The frequencies of factor V Leiden and/or prothrombin variant G20210A are increased in patients age <50 years with normal or near normal coronary arteries after MI.

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Haemophilia B Leyden is characterized by severe factor IX deficiency during childhood with partial resolution at puberty or following the administration of anabolic steroids. The disorder has recently been associated with point mutations in the putative factor IX promoter region, which contains an imperfect direct repeat spanning a possible start site of transcription. We have identified a T to C transition at position +8 in the promoter region of a patient with the haemophilia B Leyden phenotype. A mutation at this site has not been previously reported and occurs within the repeat consensus sequence in the transcribed but untranslated portion of the gene. There is no family history of haemophilia and sequence analysis of his mother and other family members indicates that the mutation has arisen de novo in this patient. This observation provides further support for a causal relationship between point mutations in the presumptive promoter region of the factor IX gene and the Leyden phenotype.

To cite this article: Van de Water NS, Tan T, May S, Browett PJ, Harper P. Factor IX polypyrimidine tract mutation analysis using mRNA from peripheral blood leukocytes. J Thromb Haemost 2004; 2: 2073-5.The production of illegitimately transcribed mRNA from peripheral blood leukocytes and its use for mutational analysis of many tissue-specific genes has been known for 15 years [1]. Two recent publications in this journal by Green et al. and Cutler et al. [2,3] have, however, highlighted the difficulty in using ÔillegitimateÕ transcripts of factor FIX mRNA isolated from white blood cells for sequence analysis. Both groups suggested that it was not possible to obtain full-length FIX transcript from peripheral lymphocytes. Green et al.[2] also postulated that because only exons g-h could be amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), this truncated transcript produced a novel serine protease which was unrelated to blood coagulation. RNA degradation in yeast proceeds predominantly via a mechanism involving decapping and 5¢-3¢ exonuclease activity. In humans this mechanism also plays a major role in RNA degradation and, in addition, a 3¢-5¢ pathway involving decapping and deadenylation is also evident [4]. The predominance of the 5¢-3¢ mechanism would explain why the 3¢ regions of transcripts are the most well preserved in cell extracts and why, when isolating low copy number transcripts, the 3¢ end is often the only portion seen by RT-PCR analysis. It is therefore possible that normal full-length FIX mRNA is produced but is quickly degraded because of the low copy number of FIX transcripts in peripheral blood cells and the inherent susceptibility of mRNA to degradation. By careful RNA preparation and nested PCR it is possible to detect FIX mRNA containing exons other than g-h. We present here a case analysis which describes a hemophilia B patient with a mutation within the polypyrimidine tract of intron b which results in exon skipping and the production of FIX mRNA with exon b spliced to exon d.The patient has mild hemophilia B with a FIX level of 2%. On examination of his genomic DNA by sequencing his FIX gene exon by exon the only defect observed was a four-base deletion of TTCT at nucleotide 6659-6662 within the polypyrimidine tract of intron b (Fig. 1a,b).RNA was extracted from 10 mL of peripheral blood collected into citrate, phosphate, dextrose (CPD) anticoagulant. The red cells were initially lyzed using NH 4 Cl/KHCO 3 lysis buffer and the leukocyte cell pellet RNA was extracted by the method of Chomczynski and Sacchi [5]. cDNA was then produced with random primers and SuperScript II (Invitrogen, Auckland, New Zeland). Thirty cycles of PCR using this cDNA as template and primers located in exons b and d at positions 6342 and 10464 were followed by a further 30 cycles using 5 lL of first-round reaction mix as template and nested primers at positions 6358 and 10424. The final RT-PCR product from normal FIX mRNA was expected to be 212 bp.We extracted RNA from our patient with hemophil...

SummaryProtein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05–3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.

This study compared colonoscopic findings in families meeting the Amsterdam criteria (A) for hereditary non-polyposis colorectal cancer (HNPCC) but stratified according to whether the familial cancers showed DNA microsatellite instability. DNA was extracted from paired samples of normal and cancer, and microsatellite instability was analysed at up to six loci. Families were termed replication error positive (RER+) when at least 50% of tumours tested per family were positive. Of 26 families studied 17 were RER+ and 9 were RER-. Cancers in the A/RER- families showed no right-sided predilection (P < 0.001). Colonoscopies have been performed on 182 at-risk members of A/RER+ families and 60 members of A/RER- families. More of the at-risk members of A/RER-families were found to have adenomas at colonoscopy (P = 0.095), but these were smaller than those of A/RER+ families (P = 0.19). The adenoma:carcinoma ratio was twice as high in A/RER- families (13:1) as in A/RER+ families (7:1). One of the A/RER- families had hyperplastic polyposis. The others do not appear to have attenuated familial adenomatous polyposis and are similar to the adenoma families or late-onset colorectal cancer families described by others. This study illustrates the importance of molecular technology in separating HNPCC from syndromes with overlapping phenotypes.

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