Summary Protein Z‐dependent protease inhibitor (ZPI) is a serpin that inhibits the activated coagulation factors X and XI. The precise physiological significance of ZPI in the control of haemostasis is unknown although a deficiency of ZPI may be predicted to alter this balance. The coding region of the ZPI gene was screened for mutations using denaturing high‐performance liquid chromatography. 16 mutations/polymorphisms within the coding region of ZPI were identified including two mutations, which generated stop codons at residues R67 and W303. We observed nonsense mutations within the ZPI gene in 4·4% of thrombosis patients (n = 250) compared with 0·8% of controls (n = 250). The difference in distribution of stop codon mutations between thrombosis patients and controls was significant (P = 0·02) with an odds ratio of 5·7 (95% confidence interval, 1·25–26·0). Our results suggest an association between ZPI deficiency and venous thrombosis and we propose that ZPI deficiency is potentially a new form of thrombophilia.

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...

1056 Background: Estrogen receptor positive (ER+) metastatic breast cancers (MBC) that express constitutively active somatic ESR1 mutations at Y537S and D538G allow tumors to progress in the presence of approved endocrine therapies. For patients with ER+ MBC, fulvestrant is the first line of treatment. Palbociclib or other CDK4/6 inhibitors are now being included. Preliminary studies show that lasofoxifene, a selective ERα modulator (SERM), was effective in reducing tumor growth in an endocrine resistant xenograph model expressing ERα mutations Y537S or D538G. Additionally, lasofoxifene more effectively inhibited the development of liver and lung metastases than fulvestrant. Lasofoxifene is currently under evaluation in a phase 2 study. Because certain combinations of a hormonal agents like fulvestrant improved efficacy, we investigated the combination of palbociclib and lasofoxifene as a potential therapeutic for mutant ESR1 MBC. We hypothesized that this combination should improve outcome and compared it to a combination with fulvestrant. Methods: We first determined the optimal dose of lasofoxifene in an intraductal (MIND) xenograph model of MCF-7 cells that express active ERα Y537S and D538G. Subsequently, we performed combination studies with lasofoxifene (10mg/kg 5/week SQ) +/- palbociclib (100mg/kg gavage, 5/week) or fulvestrant (5mg/mouse/week, SQ) +/- palbociclib. Results: Lasofoxifene alone was significantly more effective than fulvestrant at inhibiting the metastasis of both MCF7 Y537S and D538G tumors to the lungs and liver. Lasofoxifene + palbociclib was more effective than fulvestrant + palbociclib at reducing primary tumor growth; both combinations demonstrated an increased response. Lasofoxifene + palbociclib was more effective at inhibiting liver metastasis than either drug alone and was more effective than fulvestrant + palbociclib at reducing metastasis to the liver and lung. Structural studies showed that lasofoxifene effectively disrupts the active conformation of the ERα Y537S ligand-binding domain. Conclusions: These results demonstrate that lasofoxifene, in combination with CDK4/6 inhibitors like palbociclib, has promise for treating endocrine therapy resistant ER+ MBC patients whose tumors express activating ESR1 mutations, more effectively than either drug alone.

The 5' untranslated region (5'UTR) of beta-globin has been well characterized and is often used as a model for eukaryotic transcription/translation, but there are still questions regarding the mechanism of translational control. Mutations affecting the Cap site at + 1 and at positions +10, +22, +33 and +40-43 have been described, and it is thought that the initiator element required for transcription stretches from -2 to +7 relative to the Cap site with a downstream element situated from +10 to +15. The influence on initiation or translation of sequences between +7 and +10 is unknown. We report here a family with beta-thalassemia (beta-thal) who have a + 8 (CT) mutation. Molecular studies indicate that this mutation leads to a reduction in mRNA levels and we discuss the implications of a CT change at this position on the transcription/translation process.

ZPI is a recently characterised inhibitory serpin present in plasma. In vitro studies have shown that ZPI inhibits both factor Xa (in the presence of protein Z) and factor XIa. Deficiency of ZPI is predicted to enhance coagulation and may be a risk factor for venous thrombosis. To test this hypothesis we carried out mutation screening within the coding region of the ZPI gene in a cohort of 150 patients with a history of DVT or PE (first event before the age of 60 years) and 150 matched controls. Five PCR products were produced for each subject. Heteroduplex analysis was performed using dHPLC and the PCR products were sequenced directly if a heteroduplex mismatch was identified. Sixteen mutations/polymorphisms were identified within the coding region of the ZPI gene. Two mutations were regarded as functionally significant as they produced stop codons at arginine 67 and tryptophan 303 and would be expected to result in the loss of ZPI activity. These stop codon mutations were found in 8 patients and 2 controls. Based on these results, a power calculation was performed that showed that an additional 100 patients and controls would be necessary to confirm that the stop codon mutations were statistically significant risk factors for thrombosis. Bidirectional allele specific PCR was developed to rapidly identify these stop codon mutations and a further 100 patients and 100 controls were examined using this technique. The stop codon mutations were found in 11 patients (W303X n=8: R67X n=3) and only 2 controls (R67X n=2) (two sided Fishers exact p=0.02) with an odds ratio = 5.7 (95%CI, 1.25 to 26.0). In addition two further non-conservative mutations were identified in two other thrombosis patients. These resulted in a serine to tyrosine (codon 122) and a phenylalanine to leucine (codon 124) change in the region homologous to the D helix of other heparin activated serpins. These changes, involving bulky aromatic amino acid residues, have the potential to disrupt the structure and function of the D helix. We identified a further 12 mutations/polymorphisms (C454G, C574T, A603G, A647G, G752A, A947T, C1276T, G1277A, G1438A, A1617C, G1789T and C1811T (italics: not previously described)). The significance of these mutations is uncertain. We have identified mutations causing stop codons, which will result in loss of ZPI function, in 4.4% of patients who present with venous thrombosis before the age of 60 years, compared with an incidence of only 0.8% in controls. Further studies are ongoing to measure the plasma concentration of ZPI in the affected individuals. Our results support an association between mutations in the ZPI gene and venous thrombosis. We propose that ZPI deficiency as a result of these mutations is potentially a new form of thrombophilia.