Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages.

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (Scl(Δ19/Δ19)) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in Scl(Δ19/Δ19) mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in Scl(Δ19/Δ19) fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements.

Methylation of the suppressor of cytokine signaling 3 gene (SOCS3) in myeloproliferative disorders.

BACKGROUND: The JAK2 V617F mutation can be found in patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Mutation or methylation of other components of JAK/STAT signaling, such as the negative regulators suppressor of cytokine signaling 1 (SOCS1) and SOCS3, may contribute to the pathogenesis of both JAK2 V617F positive and negative myeloproliferative disorders. DESIGN AND METHODS: A cohort of patients with myeloproliferative disorders was assessed for acquired mutations, aberrant expression and/or CpG island hypermethylation of SOCS1 and SOCS3. RESULTS: No mutations were identified within the coding region of either gene in 73 patients with myeloproliferative disorders. No disease-specific CpG island methylation of SOCS1 was observed. SOCS1 expression was raised in myeloproliferative disorder granulocytes but the level was independent of JAK2 V617F status. Hypermethylation of the SOCS3 promoter was identified in 16 of 50 (32%) patients with idiopathic myelofibrosis but not in patients with essential thrombocythemia, polycythemia vera or myelofibrosis preceded by another myeloproliferative disorder. Confirmation of methylation status was validated by nested polymerase chain reaction and/or bisulphite sequencing. SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F positive myeloproliferative disorders, consistent with SOCS3 being a target gene of JAK2/STAT5 signaling. There was a trend towards an association between SOCS3 methylation and lower SOCS3 expression in JAK2 V617F negative patients with idiopathic myelofibrosis but not in JAK2 V617F positive ones. Finally, SOCS3 methylation was not significantly correlated with survival or other clinical variables. CONCLUSIONS: SOCS3 promoter methylation was detected in 32% of patients with idiopathic myelofibrosis suggesting a possible role for SOCS3 methylation in this disorder. The pathogenetic consequences of SOCS3 methylation in idiopathic myelofibrosis remain to be fully elucidated.

An acquired translocation in JAK2 Val617Phe-negative essential thrombocythemia associated with autosomal spread of X-inactivation.

The acquired mutation Val617Phe in the tyrosine kinase JAK2 was recently identified in most but not all patients with classical myeloproliferative disorders. We describe a cytogenetic and molecular study of a JAK2Val617Phe-negative case of essential thrombocythemia harboring the acquired translocation t(X;5)(q13;q33). We show that this involves the inactive X-chromosome and is associated with silencing of autosomal genes within the adjacent 5q minus syndrome common deleted region. This is the first documented example of autosomal gene silencing adjacent to an X-autosome breakpoint in human malignancy and such a mechanism may underlie the pathogenesis of related disorders with translocations involving Xq13.

Size matters: the prognostic implications of large and small deletions of the derivative 9 chromosome in chronic myeloid leukemia.

Deletions of the derivative 9 chromosome (der(9)) are associated with poor prognosis in chronic myeloid leukemia (CML). Several models have been proposed to account for this association. To distinguish between the various models we mapped the deletion in 69 Philadelphia-positive CML patients carrying a der(9) deletion and compared the size of the deletion with the patients' outcome. Our results demonstrate that patients with large deletions had a significantly worse survival than those with small deletions whereas the outcome for patients with small deletions was similar to that of patients lacking a deletion. These results support the tumor suppressor gene model for the pathogenesis of der(9) deletions, argue against alternative models and provide insight into candidate gene location.

Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.

