Glutathione S-transferase theta 1 (GSTT1) gene defect in myelodysplasia and acute myeloid leukaemia.
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Acute Disease↗
Biomedical subjects
R E Gale
Publications and source records attributed to R E Gale.
Frequency of CBF beta/MYH11 fusion transcripts in patients entered into the U.K. MRC AML trials. The MRC Adult Leukaemia Working Party.
It has been established that cytogenetic findings at diagnosis of acute myeloid leukaemia (AML) are a powerful prognostic indicator. Patients who have the inv(16)(p13q22), closely associated with the FAB subtype M4Eo. are deemed to have good-risk disease. This subtle translocation may be difficult to detect in poor-quality metaphase preparations and if missed could lead to the incorrect assignment of risk group and influence further treatment strategies. We studied 321 patients with AML at diagnosis for the presence of inv(16)(p13q22) and CBF beta/MYH11 fusion transcripts by cytogenetic and RT-PCR techniques respectively. Karyotypic analysis detected 21 cases of inv(16) (p13q22), all of which were PCR positive. A further 12 cases were detected at the molecular level only, in FAB types other than M4Eo. The observed frequencies of CBF beta/MYH11 fusion transcripts in our study have been adjusted for the reported incidence of each FAB subtype and we calculate that 10.1% of all new cases of AMLs have molecular evidence of inv(16)(p13q22). only half of which are of the M4Eo subtype. We conclude that molecular screening for the presence of CBF beta/MYH11 fusion transcripts should be mandatory in all case of AML at diagnosis.
Adolescent↗
Analysis of the coding sequence for the GM-CSF receptor alpha and beta chains in patients with juvenile chronic myeloid leukemia (JCML).
Peripheral blood granulocyte-macrophage progenitors (CFU-GMs) from patients with juvenile chronic myeloid leukemia (JCML) are able to proliferate spontaneously in the absence of exogenous growth factors and studies have shown that these progenitors display a hypersensitive growth response to GM-colony-stimulating factors (CSFs) in culture. We screened RNA from six patients with JCML for mutations in the GM-CSF receptor (GM-CSFR) coding sequence using RT-PCR-SSCP. Altered patterns were found in five patients for the GM-CSFR alpha chain, and in five patients for the beta chain. Two nucleotide substitutions accounted for all alpha chain abnormalities; one was conservative for Val333 and the other caused an Ala17-->Gly substitution. Both had previously been detected in normal controls. Sequencing of beta chain abnormalities revealed four base substitutions. Three were previously described polymorphisms, one causing a Pro603-->Thr substitution and two conservative point mutations involving Ser426 and Pro648. A further nucleotide mutation, which resulted in a Glu249-->Gln substitution, was also found but is not thought to be of pathological significance. Polymorphisms of the GM-CSFR alpha and beta chains are common, but pathogenic point mutations of the GM-CSFR are infrequent in patients with JCML and are unlikely to be involved in the hypersensitivity to GM-CSF demonstrated by progenitor cells.
Humans↗
Demonstration of developing myelodysplasia/acute myeloid leukaemia in haematologically normal patients after high-dose chemotherapy and autologous bone marrow transplantation using X-chromosome inactivation patterns.
Autologous bone marrow or peripheral blood stem cell transplantation may carry an increased risk of secondary myelodysplasia (MDS) and acute myeloid leukaemia (AML), which are already recognized as complications of conventional treatment for lymphoid malignancies. In order to ascertain whether it is possible to detect the evolution of such a clone at an early stage in its development we have studied X-chromosome inactivation patterns (XCIPs) in three informative females who developed abnormal myelopoiesis after high-dose chemotherapy and ABMT. In one patient transplanted for relapsed Hodgkin's disease a leukaemic clone comprising approximately 50% of the patient's myeloid cells was detectable by comparison of peripheral blood granulocyte and T-cell XCIPs when the full blood count and morphology were normal. She presented with AML 7 months later. In two patients transplanted for AML, XCIP analysis was complicated by constitutively skewed Lyonization patterns, nevertheless a progressive alteration could be demonstrated by serial analyses. In one patient a difference was detectable 28 months before presentation with MDS. In the other patient, despite evident mild pancytopenia and alterations in her XCIPs over the past 4 years, she has developed no definitive myelodysplastic features and oligoclonality due to stem cell failure cannot be excluded. These studies show that XCIPs can be used to predict development of MDS/AML in some patients, but the technique is limited by technical variability and frequent constitutional skewing in the haemopoietic system.
