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X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

Objective: To describe the analysis of over 5300 patient samples for the HFE genotype. Methods: Blood samples received from hospitals in England, Wales and Ireland were analysed with a single, multiplex PCR using heteroduplex generators for the C282Y, H63D and S65C variants of the HFE gene. PCR products labelled with fluorescent dyes were analysed by capillary electrophoresis. Genotype frequencies were analysed according to the reasons given for testing. Results: Analysis of 400 samples sent in duplicate revealed one error that was associated with reporting rather than the methodology. Of 5327 samples received, 1122 were for family testing, 2470 for diagnostic testing and in 1735 cases no reason was given. Overall, homozygosity for C282Y was found in 14% of samples received for family testing and in 16% of the remaining samples. Clinical indications such as “liver disease” were of little predictive value for homozygosity for C282Y, but this increased if a raised serum ferritin concentration or transferrin saturation was indicated. When the diagnosis was iron overload, 39% of subjects tested were homozygous for C282Y. Compound heterozygosity (C282Y/H63D) was more frequent than in the general population but the frequency was not further increased in subjects for whom there was a diagnosis of iron overload. The frequencies of heterozygosity for H63D or S65C and homozygosity for H63D were not significantly increased in any group compared with the general population frequency. Conclusion: These results demonstrate the reliability of the methodology and confirm the difficulty of identifying genetic haemochromatosis purely on the basis of clinical suspicion that haemochromatosis may be responsible for liver disease, diabetes or arthritis.

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]–independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34+ erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all-trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto-oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA-resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA-sensitive and -resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.

Background Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved.Design and Methods In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene.Results Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders.Conclusions Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein.

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1α,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to `anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

The most common single mutation observed in approximately one-third of AML cases, is an activating mutation of the tyrosine kinase receptor FLT3. The presence of the mutation is associated with adverse prognosis in terms of increased risk of disease relapse. The majority of mutations (25–35% of AML patients) occur as an internal tandem duplication (ITD) within the juxtamembrane region. A further 3–7% of patients have a mutation within the tyrosine kinase domain (TKD) of FLT3. The type of mutation, and its population size, may have prognostic implications. We have used the Agilent 2100 Bioanalyser to assay PCR reactions for the ITD and the TKD mutations. The “chips” used within the Bioanalyser separate RNA or DNA through glass etched micro channels filled with gel matrix, nucleic acid is stained and laser detected. The PCR primers and protocol for the ITD analysis were as previously published (Kottaridis et al. Blood. 98, 2001), but within our laboratory the reactions were analysed on the Bioanalyser using a DNA 500 chip. We detected the presence of an ITD in 19.9% of the 291 AML samples, with percent of mutant RNA varying from 2.9% to 97.5% (median 27%) and with the size of the ITD varying from 15–212 bp (median 41 bp). A separate PCR reaction using primers flanking the TKD domain was used, in combination with an Eco RV or Hinf I (Smith et al, MTAL Workshop, 2003) digestion, to detect and quantitate the different mutations. Distinctive digestion and/or heteroduplex patterns were observed that identified D835, D835del, I836del or D839G mutation. TKD mutations were observed in 12.9% (10.1% D835 mutations, 1.4% I836del, 1.4% D839G) of samples examined, with the mutant RNA ranging from 1.0% to 52.8%. The D835/6 mutation rate was higher than previously reported and may be due to the efficiency and sensitivity of the 2100 Bioanalyser for the analysis of FLT3 mutations. This gave an overall FLT3 mutation rate in the AML population studied of approximately 32.8%; females had a higher overall incidence of 39.1% compared to 27.2% for males. The samples were derived from two NCI trials, AML 14 for patients over 60 years old and AML15 for younger patients. The rate of ITD mutations was 15.3% in AML14 patients compared to 27.5% in AML15, although the incidence of TKD mutations was similar (13.5% v 14.3%). The incidence of ITD mutations was over 45% in patients in the 30–40 year age group at diagnosis. A large percentage of these patients have also been gene expression profiled and the molecular effect of the FLT3 ITD mutations across the age groups has also been examined.