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Letters to the Editor |
Diagnostic Laboratory, Becker, Olgemöller und Kollegen, München, Germany
aAddress correspondence to this author at: Becker, Olgemöller und Kollegen, Führichstrasse 70, 81671 München, Germany. Fax 49-89-450917-6463; e-mail sburggraf{at}labor-bo.de.
To the Editor:
The protein Z-dependent protease inhibitor (ZPI, gene symbol SERPINA10, serine peptidase inhibitor, clade A, member 10) inhibits the hemostatic factors Xa and XIa, and in vitro studies indicate that it is an anticoagulant (1)(2). However, the exact role of ZPI in the coagulation system is still unknown. In a recent study, 1018 patients with an episode of venous thromboembolism and 1018 controls with no history of thromboembolic disease were screened for different ZPI polymorphisms(3). A multivariant analysis, which included the Factor V Leiden mutation and the prothombin 20210 G>A sequence alteration, showed that the R67X change is an independent risk factor for venous thrombosis. The calculated odds ratios were 3.32, 5.32, and 4.24 for ZPI-R67X, Factor V Leiden, and prothombin 20210 G>A, respectively(3).
We designed an assay for rapid detection of the R67X mutation that involves real-time PCR and melting point analysis on a LightCycler 1.5 Instrument. Primers (5'-CAG CTT GCC AAG GAG AC-3' and 5'-CTC TCT TGA TCT GGG TTT CAG T-3'; NM 016186 position 686–702 and 845–824, respectively) and probes (anchor probe, 5'-CY5.5-TGG CAA CAT GGT CTT CTC TCC ATT TGG CA-phosphate-3', and wild-type detection probe, 5'-CTG CGA AAG ATC TCC ATG AGG CAC-fluorescein-3'; position 751–779 and 725–748, respectively) were designed using LightCycler probe design software (Roche Molecular Biochemicals). PCRs were performed in a final volume of 10 µL in glass capillaries (Roche Molecular Biochemicals). Each reaction mixture contained 4 pmol forward primer, 10 pmol reverse primer, 2 pmol each of anchor and detection probe, 2 µL LightCycler FastStart DNA MasterPLUS Hybridization Probes, and 2.5 µL genomic DNA. The thermocycling conditions were: initial denaturation and activation of Taq polymerase, 95 °C for 10 min, followed by 40 amplification cycles (95 °C for 0 s, 55 °C for 10 s, and 72 °C for 20 s, with a ramping rate of 20 °C/s). For the subsequent melting point analysis, the capillary was heated at 95 °C for 10 s, incubated at 40 °C for 1 min, and then slowly (0.1 °C/s) heated to 85 °C.
To validate the new LightCycler assay, we screened 150 different anonymous blood samples. DNA was extracted from 200 µL of EDTA-treated blood with the QIAamp DNA Blood Mini Kit (Qiagen).
In the melting point analysis, the majority (147) of the samples showed only 1 melting peak at 
68 °C (Fig. 1
), indicating they contained only the wild-type allele, arginine at position 67. Sequencing of 2 of the wild-type samples confirmed the LightCycler result (data not shown).
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Three samples showed 2 melting peaks, at 62 °C and 68 °C (Fig. 1
), the result expected for a heterozygous R67X mutation. However, sequence analysis revealed that only 1 of the 3 heterozygous samples contained the R67X (728 C>T) mutation. The other 2 were heterozygous for R67Q (729 G>A) (data not shown), a mutation that is not listed in the NCBI SNP database.
The R67X and the R67Q mutations could not be clearly differentiated in our melting point assay (Fig. 1
). To differentiate R67X and R67Q, all samples exhibiting a melting profile other than wild type were subjected to an RFLP analysis. We transferred 2 µL of the PCR product to a tube containing 2 units of restriction endonuclease ItaI in 1x restriction buffer H (both from Roche Molecular Biochemicals); the total assay volume was 10 µL. After samples were incubated at 37 °C for 60 min, the restriction fragments were separated by agarose gel electrophoresis. The digestion product from the sample with a heterozygous R67X mutation contained 3 fragments (160, 140, and 20 bp; Fig. 1
), whereas the digestion product from either the R67Q mutation or the wild type contained only 2 bands (140 and 20 bp; Fig. 1
). An alternative approach would be to reanalyze non–wild-type samples using a LightCycler assay with an R67Q- or R67X-specific detection probe.
Venous thrombosis is a multigenic disease. Only 2 polymorphisms, however, Factor V Leiden and prothrombin 20210 G>A, have been established as clear genetic risk factors for thrombosis, despite considerable efforts to identify and validate new polymorphisms affecting hemostatic proteins. The R67X mutation might be a new candidate for inclusion in thrombotic risk assessment (1)(3). Results of another study, however, do not support a role of the R67X mutation in thrombosis(4).
It remains to be determined if any of these studies have been affected by the R67Q mutation, a polymorphism whose physiological role in hemostasis is not yet known.
Acknowledgments
Grant/funding support: None declared.
Financial Disclosures: None declared.
Acknowledgments: We thank Charalampos Aslanidis and Javier Corral for valuable comments and support during sequence analysis of the ZPI gene.
References
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