Acute Disease↗
Analysis of granulocyte colony stimulating factor receptor isoforms, polymorphisms and mutations in normal haemopoietic cells and acute myeloid leukaemia blasts.
Deletion mutants of the intracytoplasmic domain of the granulocyte colony stimulating factor receptor (G-CSFR) have shown that it contains a membrane-proximal region which must be conserved to allow the receptor to transduce a mitotic signal, and a C-terminal region necessary for transduction of cell differentiation. Changes in the intracytoplasmic domain may result in the uncoupling of these two processes, as in acute leukaemia, and such alterations could occur either as isoforms or mutations. We have studied the transmembrane domain and intracytoplasmic tail of the G-CSFR in RNA from blood or bone marrow of 11 haematologically normal controls and 40 patients with acute myeloid leukaemia (AML). Two novel transcripts of the receptor were identified, both were minor components and are unlikely to be of major physiological significance. We could find no evidence for altered levels of expression of these transcripts in the AML patients. In addition, only one point mutation was detected in the 40 patients screened by RT-PCR-SSCP, a C-->A substitution at nucleotide 2088 which changes a threonine to asparagine in the transmembrane domain and is probably a polymorphism. These results suggest that abnormalities in the G-CSFR are uncommon in AML.
Alternative Splicing↗
The retinoblastoma gene (rb1) in acute myeloid leukaemia: analysis of gene rearrangements, protein expression and comparison of disease outcome.
The occurrence of retinoblastoma gene abnormalities in a large subset of various malignancies suggests an important role for this tumour suppressor gene in carcinogenesis, but this varies considerably from one tumour type to another and results in patients with acute myeloid leukaemia (AML) have been controversial. We analysed 106 AML patients and 18 normal controls for RB1 gene rearrangements and 86 AML patients for RB protein (pRB) expression. Southern blot analysis detected no gross gene rearrangements, but several restriction enzyme polymorphisms were observed. By Western blot analysis, 20 patients (23%) had no detectable pRB protein and seven (8%) had truncated pRB bands. Discordance between the DNA and protein data suggests that there may be minor deletions and point mutations in the RB1 gene or abnormalities in the proteins regulating the expression of pRB. No significant differences in the frequency of attainment of complete remission or length of survival were observed between patients with normal and abnormal pRB.
Acute Disease↗
Amplification of mitochondrial DNA in acute myeloid leukaemia.
There is a long-standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML). We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2-50 were observed in 25 AML samples and 13 of these showed 8-fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3-10-fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.
Acute Disease↗
The beta subunit common to the GM-CSF, IL-3 and IL-5 receptors is highly polymorphic but pathogenic point mutations in patients with acute myeloid leukaemia (AML) are rare.
The intracytoplasmic tail of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) beta c chain is essential for the activation of ligand-mediated signal transduction pathways in myeloid cells. Alterations in this region could deregulate normal signalling processes. We have therefore used RT-PCR-SSCP analysis of the receptor tail to look for point mutations in RNA from 35 patients with acute myeloid leukaemia (AML) and 10 haematologically normal controls. Patterns differing from those of the haemopoietic cell line TF-1 were detected in 25/35 (71%) AML patients and 8/10 (80%) normal controls. A total of six base substitutions were identified by sequencing. Three were conservative for the amino acid involved, three led to amino acid differences, valine652-->methionine, glycine647-->valine and proline603-->threonine. One alteration was found only in a normal control, the other five were all found in both AML patients and normal controls suggesting that they were DNA polymorphisms. Two substitutions were particularly common with allele frequencies of 0.23 (G1972-->A, unchanged proline648) and 0.13 (C1306-->T, unchanged serine426). These results indicate that the GM-CSFR beta c chain is highly polymorphic but point mutations of the intracytoplasmic tail do not appear to contribute frequently to the pathogenesis of AML.
