T. R. Rutherford

The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking

Fanconis anaemia (FA) is characterized by increased spontaneous and induced chromosome fragility. This has been widely regarded to be due to a defect in DNA crosslink repair, because of the sensitivity of cells to known DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Although Fanconi cells are also sensitive to molecular oxygen, and may be protected by antioxidants, this has generally been considered to be a secondary phenomenon. However, it has recently been demonstrated that the FAC protein, coded for by the Fanconi anaemia gene for complementation group C, is strictly cytoplasmic and does not enter the nucleus even after DNA damage, which seems inconsistent with a role in DNA repair.

The role of occupational and environmental exposures in the aetiology of acquired severe aplastic anaemia: a case control investigation

Summary.  Aplastic anaemia is a rare but serious disorder with a high morbidity and mortality rate. The causes of aplastic anaemia are, for the most part, unknown. We report on the hypothesis that aplastic anaemia may be caused by occupational and/or environmental exposures to certain chemicals. The UK Aplastic Anaemia Study was an interview‐based case–control study covering the whole of Great Britain. Those patients diagnosed between 1 July 1993 and 20 October 1997, aged ≤75 years and born and diagnosed in the UK were eligible for the study. Two hundred eligible cases of aplastic anaemia were compared with 387 age‐ and sex‐matched controls. A number of occupational exposures showed increases in risk. In a multivariate model of these exposures the odds ratios (ORs) for solvents/degreasing agents, pesticides and radiation were >2 and statistically significant. Reported chemical treatment of houses within 5 years of diagnosis had a significantly raised risk for adults [OR = 2·51, 95% confidence interval (CI) 1·02–12·01], particularly for woodworm treatment (OR = 5·1, 95% CI 1·5–17·4). This study identified significant risks associated with self‐reported exposure to solvents, radiation and pesticides in the workplace. Self‐reported chemical treatment of houses was also associated with an increased risk of developing aplastic anaemia, in keeping with previous literature.

Bcl‐2 and Bcl‐x expression in the CD34+ cells of aplastic anaemia patients: relationship with increased apoptosis and upregulation of Fas antigen

Aplastic anaemia (AA) is a syndrome of haemopoietic failure involving increased apoptosis in stem cells. AA CD34+ cells often have upregulated Fas antigen, but this does not explain the increased apoptosis in all patients. To examine whether abnormal expression of the apoptotic modulators Bcl‐2 and Bcl‐x is involved in increased apoptosis in the CD34+ cells of patients, we examined cells from 19 AA patients and 18 normal controls by triple staining for CD34, Bcl‐2 or Bcl‐x, together with 7‐amino actinomycin D to determine viability or with staining for Fas antigen. We confirmed increased apoptosis of CD34+ cells in patients. All CD34+ cells in patients and controls expressed Bcl‐2 and Bcl‐x with no significant difference between the groups. In patients, viability of CD34+/Bcl‐2hi cells was similar to that of CD34+/Bcl‐2lo cells, but CD34+/Bcl‐xhi cells were significantly more viable than CD34+/Bcl‐xlo cells. CD34+ cells from AA patients expressed upregulated Fas antigen, but this did not correlate with Bcl‐2 or Bcl‐x expression. These results suggest a more significant role for Bcl‐x as an anti‐apoptotic regulator in CD34+ cells in AA than Bcl‐2. The induction of death by Fas antigen may bypass the anti‐apoptotic effect of Bcl‐2 and Bcl‐x in CD34+ cells in AA.

Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia

Defects in stromal cell function have been demonstrated in a number of aplastic anaemia (AA) patients. Here we have studied a patient with severe AA and abnormal stromal cell function who underwent bone marrow transplantation (BMT). The objective of this study was to investigate the timing and the mechanism of correction of the stromal defect after transplantation. The patient, a 25‐year‐old woman with severe AA, underwent BMT from her brother. BM was obtained from the patient on five occasions: 2 weeks pre BMT, and 3, 8, 16 and 21 months post BMT. Stromal cells were grown to confluence and recharged with purified CD34+ cells from normal donors. The support of such cells, as assessed by weekly colony‐forming assay (CFU) of non‐adherent cells, was compared with that of stromal layers grown from normal BM. A novel technique of combined fluorescence in situ hybridization (FISH) and immunocytochemistry was used to determine the origin of specific stromal cell types on cytospins of stroma post BMT. Stromal function was defective at 2 weeks pre BMT and at 3 months post BMT, but returned to normal at 8 and 16 months post BMT. At 21 months post BMT, stromal fibroblasts and endothelial cells were shown to be of recipient origin, and macrophages and T cells were of donor origin. We present here evidence in a case of severe AA for defective stromal function before BMT and delayed normalization of function after BMT. This correlated with engraftment of donor macrophages and T cells, but not fibroblasts and endothelial cells.