BACKGROUND: Human myeloproliferative disorders form a range of clonal haematological malignant diseases, the main members of which are polycythaemia vera, essential thrombocythaemia, and idiopathic myelofibrosis. The molecular pathogenesis of these disorders is unknown, but tyrosine kinases have been implicated in several related disorders. We investigated the role of the cytoplasmic tyrosine kinase JAK2 in patients with a myeloproliferative disorder. METHODS: We obtained DNA samples from patients with polycythaemia vera, essential thrombocythaemia, or idiopathic myelofibrosis. The coding exons of JAK2 were bidirectionally sequenced from peripheral-blood granulocytes, T cells, or both. Allele-specific PCR, molecular cytogenetic studies, microsatellite PCR, Affymetrix single nucleotide polymorphism array analyses, and colony assays were undertaken on subgroups of patients. FINDINGS: A single point mutation (Val617Phe) was identified in JAK2 in 71 (97%) of 73 patients with polycythaemia vera, 29 (57%) of 51 with essential thrombocythaemia, and eight (50%) of 16 with idiopathic myelofibrosis. The mutation is acquired, is present in a variable proportion of granulocytes, alters a highly conserved valine present in the negative regulatory JH2 domain, and is predicted to dysregulate kinase activity. It was heterozygous in most patients, homozygous in a subset as a result of mitotic recombination, and arose in a multipotent progenitor capable of giving rise to erythroid and myeloid cells. The mutation was present in all erythropoietin-independent erythroid colonies. INTERPRETATION: A single acquired mutation of JAK2 was noted in more than half of patients with a myeloproliferative disorder. Its presence in all erythropoietin-independent erythroid colonies demonstrates a link with growth factor hypersensitivity, a key biological feature of these disorders. RELEVANCE TO PRACTICE: Identification of the Val617Phe JAK2 mutation lays the foundation for new approaches to the diagnosis, classification, and treatment of myeloproliferative disorders.

Characterization of the imprinted polycomb gene L3MBTL, a candidate 20q tumour suppressor gene, in patients with myeloid malignancies.

Chromosome 20q deletion is a recurrent chromosomal abnormality associated with myeloid malignancies. L3MBTL represents a strong candidate tumour suppressor gene since it lies within the common deleted region, is a member of the Polycomb-like family, encodes the human homologue of a Drosophila tumour suppressor and is expressed within haematopoietic progenitor cells. We describe the structure of L3MBTL, identify two putative promoters each associated with two CpG islands and characterize a complex pattern of alternative splicing events. Mutation analysis of the gene in patients with and without a 20q deletion identified several polymorphisms but no acquired mutations. The two CpG islands spanning promoter 2 undergo monoallelic methylation in normal haematopoietic cells consistent with imprinting of L3MBTL. Samples from patients with a 20q deletion retained either the methylated or unmethylated allele but retention of the methylated allele did not correlate with reduction in L3MBTL mRNA levels. The absence of a correlation between L3MBTL methylation and transcription could be shown to reflect loss of imprinting in one patient. In addition, our results demonstrate that inactivation of L3MBTL is not a common occurrence in patients with a 20q deletion or in cytogenetically normal patients with polycythaemia vera.

Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies.

L3MBTL encodes a member of the Polycomb family of proteins, which, together with Trithorax group proteins, is responsible for the coordinated regulation of patterns of gene activity. Members of the Polycomb family also regulate self renewal of normal and malignant hematopoietic stem cells. L3MBTL lies in a region of chromosome 20, deletion of which is associated with myeloid malignancies and represents a good candidate for a 20q target gene. However, mutations of L3MBTL have not been identified in patients with 20q deletions or in cytogenetically normal patients. Here we demonstrate that monoallelic methylation of two CpG islands correlates with transcriptional silencing of L3MBTL, and that L3MBTL transcription occurs from the paternally derived allele in five individuals from two families. Expression of the paternally derived allele was observed in multiple hematopoietic cell types as well as in bone marrow derived mesenchymal cells. Deletions of 20q associated with myeloid malignancies resulted in loss of either the unmethylated or methylated allele. Our results demonstrate that L3MBTL represents a previously undescribed imprinted locus, a vertebrate Polycomb group gene shown to be regulated by this mechanism, and has implications for the pathogenesis of myeloid malignancies associated with 20q deletions.