Base Sequence↗
Variable expression of p16 protein in patients with acute myeloid leukemia without gross rearrangements at the DNA level.
The p16 protein is an inhibitor of cyclin-dependent kinases (cdk) 4 and 6. Both cdk4 and cdk6 phosphorylate the retinoblastoma protein (pRb) and are thought to be required for cell proliferation. Mutations and homozygous deletions in the p16 gene have been found in a number of cell lines but the incidence of abnormalities in primary tumours is controversial. We have studied the p16 gene in 76 cases of acute myeloid leukemia (AML) and 18 hematologically normal controls by quantitative Southern blotting. No deletions or rearrangements were detected. Twenty-five cases also showed no abnormal band patterns by RT-PCR-SSCP analysis of the coding sequence. We analyzed 60 AML samples for p16 protein expression by Western blot analysis. A reduced level of p16 was observed in six cases, however, none of them showed methylation of the 5'-CpG island. Over-expression of p16 protein was detected in six cases. Studies in cell lines have suggested a feedback loop between p16 and pRb with a futile overproduction of p16 protein in cells containing abnormal pRb. In accordance with these findings, four of the AML cases with high levels of p16 had abnormal pRb (two alteration in band size, two absent). From our data we suggest that gross abnormalities in the p16 gene are rare in AML, but that p16 levels are variable and high levels are associated with pRb abnormalities in a subset of cases.
Acute Disease↗
Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA.
The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies.
Acute Disease↗
Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay.
Quantification of X-chromosome inactivation patterns (XCIPs) using PCR amplification of the human androgen receptor (HUMARA) locus is potentially valuable in a range of haematological disorders. Of 236 females screened, 203 (86%) were heterozygous. For quantitative XCIPs it was necessary to limit the number of PCR cycles to 20 to reduce preferential amplification of shorter alleles. The optimized PCR method was compared with Southern blotting results using either PGK, HPRT or M27beta in 51 haematologically normal females and blast cells from 27 patients with acute myeloid leukaemia (AML). Reproducible XCIP results were obtained in all 78 samples using digestion with Hpa II prior to amplification (median difference in duplicate values 3%, range 0-17%) and they correlated well with Southern blotting results, r=0.966. Greater variability was observed in the results using Hha I digestion (median difference 4%, range 0-48%). There were marked inconsistencies in repeated analyses of three AML samples and although the HUMARA-Hha I results correlated well overall with Southern blotting in the remaining 75 samples (r=0.922), in nine samples there were still discrepancies with > or = 20% difference between the two values. These results suggest that PCR analysis of the HUMARA locus in Hpa II-digested DNA is suitable for the quantification of XCIPs in haematological samples but results with Hha I should be treated with caution.
Alleles↗
Interpretation of X-chromosome inactivation patterns.
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Clone Cells↗
Tissue specificity of X-chromosome inactivation patterns.
The analysis of X-chromosome inactivation patterns has been used in a number of clinical situations such as the identification of carrier status in X-linked genetic disorders and the establishment of the monoclonal origin of tumors. Interpretation of the result obtained requires comparison with the constitutive pattern for the individual, and for hematopoietic malignancies, skin biopsies or cultured fibroblasts have often been used as the control tissue because normal cells of the same lineage as the malignancy are not generally available. However, this assumes that patterns in the different tissues are constitutionally the same. We have therefore compared X-chromosome inactivation patterns from peripheral blood (granulocytes, E- cells, and T cells), skin, and muscle from 20 hematologically normal females, and colonic mucosa from 9 individuals. In 11 patients (55%), the results obtained were similar for all tissues of an individual, but in 9 patients, significant differences were observed between tissues. The most consistent feature was a skewing in peripheral blood (> 75% expression of one allele) but not skin and/or muscle. These studies suggest that skin cannot be used as a control tissue for the interpretation of X-chromosome inactivation patterns in hematopoietic cells.
