The Effect of p.G2019S Mutation in the LRRK2 Gene on the Activity of Lysosomal Hydrolases and the Clinical Features of Parkinson's Disease Associated with p.N370S Mutation in the GBA1 Gene.

BACKGROUND: Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). METHODS: One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. RESULTS: Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). CONCLUSIONS: Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.

Whole Transcriptome Analysis of Substantia Nigra in Mice with MPTP-Induced Parkinsonism Bearing Defective Glucocerebrosidase Activity.

Mutations in the GBA1 gene represent the major genetic risk factor for Parkinson's disease (PD). The lysosomal enzyme beta-glucocerebrosidase (GCase) encoded by the GBA1 gene participates in both the endolysosomal pathway and the immune response. Disruption of these mechanisms is involved in PD pathogenesis. However, molecular mechanisms of PD associated with GBA1 mutations (GBA-PD) are unknown today in particular due to the partial penetrance of GBA1 variants in PD. The modifiers of GBA1 penetrance have not been elucidated. We characterized the transcriptomic profiles of cells from the substantia nigra (SN) of mice with co-injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and selective inhibitor of GCase activity (conduritol-ß-epoxide, (CBE)) to mimic PD bearing GCase dysfunction (MPTP+CBE), mice treated with MPTP, mice treated with CBE and control mice treated with injection of sodium chloride (NaCl) (vehicle). Differential expression analysis, pathway enrichment analysis, and outlier detection were performed. Functional clustering of differentially represented transcripts revealed more processes associated with the functioning of neurogenesis, inflammation, apoptosis and autophagy in MPTP+CBE and MPTP mice than in vehicle mice, with a more pronounced alteration of autophagy processes in MPTP+CBE mice than in MPTP mice. The PI3K-Akt-mTOR signaling pathway may be considered a potential target for therapy in PD with GCase dysfunction.

Fraction of plasma exomeres and low-density lipoprotein cholesterol as a predictor of fatal outcome of COVID-19.

Transcriptomic analysis conducted by us previously revealed upregulation of genes involved in low-density lipoprotein particle receptor (LDLR) activity pathway in lethal COVID-19 caused by SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2). Last data suggested the possible role of extracellular vesicles in COVID-19 pathogenesis. The aim of the present study was to retrospectively evaluate parameters of cholesterol metabolism and newly identified EVs, exomeres, as possible predictors of fatal outcome of COVID-19 patients infected by the Alpha and the Delta variants of SARS-CoV-2 virus. Blood from 67 patients with severe COVID-19 were collected at the time of admission to the intensive care unit (ICU) and 7 days after admission to the ICU. After 30 days patients were divided into two subgroups according to outcome-34 non-survivors and 33 survivors. This study demonstrated that plasma low- and high-density lipoprotein cholesterol levels (LDL-C and HDL-C) were decreased in non-survivors compared to controls at the time of admission to the ICU. The conjoint fraction of exomeres and LDL particles measured by dynamic light scattering (DLS) was decreased in non-survivors infected by the Alpha and the Delta variants compared to survivors at the time of admission to the ICU. We first showed that reduction of exomeres fraction may be critical in fatal outcome of COVID-19.

Altered Sphingolipid Hydrolase Activities and Alpha-Synuclein Level in Late-Onset Schizophrenia.

