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Technical Briefs |
1 Dipartimento di Medicina, Chirurgia, Odontoiatria–San Paolo–Università degli Studi di Milano, Milan, Italy;2 Laboratorio Analisi Chimica Clinica e Microbiologia, Ospedale San Paolo, Milan, Italy
aaddress correspondence to this author at: Dipartimento di Medicina, Chirurgia, Odontoiatria–San Paolo–Università degli Studi di Milano, Milan, Italy; fax 39-0250-323089, e-mail giancarla.gerli{at}unimi.it
Stromal cell-derived factor-1 (SDF-1), a member of the CXC chemokine family, is constitutively produced by bone marrow stromal cells, and it induces chemotaxis on a variety of cell types that contain the G-protein–linked receptor CXCR4. SDF-1 plays a key role in hematopoiesis, and its involvement in migration, homing, and survival of hematopoietic progenitors has been well established. It is the first chemoattractant agent reported for human CD34+ progenitor cells (1), and it promotes CD34+ cell survival by counteracting apoptosis (2).
SDF-1 may also have a role in neoangiogenesis. It increases production of vascular endothelial growth factor (VEGF), the main angiogenic factor, which in turn enhances the expression of CXCR4 receptor in endothelial cells, in an autocrine loop (3).
SDF-1 may participate in atherothrombosis. It is a potent chemoattractant of monocytes and lymphocytes (4) and has a direct effect on platelet activation (5). SDF-1 is also involved in the neovascularization and angiogenesis response that occurs during formation of unstable atherosclerotic plaques (6), in which increased expression of this chemokine has been found (5).
The 2 isoforms, SDF-1
and SDF-1ß, arise from a single gene by alternative splicing (7). Sequence analysis reveals a common polymorphism in the 3'-untranslated region (3'-UTR), implicated in mRNA turnover regulation, of the SDF-1ß gene transcript, which contains a G
A transition at position 801, designated SDF1-3'UTR-801G-A, abbreviated as SDF1-3'A (8). It has been reported that SDF1-3'A genotype action involves up-regulation of the quantity of SDF-1 protein available to bind CXCR4 (7). In addition, the SDF1-3'A polymorphism has been associated with high mobilization of CD34+ progenitor cells into peripheral blood (9).
Philadelphia-negative chronic myeloproliferative disorders (CMDs) are characterized by clonal proliferation of hematopoietic progenitor cells in the bone marrow and by extramedullary hematopoiesis associated with increased CD34+ circulating cells (10)(11)(12). Angiogenesis in bone marrow and spleen is an integral component of myeloproliferation (13)(14), and CMDs are characterized by a high incidence of thrombotic events, the mechanisms of which are undefined.
We studied SDF-1 gene polymorphisms in polycythemia vera (PV) and essential thrombocythemia (ET) to determine the frequency of the SDF1-3'A polymorphism and to evaluate its impact on susceptibility to these diseases and on the occurrence of thrombotic complications. We also investigated possible associations between SDF1-3'A, the number of circulating CD34+ progenitor cells, and clinical and hematologic features of PV and ET.
We studied 73 consecutive patients with Philadelphia-negative CMDs, 45 with PV [mean age (SD), 71 (10) years] and 28 with ET [mean age, 70 (11) years], diagnosed between 1984 and 2002, in our Hematology Outpatient Clinic. We also studied 139 healthy individuals [mean age, 68 (4) years] as controls. Informed consent was obtained from patients and controls. The diagnoses of PV and ET were made according to the diagnostic criteria established by the Polycythemia Vera Study Group (15)(16).
Mean (SD) hemoglobin was 154 (15) g/L (range, 121–194 g/L) for PV patients and 129 (20) g/L (range, 65–154 g/L) for ET patients, and the platelet count was 410 (201) x 109/L (range, 126–950 x 109/L) for PV patients and 674 (577) x 109/L (range, 364-3516 x 109/L) for ET patients. Twenty-three patients with PV and 5 patients with ET had splenomegaly, with a spleen index 
84 cm2 (length of the longitudinal axis times the length of the transverse axis measured by ultrasonography) (17).
Time from diagnosis to the investigation was 3–241 months. Twenty-three PV patients underwent chronic treatment with phlebotomy; 41 patients (18 PV and 23 ET) had cytoreductive treatment with hydroxyurea; and 43 PV and 26 ET patients were treated with aspirin. Twenty-eight patients had had previous thrombotic complications (10 stroke, 6 transient ischemic attack, 10 acute myocardial infarction, 1 peripheral arterial thrombosis, and 1 acute intestinal infarction).
In our patients we also evaluated the presence of major risk factors (sex, hypertension, cigarette smoking, diabetes mellitus, and hypercholesterolemia) commonly associated with thrombotic events.
DNA was prepared with a Nucleo Spin extraction reagent set (Macherey-Nagel).