Adolescent↗
Lineage involvement in chronic myeloproliferative disorders.
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Chronic Disease↗
Analysis of mutations in the GM-CSF receptor alpha coding sequence in patients with acute myeloid leukaemia and haematologically normal individuals by RT-PCR-SSCP.
Mutations of signal transducing molecules such as Ras have been shown to confer a growth advantage in leukaemic blasts and contribute to the pathogenesis of the disease. Alterations of signal transducing molecules other than Ras may play a role in leukaemogenesis. Knowledge of such mutations could also further our understanding of the normal signalling processes. We have therefore studied the coding sequence of the GM-CSF receptor alpha chain (GM-CSFR alpha) in patients with acute myeloid leukaemia (AML) and non-AML controls using single strand conformation polymorphism (SSCP) analysis. Abnormalities were detected in 4/32 AML patients (13%) and 2/15 non-AML controls (13%). Direct sequencing of PCR products revealed five different base substitutions. Three were conservative, two caused amino acid changes. The base substitution leading to amino acid change alanine to glycine at position 17 was found in both an AML patient and a control. It lies in the signal sequence and does not affect the mature protein. The other base change altering arginine to glutamine at position 164 is unlikely to influence the receptor structure as this structural position in the chain is not well conserved in members of the cytokine receptor family. Both amino acid changes were constitutive alterations as they could be demonstrated in the patients' children. The base changes described in the AML patients thus represent polymorphisms and do not contribute to the pathogenesis of AML.
Adolescent↗
Clonality studies in haemopoiesis.
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Animals↗
Investigation of methylation at Hha I sites using the hypervariable probe M27 beta allows improved clonal analysis in myeloid leukaemia and demonstrates differences in methylation between leukaemic and remission samples.
The methylation-sensitive enzyme Hha I has been used to assess the differentially methylated patterns on active and inactive X-chromosomes at the DXS255 locus recognized by the hypervariable probe M27 beta. The X-chromosome inactivation ratios obtained from 37 haematologically normal females using PstI and HhaI and correlated well with results obtained using PstI Hpa II (r = 0.97), and in 19 individuals with values obtained probing for either phosphoglycerate kinase or hypoxanthine phosphoribosyl transferase (r = 0.92). At least one Hha I site was found to be unmethylated on all alleles on inactive X-chromosomes. A monoclonal or oligoclonal pattern could be obtained by digestion with Hha I in 18/22 (82%) patients with acute myeloid leukaemia who had previously shown hypermethylation of both alleles using Hpa II, although in six of these patients differences in methylation could still be demonstrated between leukaemic and remission samples.
Acute Disease↗
Frequency of clonal remission in acute myeloid leukaemia.
Analysis of X-chromosome inactivation patterns in females has been used to assess clonality of various tumours and for prenatal diagnosis of X-linked disorders. Studies with these methods in acute myeloid leukaemia suggest that a significant proportion of cases have clonal remissions (ie, persistence of the malignant clone), which may represent return to a preleukaemic state. We therefore analysed X-chromosome inactivation patterns with differential methylation patterns of heterozygotes for three DNA probes, HPRT, PGK, and M27 beta, in leukaemic patients and normal controls. As expected, blast cells from 67 of 68 analysable samples (99%) were monoclonal or had a skewed X-inactivation pattern. A skewed pattern in remission was also found in 26 of 77 patients (34%), proportion only slightly greater than control (16/75, 21%). In 7 of 10 patients with a skewed pattern in myeloid cells there was similar skewing in the T cells, which is compatible with the concept of a constitutively skewed X-chromosome inactivation pattern of haemopoietic cells in these patients. Our study illustrates the difficulty of interpreting clonality in individual tumour samples and emphasises the importance of comparisons with non-malignant tissue of the same cell type from that individual and from normal control populations.
Acute Disease↗