Recent data described that patients with lysosomal storage disorders (LSDs) may have clinical schizophrenia (SCZ) features. Disruption of lipid metabolism in SCZ pathogenesis was found. Clinical features of schizophrenia (SCZ) have been demonstrated in patients with several lysosomal storage disorders (LSDs). Taking into account the critical role of lysosomal function for neuronal cells' lysosomal dysfunction could be proposed in SCZ pathogenesis. The current study analyzed lysosomal enzyme activities and the alpha-synuclein level in the blood of patients with late-onset SCZ. In total, 52 SCZ patients with late-onset SCZ, 180 sporadic Parkinson's disease (sPD) patients, and 176 controls were recruited. The enzymatic activity of enzymes associated with mucopolysaccharidosis (alpha-L-Iduronidase (IDUA)), glycogenosis (acid alpha-glucosidase (GAA)) and sphingolipidosis (galactosylceramidase (GALC), glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase)) and concentration of lysosphingolipids (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), and lysosphingomyelin (LysoSM)) were measured using LC-MS/MS. The alpha-synuclein level was estimated in magnetically separated CD45+ blood cells using the enzyme-linked immunosorbent assay (ELISA). Additionally, NGS analysis of 11 LSDs genes was conducted in 21 early-onset SCZ patients and 23 controls using the gene panel PGRNseq-NDD. Decreased ASMase, increased GLA activities, and increased HexSpn, LysoGb3, and LysoSM concentrations along with an accumulation of the alpha-synuclein level were observed in late-onset SCZ patients in comparison to the controls (p < 0.05). Four rare deleterious variants among LSDs genes causing mucopolysaccharidosis type I (IDUA (rs532731688, rs74385837) and type III (HGSNAT (rs766835582)) and sphingolipidosis (metachromatic leukodystrophy (ARSA (rs201251634)) were identified in five patients from the group of early-onset SCZ patients but not in the controls. Our findings supported the role of sphingolipid metabolism in SCZ pathogenesis. Aberrant enzyme activities and compounds of sphingolipids associated with ceramide metabolism may lead to accumulation of alpha-synuclein and may be critical in SCZ pathogenesis.

Transcriptomic Profiles Reveal Downregulation of Low-Density Lipoprotein Particle Receptor Pathway Activity in Patients Surviving Severe COVID-19.

To assess the biology of the lethal endpoint in patients with SARS-CoV-2 infection, we compared the transcriptional response to the virus in patients who survived or died during severe COVID-19. We applied gene expression profiling to generate transcriptional signatures for peripheral blood mononuclear cells (PBMCs) from patients with SARS-CoV-2 infection at the time when they were placed in the Intensive Care Unit of the Pavlov First State Medical University of St. Petersburg (Russia). Three different bioinformatics approaches to RNA-seq analysis identified a downregulation of three common pathways in survivors compared with nonsurvivors among patients with severe COVID-19, namely, low-density lipoprotein (LDL) particle receptor activity (GO:0005041), important for maintaining cholesterol homeostasis, leukocyte differentiation (GO:0002521), and cargo receptor activity (GO:0038024). Specifically, PBMCs from surviving patients were characterized by reduced expression of PPARG, CD36, STAB1, ITGAV, and ANXA2. Taken together, our findings suggest that LDL particle receptor pathway activity in patients with COVID-19 infection is associated with poor disease prognosis.

Comparative Transcriptome Analysis in Monocyte-Derived Macrophages of Asymptomatic GBA Mutation Carriers and Patients with GBA-Associated Parkinson's Disease.

Mutations of the GBA gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are the greatest genetic risk factor for Parkinson's disease (PD) with frequency between 5% and 20% across the world. N370S and L444P are the two most common mutations in the GBA gene. PD carriers of severe mutation L444P in the GBA gene is characterized by the earlier age at onset compared to N370S. Not every carrier of GBA mutations develop PD during one's lifetime. In the current study we aimed to find common gene expression signatures in PD associated with mutation in the GBA gene (GBA-PD) using RNA-seq. We compared transcriptome of monocyte-derived macrophages of 5 patients with GBA-PD (4 L444P/N, 1 N370S/N) and 4 asymptomatic GBA mutation carriers (GBA-carriers) (3 L444P/N, 1 N370S/N) and 4 controls. We also conducted comparative transcriptome analysis for L444P/N only GBA-PD patients and GBA-carriers. Revealed deregulated genes in GBA-PD independently of GBA mutations (L444P or N370S) were involved in immune response, neuronal function. We found upregulated pathway associated with zinc metabolism in L444P/N GBA-PD patients. The potential important role of DUSP1 in the pathogenesis of GBA-PD was suggested.

Mutation analysis of Parkinson's disease genes in a Russian data set.

Common variants and risk factors related to familial and sporadic cases of Parkinson's disease (PD) in diverse populations have been identified at numerous genomic loci. In this study, genetic analysis was performed through a screening of LRRK2 G2019S, GBA mutations (L444P, N370S), and common variants (E326K, T369M) in 762 PD patients and in 400 controls. Next-generation sequencing analysis of 22 PD-related genes in 28 early-onset PD cases from North-Western region of Russia was performed. The frequency of LRRK2 G2019S mutation was 5.8% in familial and 0.5% in sporadic PD cases. The frequency of GBA mutations (L444P, N370S) in PD patients was higher compared to controls (odds ratio [OR] = 6.9, 95% confidence interval [CI], 0.9-53.13, p = 0.031), particularly in patients with early-onset compared to late-onset PD (OR = 3.90 [95% CI, 1.2-13.2], p = 0.009). The frequency of E326K and T369M was twice higher among PD patients than in controls (OR = 2.24, 95% CI 1.05-4.79, p = 0.033). However, the screening of 22 PD-related genes using our novel panel of gene resequencing in our series of 28 early-onset PD failed to identify any mutations. LRRK2 and GBA mutations were found to be common risk factors for PD in North-Western region of Russia.

