Graham R. Standen

Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Preliminary observations on possible premalignant changes in bone marrow adjacent to worn total hip arthroplasty implants

Previous epidemiologic studies have suggested that there may be a risk of malignancy, especially lymphoma and leukemia, after joint replacement, but the followup has been relatively short. This is a preliminary study to see if there is any biologic basis for such a risk. Blood and bone marrow samples from 71 patients at revision arthroplasty of a loose or worn prosthesis and 30 control patients at primary arthroplasty were analyzed with cytogenetic techniques and molecular biology. There was a higher chromosomal aberration rate in cells adjacent to the prosthesis at revision surgery compared with iliac crest marrow from the same patients or with femoral bone marrow at primary arthroplasty. Clonal expansion of lymphocytes without a serum paraprotein was seen in 2 of 21 patients at revision arthroplasty performed more than 10 years after primary arthroplasty. The results of this preliminary study suggest that future epidemiologic studies should concentrate on patients with longer postoperative intervals to see if there is any risk that would be pertinent to a young patient at primary arthroplasty.

An investigation of the association of the factor V Leiden mutation and inflammatory bowel disease

BACKGROUND A thrombotic aetiology for inflammatory bowel disease (IBD) has been proposed as a result of its association with thrombo-embolic complications, smoking, the oral contraceptive pill and the response of ulcerative colitis (UC) patients to heparin. The factor V Leiden (FVL) mutation is the commonest inherited risk factor for thrombo-embolism. AIM The aim of the study was to investigate the hypothesis that the pro-thrombotic state associated with the FVL mutation is involved in the aetiology of IBD. PATIENTS AND METHODS A prospective cohort study of patients attending the Bristol Royal Infirmary IBD outpatient clinic was performed. Fifty-four patients with IBD (30 with Crohns disease (CD) and 24 with UC) and 55 historical controls were screened for the presence of FVL using the activated protein C (APC) ratio. Abnormal APC ratios were confirmed to be due to FVL using a heteroduplex-based polymerase chain reaction (PCR) technique. RESULTS Five patients had the FVL mutation, compared to two controls. One of the patients was homozygous. Two of the patients had CD and three UC. The differences between controls and IBD patients was significant when the allelic frequency of the FVL mutation in patients with UC was compared with controls, with a risk ratio of 2.27, but with limited data. CONCLUSION There appears to be a weak association between FVL and UC. This association is not strong enough to imply a causal relationship, but may be responsible for some of the thrombo-embolic complications.

Reduced CD38 expression on CD34+ cells as a diagnostic test in myelodysplastic syndromes

This report indicates that a reduced mean fluorescence intensity of CD38 expression on CD34+ cells can be used as a surrogate marker for abnormalities in the CD34+ compartment of patients with myelodysplastic syndrome. See related perspective article on page 1041. Diagnosis of myelodysplastic syndrome can be difficult especially in cases with a low blast count and a normal karyotype. Flow cytometry has been used to distinguish myelodysplastic syndrome from non-clonal cytopenias. No one single simple flow cytometric parameter has been proposed to be diagnostic of myelodysplastic syndrome. We have studied samples from 100 myelodysplastic syndrome patients and as control samples; 70 non-clonal cytopenias, 5 subjects with normal hematology, 31 patients with acute myeloid leukemia and 11 with chronic myelomonocytic leukemia or myeloproliferative disorder. We show that reduced relative mean fluorescence of CD38 below a threshold value on CD34+ cells diagnosed low-grade myelodysplastic syndrome with 95% sensitivity (95% confidence interval, 87–99%) and 92% specificity (95% confidence interval, 82–97%). This simple flow cytometric test may be of value in the routine clinical diagnosis of myelodysplastic syndrome, especially in cases with a low blast count and normal karyotype.

Factor XIIIA calgary: a candidate missense mutation (Leu667Pro) in the beta barrel 2 domain of the factor XIIIA subunit

Summary. Molecular analysis performed on a Canadian family with congenital factor XIII deficiency revealed a homozygous missense mutation (Leu667Pro) in exon 14 of the A subunit gene in three affected siblings. The mutation results from a T‐to‐C transition at nucleotide position 2087 and generates a new Msp1 restriction site. Digestion of an amplified fragment containing exon 14 with this restriction enzyme enabled the heterozygous allele to be identified in both parents (who were third cousins) and three other family members. SSCP analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. The mutant nucleotide substitution was absent in 60 normal alleles and 10 unrelated patients with XIIIA deficiency. Leu667 is located in the carboxyl terminal beta barrel 2 domain of the A subunit molecule. Computer modelling based on 3D crystallographic data predicts that the mutant protein has aberrant folding and is likely to be rapidly degraded following translation.

