Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Pathogenesis of polycythaemia vera.

Polycythaemia vera (PV) is thought to result from clonal expansion of a transformed multipotent stem cell. Progenitors from patients with PV display abnormal responses to several growth factors, suggesting the presence of a defect in a signalling pathway common to different growth factors. A number of approaches are now focused on defining the molecular lesion or lesions. Identification of causal genes will be of considerable interest both to clinicians, who currently lack a specific and sensitive diagnostic test, and to scientists interested in fundamental issues of stem cell behaviour.

Myeloproliferative disorders.

The myeloproliferative disorders (MPDs) are a group of pre-leukaemic disorders characterized by proliferation of one or more lineages of the myelo-erythroid series. Unlike the Philadelphia chromosome in chronic myeloid leukaemia, there is no pathognomonic chromosomal abnormality associated with the MPDs. Chromosomal abnormalities are seen in 30-40% of patients with polycythaemia vera (PV) and idiopathic myelofibrosis (IMF) and seem to indicate a poor prognosis. On the other hand, chromosomal abnormalities are rare in essential thrombocythaemia. Consistent acquired changes seen at diagnosis include deletion of the long arm of chromosome 20, del(13q), trisomy 8 and 9 and duplication of parts of 1q. Furthermore del(20q), trisomy 8 and dupl(lq) all arise in multipotent progenitor cells. Molecular mapping of 20q deletions and, to some extent, 13q deletions has identified a number of candidate target genes, although no mutations have yet been found. Finally, translocations associated with the rare 8p11 myeloproliferative syndrome and other atypical myeloproliferative disorders have permitted the identification of a number of novel fusion proteins involving fibroblast growth factor receptor-1.

The role of molecular genetic analysis within the diagnostic haemato-oncology laboratory.

The identification of the molecular genetic basis to many haematological malignancies along with the increased use of molecularly targeted therapy has heralded an increasing role for molecular genetic-based techniques. Demonstration of acquired changes such as the JAK2 V617F mutation within myeloproliferative neoplasms has quickly moved from a research setting to the diagnostic laboratory. Disease-specific genetic markers, such as the BCR-ABL1 fusion gene in chronic myeloid leukaemia, enable sensitive molecular genetic methods to be applied for the detection and quantification of low-level residual disease, allowing early identification of relapse. Consequently, molecular genetics now plays a crucial role in diagnosis, the identification of prognostic markers and monitoring of haematological malignancies. The development of high-throughput whole-genome approaches offers the potential to rapidly screen newly diagnosed patients for all disease-associated molecular genetic changes.

Molecular genetic analysis of haematological malignancies II: Mature lymphoid neoplasms.

Molecular genetic techniques have become an integral part of the diagnostic assessment for many lymphomas and other chronic lymphoid neoplasms. The demonstration of a clonal immunoglobulin or T cell receptor gene rearrangement offers a useful diagnostic tool in cases where the diagnosis is equivocal. Molecular genetic detection of other genomic rearrangements may not only assist with the diagnosis but can also provide important prognostic information. Many of these rearrangements can act as molecular markers for the detection of low levels of residual disease. In this review, we discuss the applications of molecular genetic analysis to the chronic lymphoid malignancies. The review concentrates on those disorders for which molecular genetic analysis can offer diagnostic and/or prognostic information.

Molecular genetic analysis of haematological malignancies: I. Acute leukaemias and myeloproliferative disorders.

Molecular genetic techniques are now routinely applied to haematological malignancies within a clinical laboratory setting. The detection of genetic rearrangements not only assists with diagnosis and treatment decisions, but also adds important prognostic information. In addition, genetic rearrangements associated with leukaemia can be used as molecular markers allowing the detection of low levels of residual disease. This review will concentrate on the application of molecular genetic techniques to the acute leukaemias and myeloprolferative disorders.

Mutation and methylation analysis of the transforming growth factor beta receptor II gene in polycythaemia vera.