Whole-Exome Sequencing in Searching for New Variants Associated With the Development of Parkinson's Disease.

Background: Parkinson's disease (PD) is a complex disease with its monogenic forms accounting for less than 10% of all cases. Whole-exome sequencing (WES) technology has been used successfully to find mutations in large families. However, because of the late onset of the disease, only small families and unrelated patients are usually available. WES conducted in such cases yields in a large number of candidate variants. There are currently a number of imperfect software tools that allow the pathogenicity of variants to be evaluated. Objectives: We analyzed 48 unrelated patients with an alleged autosomal dominant familial form of PD using WES and developed a strategy for selecting potential pathogenetically significant variants using almost all available bioinformatics resources for the analysis of exonic areas. Methods: DNA sequencing of 48 patients with excluded frequent mutations was performed using an Illumina HiSeq 2500 platform. The possible pathogenetic significance of identified variants and their involvement in the pathogenesis of PD was assessed using SNP and Variation Suite (SVS), Combined Annotation Dependent Depletion (CADD) and Rare Exome Variant Ensemble Learner (REVEL) software. Functional evaluation was performed using the Pathway Studio database. Results: A significant reduction in the search range from 7082 to 25 variants in 23 genes associated with PD or neuronal function was achieved. Eight (FXN, MFN2, MYOC, NPC1, PSEN1, RET, SCN3A and SPG7) were the most significant. Conclusions: The multistep approach developed made it possible to conduct an effective search for potential pathogenetically significant variants, presumably involved in the pathogenesis of PD. The data obtained need to be further verified experimentally.

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell-derived inflammatory signals. Donor T cell-derived tumor necrosis factor-α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell-derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell-derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence.

Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures. Intra-colony analysis revealed radial organization of CD34 and VECAD expression levels, with CD45+ blood cells emerging primarily from the colony centroid area. We identify the induced interferon gamma protein (IP-10)/p-38 MAPK signalling pathway as the mechanism for haematopoietic inhibition in our culture system. Our results highlight the role of spatial organization in hPSC-derived blood generation, and provide a quantitative platform for interrogating molecular pathways that regulate human haematopoiesis.

Loss of VPS13C Function in Autosomal-Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy.

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

Plasma oligomeric alpha-synuclein is associated with glucocerebrosidase activity in Gaucher disease.

BACKGROUND: The link between Parkinson's disease (PD) and Gaucher disease (GD), the most common lysosomal storage disease associated with loss of glucocerebrosidase (GBA) activity, can be explained by abnormal accumulation of oligomeric alpha-synuclein (α-Syn) species resulting from mutations in the GBA gene. However, in GD, the relationship between GBA activity and α-Syn accumulation in biological fluids has not been investigated. METHODS: We analyzed plasma oligomeric α-Syn levels, leucocyte GBA activity, and plasma chitotriosidase activity in 21 patients with GD. RESULTS: Negative correlation between plasma oligomeric α-Syn levels, and leucocyte GBA activity was observed in patients with GD (R(2) = 0.487; P < 0.001). CONCLUSION: The decrease in GBA activity may influence α-Syn oligomerization, explaining the high risk of PD development in GD patients. © 2015 International Parkinson and Movement Disorder Society.

Leukemogenic Ptpn11 allele causes defective erythropoiesis in mice.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by PTPN11, regulates signaling networks and cell fate in many tissues. Expression of oncogenic PTPN11 in the hematopoietic compartment causes myeloproliferative neoplasm (MPN) in humans and mice. However, the stage-specific effect(s) of mutant Ptpn11 on erythroid development have remained unknown. We found that expression of an activated, leukemogenic Ptpn11 allele, Ptpn11D61Y, specifically in the erythroid lineage causes dyserythropoiesis in mice. Ptpn11D61Y progenitors produce excess cKIT+ CD71+ Ter119- cells and aberrant numbers of cKITl° CD71+ erythroblasts. Mutant erythroblasts show elevated activation of ERK, AKT and STAT3 in response to EPO stimulation, and MEK inhibitor treatment blocks Ptpn11D61Y-evoked erythroid hyperproliferation in vitro. Thus, the expression of oncogenic Ptpn11 causes dyserythropoiesis in a cell-autonomous manner in vivo.

