrated that the JAK2 V617F mutation is not the initial and causative somatic event in PV pathogenesis. One of the major challenges of studying the molecular events in PV is to isolate and expand the disease-initiating HSC clones in vitro. To overcome this hurdle, we have utilized the recently developed induced pluripotent stem cell (iPSC) technology to generate disease-specific iPSC lines that preserve the genetic identities of patient HSC clones. We previously demonstrated that interferon (IFN) a is the only therapy that converts PV hematopoiesis from clonal to polyclonal (Liu, Blood 2003). A female patient with typical PV and a high allele burden (99%) of JAK2 V617F, and ∼1% of wild-type JAK2 was treated with peg-IFNa. JAK2 allele burden decreased to ∼65%, yet the majority of her myeloid cells remained clonal. Using her blood and bone marrow progenitors as well as blood samples from other PV patients, we generated dozens of iPSC clones by retroviral or episomal vectors with several distinct JAK2 genotypes (see Table below). We examined the erythroid differentiation of 6 representative PV-iPSC lines and normal control iPSCs. The hematopoietic progenitor cells (HPCs) derived from JAK2 V617F iPSCs had enhanced erythropoiesis compared to wild-type JAK2 iPSC cells. Additionally, some EPO-independent BFU-Es also formed from homozygous JAK2 V617F iPSCs, the hallmark of PV erythropoiesis. Using a quantitative X-chromosome transcriptional assay (Swierczek, Blood 2008), we examined the clonality of the iPSC clones (with and without the JAK2 mutation) derived from this single female patient and showed the same single X-chromosome usage in all clones as in her native PV granulocytes and platelets. These data indicate that epigenetic X-chromosome silencing is not reverted in the process of generating iPSC clones. Whether these two JAK2 V617F-negative iPSC lines originated from the same PV clone or from dormant normal HSC cells cannot be yet discerned but their EPO sensitivity is currently under analysis. Analyses of whole exome sequencing of these iPS clones as well as their germ-line control are currently underway. Additionally, whole genome and epigenome analyses and high density expression array of these iPSC clones would further characterize the clonal evolution of PV. These data underscore the heterogeneity of somatic mutations within single PV patient. These studies will lead to a better understanding of the genetic lesions in PV-initiating clones. (Note: The last two authors are both considered senior authors for this work)Table.Human iPSC lines from a female PV patient and healthy donorsRepresentative iPS clone (# of clones characterized)DonorParental cell typeJAK2 WT alleleJAK2 V617F alleleKaryotypePVB1.4 (3)PVPB MNC0246, XXPVB1.1 (4)PB MNC1247, XX, +der(1;9)(q10;p10)PVB1.11 (2)PB MNC2046, XXPVM1.1 (2)BM MSC2046, XXBC1 (〉5)Healthy donorBM CD34+2046, XYPB: peripheral blood; BM: bone marrow; MSC: mesenchymal stem cell. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2073 We have previously described a unique etiology of β-thalassemia due retrotransposon LINE-1 (L1) insertion into the intron-2 of the β-globin gene in a mother and daughter of Ukrainian descent who exhibited typical laboratory features of β-thalassemia trait (1, 2). The affected β-globinL1 gene generated roughly 10–15% of the total β−globin message (1). In order to unravel how the intronic insertion of transposable element attenuates the β-globin expression, interspecific hybrids of the propositus′ lymphocytes and mouse erythroleukemia (MEL) cells were generated (laboratory of Dr. T. Papayannopoulou, Seattle, WA) and used for expression studies. The total RNA from differently treated (emetine, 5-aza-2′-deoxycytidine (Sigma)) in vitro cultured cell hybrids were isolated and β-globin transcripts were analyzed by Real-Time qPCR with gene-specific primers. Nuclei from hybrid MEL cells were isolated to performed nuclear run-on assay. Bisulfite modification was done on genomic DNA from MEL hybrids and the promoter and enhancer regions of β-globin gene were amplified, PCR products were subcloned and sequenced. We demonstrated that the observed reduction in steady-state level of β-globin mRNA is partially caused by aberrant splicing followed by activation of nonsense-mediated decay (NMD) pathway, leading to increased degradation of aberrant β-globin mRNA variants. Reduction in expression of β-globin mRNA from β-globinL1 allele comes also from altered rate of transcription. We performed PCR-based nuclear run-on assay and forty minutes of in vitro transcription revealed 30% decrease in β-globinL1 allele transcription rate compared to wild-type β-globin allele. It is known that L1 regulatory regions are highly methylated (3) but there is no evidence so far demonstrating