The G1456 to T mutation in the thrombomodulin gene is not frequent in patients with venous thrombosis.

To the Editor: CAGC-3* and 5*-TGGACGGAGCCAGGCTCCTG-3*) were used to produce two PCR products with overlapping sequences at the mutagenized end. After the first round of PCR, 1 mL of 1:100 dilution Thrombomodulin is a major component of the protein C anticoagulant pathway. Biochemical detection of thrombomodulin defects of the PCR products were used to amplify, in a second round of PCR, a 153-bp fragment using the TM 23 and TM 24B primers. are hampered by its location in the endothelial cell. The characterization of molecular alterations in the thrombomodulin gene of selected The Rsa I digestion pattern of the mutagenized PCR products showed the introduction of a G to T substitution at nucleotide 1456. Soluble patient populations represents an alternative diagnostic approach. Recently, a point mutation G to T in the thrombomodulin gene thrombomodulin levels were also measured in patients and controls by enzyme-linked immunosorbent assay (Asserachrom Thromboof a 45-year-old man with venous thrombosis and that of his son was detected. The mutation predicts an Asp to Tyr amino acid modulin; Diagnostica Stago, Asnière, France), because the reported patient with the mutation had soluble thrombomodulin levels less substitution and creates a novel restriction site for Rsa I digestion in the gene. To assess the frequency of this mutation in patients with than the normal laboratory range. We could not find the G to T mutation in any of the patients venous thrombosis, we screened 100 Italian patients consecutively referred to the Thrombosis Center for a documented episode of and controls; two of the patients and four control individuals had soluble thrombomodulin levels less than the normal laboratory range venous thrombosis and 100 ageand sex-matched asymptomatic controls. The most frequent thrombotic episode was deep vein (mean 0 2 SD calculated from 100 determinations in healthy subjects; 7 ng/mL). We conclude that the G to T thrombomodulin thrombosis of the lower limbs (54%), followed by superficial thrombophlebitis (21%), pulmonary embolism (14%), cerebral vein thromgene mutation is not frequently associated with venous thrombosis in Italy and that it does not account for a significant proportion of bosis (7%), and visceral vein thrombosis (4%). Characteristics of the patients are shown in Table 1. the thrombotic episodes that remain still unexplained by the known causes of hereditary thrombophilia. The low levels of soluble thromThe G to T mutation was searched for by amplification and restriction analysis. Oligonucleotide primers employed were as origibomodulin antigen found in two patients and four control individuals may be due to acquired causes such as an increased turnover or to nally described by Ohlin and Marlar, ie, 5*-CGGTACCTTCGAGTGCATCT-3* (TM 23) and 5*-ACGGCCGGAGGAGTCAAGGTa different and as yet unidentified genetic defect in the thrombomodulin gene. 3* (TM 24B). The original polymerase chain reaction (PCR) method was used with minor modifications (50 mL reaction mixture, with annealing at 657C). In addition, a DNA fragment containing the Elena M. Faioni G to T mutation was constructed as a positive control. Briefly, Giuliana Merati two sets of oligonucleotides carrying the mutation of interest (5*Flora Peyvandi CAGGTGCCAGATGTTTTGCA-3* and 5*-TGTCGCCACCGTPaola M. Bettini ACACCTTGCCGGA-3*; 5*-CGGCAAGGTGTACGGTGGCGAPier Mannuccio Mannucci Angelo Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Maggiore Hospital and Institute of Internal Medicine University of Milano Table 1. Characteristics of 100 Patients With Venous Thrombosis Milan, Italy Median age (range) of patients* 44 (17-73) Sex (Males/Females) 40/60 REFERENCES Median age (range) at first thrombotic episode 35 (12-65) 1. Esmon CT: The roles of protein C and thrombomodulin in the Patients with 1 thrombotic episode* (%) 54 regulation of blood coagulation. J Biol Chem 264:4743, 1989 Patients with 2 thrombotic episodes* (%) 26 2. Ohlin A-K, Marlar RA: The first mutation identified in the Patients with 3 or more thrombotic episodes* (%) 20 thrombomodulin gene in a 45-year-old man presenting with thromMedian (range) soluble thrombomodulin (ng/mL) boembolic disease. Blood 85:330, 1995 Patients 16 (5-62) 3. De Stefano V, Finazzi G, Mannucci PM: Inherited thrombophiControls 16 (3-47) lia: Pathogenesis, clinical syndromes and management. Blood 87:3531, 1996 * At time of referral.