Blood stem cell fate regulation by Delta-1-mediated rewiring of IL-6 paracrine signaling.

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1), a key component of the stem cell niche, regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that, although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6), it has opposite effects on myeloid cell production. Mechanistically, these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression, converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells, altering feedback networks and their impact on stem cell fate.

Enrichment of Sca1+ hematopoietic progenitors in polycythemic mice inhibits leukemogenesis.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by a pronounced increase in the number of erythroid cells. However, despite this aberrant proliferation, the incidence of erythroleukemia is paradoxically rare in PV patients. In this study, we show that the progression of Friend virus-induced erythroleukemia is delayed in a mouse model of primary familial congenital polycythemia in which the wild-type Epo-receptor (EpoR) gene is replaced with a truncated human EPOR gene. Herein, we show that these mice exhibit enrichment of Sca1(+)/cKit(-) progenitors and several mature immune cells, such as dendritic cells and macrophages. In cotransplantation experiments, Sca1(+)/cKit(-) progenitors inhibit the tumorigenicity of Sca1(-)/cKit(+) erythroleukemic cells. A cell line established from Sca1(+)/cKit(-) progenitors is also capable of inhibiting leukemic proliferation in culture and in mice. This phenomenon of leukemic inhibition, also detected in the serum of PV patients, is partially attributed to increased nitric oxide secretion. In addition, the administration of erythropoietin into leukemic mice induces a polycythemia-like state associated with the expansion of Sca1(+)/cKit(-) progenitors and derivative immune cells, thereby inhibiting leukemia progression. This study indicates that a combination therapy incorporating the enrichment of Sca1(+)/cKit(-) progenitors may serve as a novel approach for the treatment of leukemia.

Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis.

PTPN11, which encodes the tyrosine phosphatase SHP2, is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis, we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11(D61Y). Expression of Ptpn11(D61Y) in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin(-)Sca1(+)cKit(+) (LSK) compartment is expanded and "right-shifted," accompanied by increased stem cell factor (SCF)-evoked colony formation and Erk and Akt activation. However, repopulating activity is decreased in diseased mice, and mice that do engraft with Ptpn11(D61Y) stem cells fail to develop MPD. Ptpn11(D61Y) common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) produce cytokine-independent colonies in a cell-autonomous manner and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Ptpn11(D61Y) megakaryocyte-erythrocyte progenitors (MEPs) yield increased numbers of erythrocyte burst-forming units (BFU-Es), but MEPs and erythrocyte-committed progenitors (EPs) produce fewer erythrocyte colony-forming units (CFU-Es), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cell-autonomous and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.

Overexpression of SOCS-2 and SOCS-3 genes reverses erythroid overgrowth and IGF-I hypersensitivity of primary polycythemia vera (PV) cells.

Polycythemia vera (PV), an acquired, chronic, clonal disorder arising in a multipotential hematopoietic progenitor cell, is characterized by hyperplasia of three major myeloid lineages, with a pronounced increase in cells of the erythroid lineage. Erythroid progenitor cells in PV are strikingly hypersensitive to insulin-like growth factor-I (IGF-I); this effect is specific and is mediated through the IGF-I receptor. To investigate the possibility that in PV the increase in number of erythroid progenitors and their hypersensitivity to IGF-I result from a defect in negative regulation of cytokine activity, we examined the expression of members of the SOCS gene family. Circulating mononuclear cells, grown in serum-free methylcellulose medium in the presence of IGF-I, produced BFU-E-derived colonies whose cells revealed a reduction of SOCS-2 and SOCS-3 expression in PV only. Overexpression of these genes in transfected PV cells reduced their erythroid overgrowth and IGF-I hypersensitivity. We hypothesize that a defect in expression of SOCS-2 and SOCS-3 genes may be crucial for the IGF-I hypersensitivity and progressive increase in erythroid cell population size characteristic of PV.