P. R. Winship

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 repacement of the codon for Asp419 which is highly conserved cross species and is located in the receptors 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 receptors extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.

A rapid method for haemophilia B mutation detection using conformation sensitive gel electrophoresis

Conformation sensitive gel electrophoresis (CSGE) was confirmed as an effective procedure for screening the factor IX (FIX) gene by detecting 10/10 previously known FIX gene mutations. The FIX genes of a further 11 haemophilia B patients with unknown mutations were then screened and an abnormal CSGE profile was identified in all cases. Subsequent DNA sequencing demonstrated one of these to be a novel mutation (31133insT, Arg338Fs), the remaining 10 having been previously reported on the haemophilia B database. Mutation screening of the FIX gene using CSGE was demonstrated to be a rapid and efficient means of carrier analysis in families with haemophilia B.

An MseI RFLP in the 5’flanking region of the factor IX gene: its use for haemophilia B carrier detection in Caucasian and Thai populations

Summary. We have identified an MseI restriction fragment length polymorphism (RFLP) in the 5’flanking region of the factor IX gene. RFLPs in the factor IX locus are routinely used as linkage markers to track defective factor IX genes through affected pedigrees, allowing diagnosis of the haemophilia B carrier state in female members of the kindred. Currently, seven intragenic polymorphic loci have been characterized allowing carrier diagnosis in 89% of cases in the Caucasian population. Additional screening with the MseI RFLP reported in this publication increases this figure to 94% of cases. In Asian populations only one of these RFLPs (HhaI) is present at any significant frequency. Hence, carrier detection rates are much reduced in comparison to the corresponding figure of 89% in Caucasians. We report that this MseI RFLP is also present in the Thai population. Indeed, when used in combination with the HhaI polymorphism, the MseI RFLP should significantly improve the carrier detection rate in the Thai population from 11% to 40% of cases.

Tribbles-1 and -2 are tumour suppressors, down-regulated in human acute myeloid leukaemia.

Constitutive MAPK signalling is observed in approximately 50% of acute myeloid leukaemia (AML) cases. JNK activation in particular is associated with treatment failure in AML. Tribbles proteins (trb-1, trb-2 and trb-3) are potent negative regulators of MAPK pathways influencing apoptosis, differentiation and cell-cycle progression. Here we aimed to examine tribbles gene expression in AML and to characterise their role in leukaemic cells. A microarray dataset was interrogated for tribbles expression levels in AML cases and healthy controls. Myeloid cell proliferation and apoptosis were assayed in response to trb-1/trb-2 gene knockdown and overexpression, as well as a physical and functional interaction between trb and C/EBPalpha. Trb-2 expression was reduced in AML compared to healthy controls (correlating with nucleophosmin (NPM1) mutations), while low trb-1 expression was associated with inactive C/EBPalpha. In vitro assays indicated that trb-1/trb-2 are growth restrictive and pro-apoptotic in Me-1 cells, each capable of inhibiting JNK activation. JNK inactivation was itself associated with reduced Bcl-2 Ser70 phosphorylation, a residue which, when phosphorylated, maintains the anti-apoptotic activity of Bcl-2. Consistent with this, tribbles-mediated dephosphorylation of Bcl-2 Ser70 was associated with subsequent apoptosis. Trb-1/trb-2 transcription appeared to be moderately C/EBPalpha-responsive, and physical interaction between C/EBPalpha and trb-1/trb-2 was observed, suggesting a potential for auto-regulation of trb-1 and trb-2 transcription. In conclusion, we propose that trb-1 and trb-2 tumour suppressor activity may be abrogated in a proportion of AML patients. This may lead to enhanced cell survival, and therefore contribute to pathogenesis of the disease. Trb-1/trb-2 may, therefore, represent useful therapeutic targets for the treatment of AML in patients with dys-regulated trb activity.

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  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.

Gene structure, expression profiling and mutation analysis of the tumour suppressor SHIP1 in Caucasian acute myeloid leukaemia

Gene structure, expression profiling and mutation analysis of the tumour suppressor SHIP1 in Caucasian acute myeloid leukaemia

von Willebrand factor variant p.Arg924Gln marks an allele associated with reduced von Willebrand factor and factor VIII levels

Summary.  Background: von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies. Objectives: To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD. Methods: One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM‐1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells. Results: c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843‐8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co‐inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF‐FVIII binding and normal clearance of VWF and FVIII. Conclusions: The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels.

Regulation of the human protein S gene promoter by liver enriched transcription factors

Protein S is expressed in a number of tissue types, one of the most physiologically relevant being the liver. However, transcriptional control of protein S gene expression is poorly understood. We have characterised a 638 bp area in the 5′ flanking region of the human protein S gene, spanning all 10 previously reported transcription initiation sites, which demonstrates promoter activity in the human liver‐derived cell line HepG2. More refined reporter gene analysis of this region enabled the identification of three transcription initiation sites whose absence is associated with significantly reduced promoter activity, together with a number of positively and negatively acting transcriptional regulatory elements. Consistent with these findings, DNaseI footprinting analysis identified eleven sites (I–XI) from within this 638 bp region that show evidence of binding nuclear proteins. We present evidence to show that the liver‐specific factors hepatocyte nuclear factor 1 (HNF1) and HNF4 bind regions of the protein S promoter, which lie within the identified protein binding sites V and VIII, respectively, and that HNF4 activates the protein S promoter. Reporter gene analysis suggests that members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors are potent activators of protein S gene transcription in HepG2 cells.

The high frequency of the — 6G A factor IX promoter mutation is the result both of a founder effect and recurrent mutation at a CpG dinucleotide

We report a new Liverpool family with a mild haemophilia B Leyden phenotype caused by a — 6G → A mutation in a CpG dinucleotide in the promoter of the clotting factor IX gene. This mutation had previously been identified in three other U.K. pedigrees and six others worldwide. To investigate whether these mutations were of independent origin, the haplotype was determined for eight polymorphic loci, within or immediately adjacent to the factor IX gene, for nine of the 10 existing patients. Six probands had identical haplotypes, including all four U.K. probands, suggesting that they arose from a common founder. The other three probands differed in haplotype from the common haplotype, and from each other. suggesting that they were independent mutations at this CpG dinucleotide.