retrotransposon-mediated epigenetic control of neighboring genes. Therefore we determined the methylation status of 6 CpGs between −415 bp and +110 bp (bases upstream and downstream of transcription start) on the promoter region and 3 CpGs between +442 bp to +592 bp on the enhancer region (bases downstream of the poly(A) signal) of the mutated and normal β−globin alleles. Enhancer region of the mutated allele was 100% methylated (100% Me-CpG versus 9% Me-CpG in control), and promoter region was partially methylated (≈ 65% Me−CpG versus 35% Me-CpG in control). Surprisingly, treatment of induced hybrid MEL cells with demethylation agent (5-aza-2′-deoxycytidine, decitabine) did not change the methylation profile and consequently did not increase the expression of β-globinL1 mRNA whereas the expression of γ-globin gene, which was used as an internal control, was increased. We therefore hypothesize that decreased rate of transcription from β-globinL1 allele is associated with altered DNA topology caused by the L1 insertion and β-globinL1 promoter-enhancer displacement. Resulting chromatin modifications lead to permanent β-globinL1 gene silencing and hypermethylation of regulatory sequence refractory to demethylating agent. Divoky V et al: Blood. 1996;88 (suppl1):148a. Kimberland ML, Divoky V et al: Hum Mol Genet. 1999;8:1557-1560. Hata K, Sakaki Y: Gene. 1997;189:227-234. Grant support: NS10281 −3/2009 Ministry of Health, Czech Republic and Internal grant agency of Palacky University grant No. LF_2010_013. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5307 Background: Thalassemias are rare disorders in Middle Europe. However, as a result of historical and recent migration, thalassemias became common cause of congenital anemia in the Czech and Slovak populations. Abnormal hemoglobin variants and red-cell enzymopathies are rare cause of congenital anemia in this region. The aim of this work was to update the original reports of this research published almost two decades ago (Indrak et al., Hum Genet 1992; 88:399–404, Xu et al., Blood 1995; 85:257–63, Lenzner et al., Blood 1997; 89:1793–9). We assessed the frequency and spectrum of β-globin gene mutations in the patients with clinical symptoms of β-thalassemia or δ,β-thalassemia, the α-globin gene status in the patients with clinical symptoms of α-thalassemia, and we characterized red cell enzymopathies on molecular level in the Czech and Slovak populations. Patients and methods: Nearly 390 cases with clinical symptoms of thalassemia or hereditary nonspherocytic hemolytic anemia from several centers of Czech and Slovak Republic were analyzed. Hematological parameters, hemoglobin electrophoresis and enzyme activities were measured by standard procedures. Genomic DNA was used for PCR-sequencing analysis. Results: We identified 22 β-thalassemia mutations in more than 260 heterozygotes; most of the mutations were of Mediterranean origin. The newly discovered insertion of transposable element L1 into the HBB gene represents a novel etiology of β-thalassemia due to a silencing effect of repressive chromatin associated with retrotransposon insertion. The list of abnormal hemoglobins now contains 14 β-globin variants, involving Heinz body hemolytic anemia variant Hb Hana (β63(E7) His-Asn), phenotype of which was worsened by concomitant partial glutathione reductase deficiency (Mojzikova et al., Blood Cells Mol Dis 2010; 45:219–22). Several G6PD and PK variants were described in the Czech and Slovak populations; the G6PD variants include G6PD Olomouc, G6PD Varnsdorf and G6PD Praha. Recently, we identified a new frameshift mutation c. 1553delG (p. Arg518fs) at the homozygous state in exon 11 of the PKLR gene of the pediatric patient who suffered from transfusion dependent hemolytic anemia with Hb=9.4 g/dL, Ret=4.5%. His red cells PK activity was 4.52 IU/gHb (normal range 13–17 IU/gHb). The mutation occurs in C domain of PK-R subunit containing the binding site for fructose-1,6-bisphosphate. The patient's extremely elevated level of growth differentiation factor 15 (GDF15, 3577 pg/mL, healthy controls 231–345 pg/mL) could explain hereditary hemochromatosis and signs of iron overload in this patient. Conclusions: In the Czech and Slovak populations, hemoglobinopathies and red-cell enzymopathies appear to be an uncommon disorder, which, however, must be considered as the prevailing cause of congenital anemia. Most of the thalassemia patients were heterozygous, manifesting thalassemia minor. Most of the hemoglobin variants were described in single families, some of them originated locally. Among hemolytic anemias due to red-cell enzymopathies is the most frequent PK deficiency. This work was supported by grants NT11208, NS10281 (Ministry of Health Czech Republic), MSM6198959205 (Ministry of Education, Youth and Sports) and student projects LF_2011_006 and LF_2011_011 of the Palacky University. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1755 Polycythemia vera (PV) is an acquired clonal hematopoietic disorder characterized by the JAK2V617F somatic mutation, which, however, constitutes only a variable part of the PV clone. Even within the JAK2V617F subclone, heterozygous and homozygous JAK2V617F progenitors coexist. Further, family clustering of PV suggests the existence of predisposing germline mutations. The nature of these pre-JAK2V617Fsomatic and germline mutations has not yet been identified, and the search for these predisposing genetic lesions using marrow and blood cells is hampered by their genetic heterogeneity. In order to overcome this obstacle, we generated 5 inducible pluripotent stem cell lines (iPSC) from the same PV female with different JAK2 genomic configuration (4 from CD34+ blood cells, one from marrow mesenchymal cells having germ-line DNA configuration). Genomic DNA was isolated from the cell lines, followed by whole genome sequencing (WGS) at 〉20X depth, and whole exome sequencing (WES) at 〉130X depth. We have developed a comprehensive WGS/WES pipeline for sequence alignment and variant calling. The major components are: a) Pre-processing and quality control of raw reads outputted from sequencing instruments; b) Align the read against the Human Genome Project reference using BWA (Burrows-Wheeler Aligner); c) Local realignment at indels using GATK (Genome Analysis Toolkit); d) Identify germline/somatic single nucleotide variants (SNVs), copy number variants (CNV) and structural variants (SV) using GATK, Varscan2, Breakdancer, CNVnator, and Pindel. The variant calls are intersected and merged. Calls detected by two or more algorithms are regarded as high confidence. Two somatic mutations, rs1047840, and rs3795677 were identified by WGS and WES in the CD34+ cell derived iPSCs. rs1047840 (E589K) is a SNP in EXO1 gene that encodes a protein with 5' to 3' exonuclease activity as well as an RNase H activity. EXO1 is involved in DNA mismatch repair and homologous recombination. rs1047840 conferred increased risk of breast, lung, and gastric cancer. rs3795677 (R64H) is a SNP in FMN2 gene, which is required for microtubule-independent chromatin positioning during metaphase. Interestingly, these two mutations are all common SNVs, and they are located in a duplicated region on 1q43. Whole genome analysis is ongoing to characterize and validate the complex mixture of SVs in this region, identify potential translocations and determine the exact genomic breakpoints. The variants will be validated in non-iPSC cells from the same individual to rule out iPSC artifacts. This work identifies somatic mutations in EXO1 and FMN2, and CNVs in 1q43 as the potential genetic defects involved in the early pathogenesis of PV. It also demonstrates the value of combining WGS and WES for the identification of somatic mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2081 There are two known homozygous VHL gene mutations causing polycythemia that are not associated with a VHL cancer syndrome - an R200W mutation endemic in Chuvashia causing the first recognized disorder of augmented hypoxia sensing in normoxia, and an H191D mutation of Croatian origin. Both are located in the distal exon 3 of the VHL gene on its C-terminal domain. As different positions of loss-of-function mutations of VHL gene are associated with different type of cancers, it has been proposed that only C-terminal domain VHL mutations would cause polycythemia. However, we now contradict this notion. We report a novel homozygous variant of the VHL gene located in the middle of coding region in exon 2; 413C〉T:P138L. The propositus is a 15 year old Punjabi female with congenital polycythemia; i.e. elevated erythropoietin (EPO) 40 mIU/mL, no JAK2 mutations, and hemoglobin 19–20 g/dl. Her parents are VHL P138L heterozygotes and no VHL tumors are reported in the extended family, in contrast to the other VHL P138 residue (P138R, P138T) mutations that have been reported in VHL syndrome and renal cancer. pVHL is a negative regulator of hypoxia inducible transcription factors (HIFs) as it degrades a subunits of HIFs. It has been proposed, that an association between mutated pVHL and suppressor of cytokine signaling 1 (SOCS1) leads to JAK2 up-regulation and enhanced erythropoiesis. We show that the VHL P138L mutation, which lies in the catalytic HIF-1a peptide ligand-binding region, perturbs pHIF-1a pVHL interaction due to a conformational effect on the W117 and S111 residues lying within 2.8 to 4.3 Å distance from the mutated L138. The effect of this single mutation on overall structure is a shift of 1.9 Å RMSD (root mean square deviation) from the wild-type