Diagnosis of sickle-cell disease with a universal heteroduplex generator

A new diagnostic technique, universal heteroduplex generator screening, has been developed to detect homozygosity and heterozygosity for sickle-cell disease. Since it is a polymerase chain reaction-based technique, it may be used to detect haemoglobin S and haemoglobin C genotypes in adults, neonates, or from coelocentesis during the first 10 weeks of pregnancy. beta-globin gene nucleotide sequences are amplified by the polymerase chain reaction, and are heteroduplexed with a beta-globulin universal heteroduplex generator. Haemoglobins S and C mutations are identified by characteristic polyacrylamide minigel banding patterns. The technique is simple and rapid to do, even by nonspecialist laboratories, and is applicable to large-scale screening for haemoglobin S and C mutations as well as prenatal diagnosis of sickle cell disease.

Factor XIII ABristol 1: detection of a nonsense mutation (Arg171→+stop codon) in factor XIII A subunit deficiency

Summary Molecular analysis has been performed on a patient with coagulation factor XIII A subunit deficiency. A previously published genomic sequence indicates that exon 3 of the factor XIII A subunit gene contains two TaqI restriction sites within which arginine (CGA)→stop (TGA) nonsense mutations are possible. Oligonucleotide primers were therefore used to amplify exon 4 by the polymerase chain reaction. TaqI digestion of the 326 base pair (bp) product derived from normal genomic DNA yielded expected fragments of 244, 73 and 9 bp in size. In the case of the patient, however, an additional fragment of 253 bp was present. Direct sequence analysis showed that the 5′ TaqI site had been lost from one allele by a C→T transition at nucleotide 598. Family studies demonstrated the mutation in the patients father but no other first‐degree relatives. This is the third independent mutation described in the factor XIII A subunit gene and the first to be identified in a patient compound heterozygous for the disorder.

Prenatal diagnosis in factor XIII-A deficiency.

Congenital factor XIII deficiency is a severe bleeding disorder that is inherited as an autosomal recessive trait. The condition is commonly due to absence of the factor XIII-A subunit protein in the plasma. The case of a baby is reported who showed typical clinical features of factor XIII-A deficiency, including recurrent bleeding from the umbilical stump and a life threatening haemorrhage after circumcision. Family studies were performed and molecular analysis, using a Short Tandem Repeat (STR) marker closely linked to the A subunit gene, allowed antenatal exclusion diagnosis to be undertaken in a subsequent pregnancy. The case highlights the importance of seeking a family history of bleeding disorders before surgery in the neonatal period, particularly if the parents are consanguineous.

Rapid genotype analysis in type 2B von Willebrand's disease using a universal heteroduplex generator

Summary. A new diagnostic technique based on DNA heteroduplex analysis has been used to identify specific point mutations in the von Willebrands factor (vWF) gene of patients with von Willebrands disease type 2B. Molecular analysis in these patients has shown previously that their mutations are clustered in a short region of sequence in exon 28 of the vWF gene. The principle of the method involves heteroduplex formation between amplified genomic sequence containing the defect and an exon 28 vWF gene universal heteroduplex generator (UHG). The UHG is a synthetic vWF gene exon 28 homologue which contains a number of sequence mismatches designed to generate allele specific heteroduplexes for each type 2B mutation. Individual mutant genotypes are identified by characteristic banding patterns following polyacrylamide minigel electrophoresis. The technique is rapid, simple, inexpensive, and is ideally suited for adoption by non‐specialist haematology laboratories for screening purposes.

Structural analysis of a missense mutation (Val414Phe) in the catalytic core domain of the factor XIIIA subunit

Molecular analysis has been performed on a Malaysian patient with a severe bleeding disorder due to factor XIIIA subunit deficiency. Total mRNA was isolated from the patients leucocytes and four overlapping segments corresponding to the entire coding region of the A subunit cDNA were amplified by RT‐PCR. The cDNA segments amplified efficiently and were of expected size. Direct sequencing of the complete reading frame revealed a single homozygous base change (nt 1327G→T) in exon 10 corresponding to a missense mutation, Val414Phe, in the catalytic core domain of the A subunit monomer. The mutation eliminates a BsaJ1 restriction site and family screening showed that both parents were heterozygous for the defect. The base substitution was absent in 55 normal individuals. Val414 is a highly conserved residue in the calcium‐dependent transglutaminase enzyme family. Computer modelling based on 3D crystallographic data predicts that the bulky aromatic side chain of the substituted phenylalanine residue distorts protein folding and destabilizes the molecule. In addition, conformation changes in the adjacent catalytic and calcium binding regions of the A subunit are likely to impair the enzymatic activity of any protein synthesized.