Polycythaemia vera (PV) is a myeloproliferative disorder (MPD) thought to result from transformation of a haemopoietic stem cell. Transforming growth factor beta1 (TGF-beta1) is a negative regulator of haemopoietic stem cells, an effect mediated by direct binding to TGF-beta receptor II (TGF-beta RII). Reduced levels of TGF-beta RII mRNA or protein have been reported in several MPDs including PV, suggesting a role for TGF-beta RII in PV. No mutational analysis of the TGF-beta RII gene has yet been performed in PV. To investigate whether genetic or epigenetic alteration of the TGF-beta RII gene contributes to the pathogenesis of PV, we performed mutation and methylation analysis in 15 PV patients. The promoter, all seven exons and all intron/exon junctions were studied using single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA). In total, three single nucleotide polymorphisms (SNPs) were identified. These were located in the promoter, intron 2 and exon 5. No acquired mutations were detected in any patient sample. We also present a novel method, termed methylation-specific strand extension (MSSE), for the detection of methylated CpG dinucleotides. The combination of bisulphite modification and MSSE permits rapid analysis of the methylation status of CpG dinucleotides in multiple samples. We analysed the methylation status of the promoter and of a CpG island within exon 1 in 15 PV patients. No aberrant methylation was detected in either of these regions. These data demonstrate that neither mutation nor abnormal methylation of the TGF-beta RII gene is associated with the pathogenesis of PV. Furthermore, MSSE is a rapid and robust approach for assessing the methylation status of a given genomic region.

Molecular genetics and cytogenetics of myeloproliferative disorders.

The myeloproliferative disorders are believed to represent clonal malignancies resulting from transformation of a pluripotent stem cell. X-inactivation patterns of peripheral blood cells have been proposed as a useful diagnostic tool but this method is limited by the finding of a clonal X-inactivation pattern in a significant proportion of normal elderly women. There is no pathognomonic chromosomal abnormality associated with the myeloproliferative disorders. However, consistent acquired cytogenetic changes include del(20q), del(13q), trisomy 8 and 9 and duplication of segments of 1q, all of which have been observed at diagnosis or before cytoreductive therapy and therefore represent early lesions which contribute to the pathogenesis of these disorders. Although, the acquired molecular defects underlying most myeloproliferative disorders have not yet been elucidated, translocations associated with the rare 8p11 syndrome have permitted identification of a novel fusion protein. The role of a number of candidate genes in the other myeloproliferative disorders has also been studied, but no mutations have been identified so far. It is likely that a number of genes will be involved, given the varied phenotypes of the diseases. Identification of causal genes will be of considerable interest to both clinicians, who currently lack a specific and sensitive diagnostic test, and scientists interested in fundamental issues of stem cell behaviour.

Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by formation of the BCR-ABL fusion gene, usually as a consequence of the Philadelphia (Ph) translocation between chromosomes 9 and 22. Large deletions on the derivative chromosome 9 have recently been reported, but it was unclear whether deletions arose during disease progression or at the time of the Ph translocation. Fluorescence in situ hybridization (FISH) analysis was used to assess the deletion status of 253 patients with CML. The strength of deletion status as a prognostic indicator was then compared to the Sokal and Hasford scoring systems. The frequency of deletions was similar at diagnosis and after disease progression but was significantly increased in patients with variant Ph translocations. In patients with a deletion, all Ph(+) metaphases carried the deletion. The median survival of patients with and without deletions was 38 months and 88 months, respectively (P =.0001). By contrast the survival difference between Sokal or Hasford high-risk and non-high-risk patients was of only borderline significance (P =.057 and P =.034). The results indicate that deletions occur at the time of the Ph translocation. An apparently simple reciprocal translocation may therefore result in considerable genetic heterogeneity ab initio, a concept that is likely to apply to other malignancies associated with translocations. Deletion status is also a powerful and independent prognostic factor for patients with CML. The prognostic significance of deletion status should now be studied prospectively and, if confirmed, should be incorporated into management decisions and the analysis of clinical trials.

Distinct 5' SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites.

The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions.

A detailed physical and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative disorders and refinement of the common deleted region.

Acquired deletions of the long arm of chromosome 20 are the most common chromosomal abnormality seen in polycythemia vera and are also associated with other myeloid malignancies. Such deletions are believed to mark the site of one or more tumor suppressor genes, loss of which perturbs normal hematopoiesis. A common deleted region (CDR) has previously been identified on 20q. We have now constructed the most detailed physical map of this region to date--a YAC contig that encompasses the entire CDR and spans 23 cM (11 Mb). This contig contains 140 DNA markers and 65 unique expressed sequences. Our data represent a first step toward a complete transcriptional map of the CDR. The high marker density within the physical map permitted two complementary approaches to reducing the size of the CDR. Microsatellite PCR refined the centromeric boundary of the CDR to D20S465 and was used to search for homozygous deletions in 28 patients using 32 markers. No such deletions were detected. Genetic changes on the remaining chromosome 20 may therefore be too small to be detected or may occur in a subpopulation of cells.