complex structure. Molecular dynamics simulations also found that the VHL P138L mutation can affect the binding of SOCS1 protein to the pVHL groove due to a strong steric effect of T124, H125 and D126 residues located close to the C-terminal pVHL. The accumulation of HIFs and up-regulated transcription of downstream target genes including those for glucose transporter-1 (SLC2A1) and transferrin (TF) were found in the propositus' granulocytes. We then analyzed the VHL P138L propositus and her heterozygous parents' erythroid progenitors and found them to be hypersensitive to EPO (a feature of primary polycythemias without EPO independent colonies). Because of reports that RUNX1/AML1 transcript levels are specifically upregulated in erythroid progenitors in polycythemia vera (PV) and allegedly responsible for PV EPO hypersensitivity, we analyzed erythroid RUNX1/AML1 transcripts in the propositus and her parents and found it increased. We demonstrate that a homozygous mutation in exon 2 of the VHL gene can also be associated with a polycythemic phenotype rather than VHL syndrome tumors. Further, we report that increased levels of RUNX1/AML1 transcripts are not specific for PV but can be seen in other primary polycythemias. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 705 Polycythemia vera (PV) is a clonally derived hematopoietic malignancy arising from a multipotent hematopoietic stem cell (HSC). Almost all patients have the acquired somatic mutation, JAK2V617F. There is strong evidence suggesting that the JAK2V617F mutation is not a PV-initiating genetic event in its pathogenesis. Thus, “pre- JAK2V617F mutation(s)” account for clonal dominance before the acquisition of the JAK2V617Fmutation. The existence of PV family clustering indicates that germline genetic variations contribute to PV genesis. To explore possible PV-initiating events, we performed deep exome sequencing of 31 JAK2V617F-positive PV patients. DNA from granulocytes (clonal cells) and their matched T-lymphocytes (polyclonal cells with germline DNA) were collected and exonic regions were sequenced to ∼130-fold coverage to identify somatic and germline base substitutions, insertions and deletions (indels), as well as copy number alterations (CNA). DNA from skin was also examined in 6 PV patients and was sequenced to ∼117-fold coverage in order to rescue any germline events that may have been missed in the T-lymphocytes. Our findings confirm the somatic loss of heterozygosity (LOH) and trisomy at 9p. The proportion of JAK2V617 as determined by the fraction of the reads harboring the mutations in the patient's granulocytes varied from almost undetectable to 100% of the reads. As previously reported, the degree of LOH at 9p correlated with the variant fraction of JAK2V617F with stronger LOH associated with higher allelic burden of JAK2V617F. We also observed single examples of LOH on 22q and 18p of which the latter is a novel finding in this study. In addition, we observed focal amplification at the PRSS1 locus in 75% of the PV granulocytes. Nine patients also harbor recurrent somatic mutations in genes with known roles in hematologic malignancies including four patients (13%) carrying IDH2 somatic mutations, 1 previously described and 2 novel. Additional somatic mutations were found in several other genes functioning in the growth signaling and chromatin remodeling processes. Remarkably, four patients exhibited novel germline variation in JAK2 itself, including one canonical JAK2V617F variant and 3 other missense variants. Moreover, nearly one half of the patients harbor novel germline variants in FAT2, a tumor suppressor essential for controlling cell proliferation. Interestingly, we also observed in 71% of patients, a germline common allele of EXO1, rs1047840, which has been linked to multiple cancers by genome-wide association studies (GWAS). Our study, using whole exome sequencing, recapitulated all of the major findings of PV genomic alteration from earlier studies. Prior to this study JAK2V617 or K539L and associated 9p LOH, were the only known somatic variation. We have extended previous observations by demonstrating recurrent germline and somatic variation that potentially could contribute to the initiation of the disease. Both CNA alterations and point mutations in important cancer genes were seen in a significant number of patients and germline variants were seen in almost all patients in the cohort. This study finds strong evidence in support of the hypothesis of a second hit contributing to PV onset and progression as well as germline predisposition of PV. Disclosures: No relevant conflicts of interest to declare.

Description: Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

Description: Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.