Circulating levels of cobalt and chromium from metal-on-metal hip replacement are associated with CD8+ T-cell lymphopenia.

We carried out a cross-sectional study with analysis of the demographic, clinical and laboratory characteristics of patients with metal-on-metal hip resurfacing, ceramic-on-ceramic and metal-on-polyethylene hip replacements. Our aim was to evaluate the relationship between metal-on-metal replacements, the levels of cobalt and chromium ions in whole blood and the absolute numbers of circulating lymphocytes. We recruited 164 patients (101 men and 63 women) with hip replacements, 106 with metal-on-metal hips and 58 with non-metal-on-metal hips, aged < 65 years, with a pre-operative diagnosis of osteoarthritis and no pre-existing immunological disorders. Laboratory-defined T-cell lymphopenia was present in 13 patients (15%) (CD8(+) lymphopenia) and 11 patients (13%) (CD3(+) lymphopenia) with unilateral metal-on-metal hips. There were significant differences in the absolute CD8(+) lymphocyte subset counts for the metal-on-metal groups compared with each control group (p-values ranging between 0.024 and 0.046). Statistical modelling with analysis of covariance using age, gender, type of hip replacement, smoking and circulating metal ion levels, showed that circulating levels of metal ions, especially cobalt, explained the variation in absolute lymphocyte counts for almost all lymphocyte subsets.

Identification and functional characterization of a polymorphic oestrogen response element in the human coagulation factor IX gene promoter.

The liver expressed procoagulant factor IX (FIX) shows inter-individual variation in levels, some of which is heritable. Raised levels of FIX are associated with a thrombotic tendency. This study demonstrated that, in females but not males, part of this variation in FIX levels is due to polymorphic genotype at a locus in the factor IX gene (F9) promoter 698 bp upstream of the major transcription initiation site (-698C/T). The -698C allele (associated with higher FIX level) shows closer homology to a canonical ORE sequence and a higher binding affinity for oestrogen receptor alpha than the -698T allele. Reporter gene vectors were constructed with elements spanning residues -738 to +50 of the F9 promoter corresponding to wild type -698C and -698T alleles. A related series of vectors comprising three copies of the F9 ORE driving expression of a minimal synthetic promoter were also created. Transfection into the liver-derived HepG2 and erythroleukaemic K562 cell lines demonstrated increased levels of expression in the presence of oestrogenic factors when compared to those found in their absence; this stimulation was more pronounced in the non-liver derived K562 cell line and from the reporter vectors containing promoter elements corresponding to the -698C allele.

The -1185 A/G and -1051 G/A dimorphisms in the von Willebrand factor gene promoter and risk of myocardial infarction.

Elevated plasma von Willebrand factor (VWF) levels are associated with coronary artery disease, although the precise mechanism for this is unclear. Recently, four linked dimorphisms in the VWF gene promoter were demonstrated to influence plasma VWF level. We conducted a case-control study of 525 acute myocardial infarction (MI) cases and 451 control subjects, all aged < or = 75 years, to assess the potential contribution of two of these dimorphisms (-1185 G/A and -1051 A/G) to the risk of MI. The frequency of the -1185A/-1051G haplotype, associated with elevated VWF levels, was similar in the case and control groups, yielding a haplotypic odds ratio for MI of 0.93 (95% CI 0.77, 1.12, P = 0.43), and there was no significant association between the -1185A/-1051G haplotype and the risk of MI in any subgroup analysed. We therefore conclude that possession of the -1185A/-1051G haplotype does not confer an increased risk for MI.

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group.

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.

c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia.

Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c-kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in-frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA --> ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in-frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in-frame deletion plus insertion mutations (n = 7) involved the loss or replacement of the codon for Asp419 which is highly conserved cross species and is located in the receptor's extracellular domain. The high frequency of the c-kit proto-oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptor's extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.

Identification of different gene expression patterns in low and high grade non-Hodgkin's lymphomas by differential display.

We have compared the patterns of gene expression in non-Hodgkin's lymphoma (NHL) biopsy samples from patients with either low grade or high grade disease, by the polymerase chain reaction (PCR) based technique of differential display. By using a combination of 30 primer pairs we estimate that we were able to survey over 3,000 genes expressed in these tissues. In this study we compared a group of three low grade follicular centre lymphomas with a group of two high grade diffuse large cell lymphomas and scored only those PCR products that were represented in all samples of each group. In doing so we were able to avoid many of the problems associated with the occurence of false PCR-positives. 139 differences were then scored as representing genes which may be differentially expressed during the transformation from low to high grade disease. However, as many of these might simply reflect changing populations of cells, we focused on only those genes which appeared to be expressed exclusively in either low grade or high grade disease. We have identified 14 such genes, of which 10 were low grade specific and 4 were high grade specific. This approach therefore appears to offer a systematic method for the identification and characterisation of differentially expressed genes, which are characteristic of different NHL sub-types.

Detection of polymorphisms at cytosine phosphoguanadine dinucleotides and diagnosis of haemophilia B carriers.

The polymerase chain reaction procedure (PCR) was used to detect a polymorphic Hha I site adjacent to the factor IX locus in a panel of 33 phenotypically normal caucasian individuals. This technique was also applied to a haemophilia B family pedigree. The Hha I polymorphic site was located 8 kb 3' to the factor IX gene, and the proportion of female subjects expected to be heterozygous at this site was 0.48. The Hha I locus was in linkage equilibrium with the other polymorphic loci on the factor IX gene. These findings, besides increasing the proportion of caucasian individuals whose haemophilia B carrier state can be diagnosed from 79% to 89%, demonstrate this widely applicable use of PCR for the detection of DNA polymorphism at cytosine phosphoguanadine dinucleotides irrespective of the methylation status.

Nullisomic deletion of the mcf.2 transforming gene in two haemophilia B patients.

The mcf.2 transforming gene sequence has been located to the region between 29 and 61 kb 3' of the factor IX gene. Two unrelated haemophilia B patients who raise antibodies to infused factor IX ('inhibitors') have deletions in excess of 273 kb encompassing the factor IX and mcf.2 genes and a CG-rich island. We believe these patients show the first nullisomic deletion of a transforming gene to be reported. No clinical condition can be attributed to the loss of the mcf.2 gene.