Clonal patterns of X‐chromosome inactivation in female patients withaplastic anaemia studied using a novel reverse transcription polymerase chain reaction method

Abstract: Conflicting results have been published on the frequency of clonal patterns of X‐chromosome inactivation in female patients with aplastic anaemia. Previous studies have used DNA methylation to measure X‐inactivation, but aberrant methylation is known to occur in some situations. We have developed a non‐radioactive reversetranscription polymerase chain reaction (RT‐PCR) method to study expression of the polymorphism at nt.1311 of the G6PD gene at the RNA level. Using this, and a similar method for the iduronate‐2‐sulfatase (IDS) gene, we have re‐evaluated X‐inactivation in AA patients. 32/35 normal individuals showed polyclonal haemopoiesis. Patients with presumed clonal diseases showed both monoclonal and polyclonal patterns, consistent with previous reports. Overall, clonal patterns were observed in granulocytes of 10/26 AA patients (38%), a significantly higher proportion than in controls (p<0.01). Two cases showed discordance between lymphocytes and granulocytes, indicating clonality arising within the myeloid lineage. Eight cases showed clonal patterns in both myeloid and lymphoid cells, indicating the involvement of a pluripotent stem cell. Clonal patterns did not correlate with age, but there appeared to be an association with duration of disease. In PNH patients, CD59‐negative cells showed clonal patterns of X‐inactivation. In two cases, however, clonal patterns were also detected in CD59‐positive cells.

Paroxysmal nocturnal haemoglobinuria due to an 88 bp direct tandem repeat insertion in the PIG‐A gene

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired stem cell abnormality which frequently develops in patients with aplastic anaemia. The disease is due to somatic mutations in the PIG‐A gene, and a variety of mutations have been reported. The majority are point mutations, or small insertions and deletions resulting in a frameshift. Previous insertions reported have all been within the range of 1–10 bp. We describe here a patient with PNH due to a large insertion of 88 bp; DNA sequencing showed this to be a tandem repeat of PIG‐A sequences. The same mutation could be found in granulocytes and lymphocytes, indicating a pluripotent stem cell origin.

Persistence of an activating N‐RAS oncogene mutation in clonogenic progenitor cells from an acute myeloid leukaemia patient in remission

Summary A patient with acute myeloid leukaemia (AML) with an activating N‐RAS oncogene mutation was studied in a haemopoietic clonogenic progenitor cell assay. Individual colonies and clusters were analysed by polymerase chain reaction and oligonucleotide hybridization for the original mutation. The mutation was detected in a majority of leukaemic clusters, but also in almost half of the differentiated colonies. After chemotherapy the patient entered clinical remission. However, the mutation could still be detected in the bone marrow. Only differentiated colonies and no leukaemic clusters were grown from the remission bone marrow, but the original mutation was still detectable in almost half of the colonies.

Childhood polyclonal T cell lymphocytosis with neutropenia : effects of antilymphocyte globulin and granulocyte colony stimulating factor in vitro and in vivo

Summary The pathogenesis of the neutropenia that occurs in some patients with chronic T cell lymphocytosis is not well understood. We have investigated a 15‐year‐old girl with this syndrome. Initial committed bone marrow progenitor numbers (CFUgm) were low but markedly increased in vitro following T cell depletion. Similarly a transient correction of neutropenia was observed following in vivo lymphocyte depletion with antilymphocyte globulin. A sustained neutrophil recovery was achieved with daily therapy using recombinant human granulocyte colony stimulating factor (rhG‐CSF) despite persistence of the lymphocytosis; during successful therapy CFUgm numbers remained low, and were not increased by the in vitro addition of rhG‐CSF. These observations suggest the possibility of an inhibitory regulatory mechanism specifically acting on neutrophil granulopoiesis.