The PCR reaction for G801A was carried out in a 25-µL final volume with 80 nM of each oligonucleotide primer (sense, 5'-CAGTCAACCTGGGCAAAGCC-3'; antisense, 5'-AGCTTTGGTCCTGAGAGTCC-3'), 100 µM of each deoxynucleotide triphosphate, 1.5 mM MgCl2, 1 U of Taq polymerase, and 5 µL of genomic DNA. The PCR conditions were as follows: 94 °C for 10 min and 35 cycles of 94 °C for 45 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were analyzed by electrophoresis in a 3% agarose gel and visualized by ultraviolet fluorescence after staining with ethidium bromide. The PCR products with the SDF1-3'G allele produced 2 discrete fragments (99 and 203 bp), whereas those with the SDF1-3'A allele produced 1 fragment (302 bp).
CD34+ peripheral cells were determined by flow cytometry.
Differences between groups were examined by the 
2 test. Odds ratios (ORs) were calculated as an index of the association of the SDF-1 genotypes (GG, AG, AA) with each disease. For each OR, 2-tailed probability and 95% confidence intervals (CIs) were calculated.
We used ANOVA to test for differences among hemoglobin values, platelet and circulating CD34+ cell counts, spleen index, and duration of disease in different genotypes. P <0.05 was considered significant. All statistical analyses were performed with SPSS software.
The allele frequencies of SDF-1 gene polymorphisms in each group were distributed according to the Hardy–Weinberg equilibrium.
The genotype distribution of SDF-1 polymorphisms was significantly different in patients and controls (2 = 11.89, P = 0.003 for PV patients; and 2 = 6.49, P = 0.039 for ET patients). The frequency of the homozygous AA genotype (Table 1
) was higher in patients with PV (16%) and ET (11%) than in controls (2%).
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Individuals homozygous for the A allele had an 8-fold higher risk for PV than did AG+GG patients (OR = 8.35; CI, 2.06–33.85; P = 0.002); the risk for ET was not statistically significant (OR = 5.44; CI, 1.04–28.50; P = 0.06; Table 1
).
We found no significant association between the SDF1-3'A polymorphism and hematologic and clinical features, such as hemoglobin concentrations, platelet counts, number of CD34+ cells in peripheral blood, splenomegaly, or duration of disease (not shown).
Thrombotic complications were more common in A-allele homozygous patients with PV (71%) and with ET (67%) than in AG+GG patients (39% of PV and 24% of ET patients, respectively); statistical significance was reached only when patients with PV or ET were analyzed jointly. Thrombotic events were more frequent in AA homozygous patients with PV or ET (7 of 10) than AG+GG individuals (21 of 63; Fig. 1
). SDF1-3'AA individuals had a 5-fold higher risk of thrombotic events than AG+GG patients (OR = 4.67; CI, 1.09–19.90; P = 0.038).
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In multiple logistic regression analysis considering sex, hypertension, cigarette smoking, diabetes mellitus, hypercholesterolemia, and A allele homozygosity, the AA genotype was the only independent predictor of thrombotic events (OR = 4.89; CI, 1.10–21.79; P = 0.037).
The SDF1-3'A polymorphism has been studied in conditions such as HIV, diabetes, and lymphoma (8)(18)(19)(20), but there are no published data about the SDF1-3'A polymorphism in patients with PV and ET. In the present study, we observed a significant difference in genotype distribution of SDF-1 gene polymorphisms in the 3'-UTR between CMD patients (PV and ET) and healthy controls; in the latter, the frequency of the SDF1-3'A allele was 21%, which was the expected value for Caucasians (21).
Concerning the association between the SDF1-3'A polymorphism and CMDs, we speculate that the SDF1-3'AA genotype might be involved in genetic susceptibility to PV. SDF-1 chemokine induces increased VEGF production, which is responsible for an angiogenic activity (3). Because the role of angiogenesis in CMDs and in their progression has been well demonstrated (14)(22), we hypothesize that the SDF1-3'A polymorphism might increase SDF-1 protein (8), which would have a role in developing angiogenesis and in the pathogenesis of the disease. Previous reports have emphasized the role of VEGF in thrombogenesis; in fact, the VEGF released by the activated platelets would promote endothelial activation with a subsequent switch to a predominant prethrombotic phenotype (23). Musolino et al.(24) suggested that increased plasma VEGF is an important signaling molecule for thrombotic risk in CMD patients.
In view of the association between the SDF1-3'A polymorphism and the positive history of thrombotic complications in our patients, we speculate that this polymorphism plays a role in thrombotic risk in CMD patients. Mirshahi et al. (3) observed that the SDF-1 chemokine induces an increase in VEGF production; we therefore hypothesize that A allele homozygosity, which can lead to increased SDF-1 and VEGF production, might be considered a genetic component that contributes to thrombotic events.
Additional studies on a larger series of patients are needed to clarify the function of the SDF1-3'A gene polymorphism in susceptibility to PV and to explain the mechanism by which the SDF1-3'AA genotype may contribute to thrombotic predisposition.
References
functional activity during differentiation of human monocytes and macrophages. J Leukoc Biol 1999;66:135-143.[Abstract]
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) and AG+GG (
)], with and without thrombotic events.