DNA-array of gene variants in venous leg ulcers: detection of prognostic indicators.

OBJECTIVE: Wound healing in venous leg ulcer (VLU) is a multi-step process involving complex pathways. Scanty knowledge at molecular level hinders clinical assessment and treatment. Anomalous handling of local iron overload, as well as unbalancing in matrix metalloproteinases (MMPs) and transglutaminase, has a recognized role in VLU establishment. We selected a number of single nucleotide polymorphisms (SNPs) in candidate genes (HFE, FPN1, MMP12, and FXIII) involved in VLU to identify potentially prognostic markers by means of DNA-array technology. METHODS AND RESULTS: The DNA-array-genotyping was assessed in 638 subjects for the following SNPs: HFE (C282Y, H63D), FPN1 (-8CG), MMP12 (-82AG) and FXIII (V34L). Of the subjects, 221 were affected by VLU (171 primary and 50 post-thrombosis), 112 by severe chronic venous disease (CVD) (CEAP, C3-C4), while 305 were matched healthy controls. The HFE and FXIII SNPs had been previously genotyped by conventional polymerase chain reaction (PCR)-methods on the same group of subjects (J Vasc Surg 2005;42:309; J Vasc Surg 2006;44:554; J Vasc Surg 2006;44:815). For the purpose of DNA-array, they were re-genotyped by means of array-techniques resulting in a 100% matching. Intergroup statistical comparisons were performed. In the risk computation, the FPN1 -8GG genotype had an overall CVD risk of 4.3 (95% CI, 1.6-12) and a VLU risk of 5.2 (95% CI, 1.9-15) virtually the same among primary VLU (4.98; 95% CI, 1.82-14.9). The MMP12-82AA genotype had a VLU risk of 1.96 (95% CI, 1.18-3.2) only in primary VLU (P = .01). In the genotype-ulcer size association studies, from a subgroup of 167 cases, we observed a smaller mean ulcer size in the MMP12 GG-genotype compared with the other genotypes (P = .001). Combining the present results with our previous published data on the same population, we suggest them to apply as tentative prognostic indicators in primary CVD. CONCLUSION: By analyzing simultaneously selected SNPs, it might be possible to glean precious information in predicting VLU onset or in stratifying patients according to their potential to heal. Although significant, our findings must be considered preliminary and the proposed prognostic indicators considered with caution, before ulterior more extensive studies in different populations can eventually confirm the present findings.

Gene polymorphisms in folate metabolizing enzymes in adult acute lymphoblastic leukemia: effects on methotrexate-related toxicity and survival.

BACKGROUND: The antifolate agent methotrexate is an important component of maintenance therapy in acute lymphoblastic leukemia, although methotrexate-related toxicity is often a reason for interruption of chemotherapy. Prediction of toxicity is difficult because of inter-individual variability susceptibility to antileukemic agents. Methotrexate interferes with folate metabolism leading to depletion of reduced folates. DESIGN AND METHODS: The aim of this study was to investigate the influence of polymorphisms for folate metabolizing enzymes with respect to toxicity and survival in adult patients with acute lymphoblastic leukemia treated with methotrexate maintenance therapy. To this purpose, we evaluated possible associations between genotype and hematologic and non-hematologic toxicity and effects on survival at 2 years of follow-up in patients with acute lymphoblastic leukemia. RESULTS: Polymorphisms in the genes encoding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and in dihydrofolate reductase (DHFR 19 bp deletion) significantly increased the risk of hepatotoxicity in single (odds ratio 5.23, 95% confidence interval 1.13-21.95 and odds ratio 4.57, 95% confidence interval 1.01-20.77, respectively) and in combined analysis (odds ratio 6.82, 95% confidence interval 1.38-33.59). MTHFR 677C>T also increased the risk of leukopenia and gastrointestinal toxicity, whilst thymidylate synthase 28 bp repeat polymorphism increased the risk of anemia (odds ratio 8.48, 95% confidence interval 2.00-36.09). Finally, patients with MTHFR 677TT had a decreased overall survival rate (hazard ratio 2.37, 95% confidence interval 1.46-8.45). CONCLUSIONS: Genotyping of folate polymorphisms might be useful in adult acute lymphoblastic leukemia to optimize methotrexate therapy, reducing the associated toxicity with possible effects on survival.

Coagulation gene predictors of photodynamic therapy for occult choroidal neovascularization in age-related macular degeneration.

PURPOSE: To determine whether different coagulation-balance genetic polymorphisms explain the variable clinical outcomes of photodynamic therapy with verteporfin (PDT-V) in Caucasian patients with occult subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). METHODS: The clinical records of consecutive patients with AMD-related occult CNV, treated with PDT-V for evidence of disease progression, were retrospectively examined. Eighty-four eligible subjects were subdivided into responders and nonresponders based on CNV responsiveness to the first PDT-V over a 3-month period. Six gene polymorphisms (i.e., factor V G1691A, prothrombin G20210A, factor XIII-A G185T, methylenetetrahydrofolate reductase C677T, methionine synthase A2756G, and methionine synthase reductase A66G) were genotyped in each patient. Logistic regression analyses were performed to explore the predictive role of phenotypic and genotypic variables for PDT-V effectiveness. RESULTS: Regression models documented that PDT-V nonresponders were more frequently patients with the hyperfibrinolytic G185T mutation of factor XIII-A (odds ratio [OR], 0.28; 95% confidence interval [CI], 0.11-0.73; P < 0.01). Univariate logistic regression was indicative of an overrepresentation of PDT-V responders among the combined carriers of thrombophilic factor V 1691A and prothrombin 20210A alleles (OR = 3.8; 95% CI: 0.94-15.6; P = 0.07). All the other predictors considered did not significantly influence the short-term CNV responsiveness to PDT-V. CONCLUSIONS: These data provide evidence of the presence of a pharmacogenetic relationship between peculiar coagulation-balance genetic backgrounds and different levels of PDT-V effectiveness in patients with AMD with occult CNV.

Time- and dose-dependent effects of chronic wound fluid on human adult dermal fibroblasts.

BACKGROUND: Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins. OBJECTIVES: We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length. METHODS: Fibroblasts were treated with 60, 240, and 720 microg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, alpha-smooth muscle actin expression by immunofluorescence, and senescence-associated beta-galactosidase activity. RESULTS: CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast-myofibroblast differentiation nor to changes in cellular senescence. CONCLUSIONS: This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing.

Predictive role of coagulation-balance gene polymorphisms in the efficacy of photodynamic therapy with verteporfin for classic choroidal neovascularization secondary to age-related macular degeneration.

OBJECTIVES: Age-related macular degeneration (AMD) represents the leading cause of blindness in Western populations. The majority of severe vision loss occurs in the exudative form of AMD, characterized by the development of choroidal neovascularization (CNV) beneath the fovea. Photodynamic therapy with verteporfin (PDT-V) represents one of the most largely employed modality that maybe achieves the subfoveal CNV inactivation in AMD patients. Although several ocular factors have been hitherto investigated as predictors, these researches have weakly contributed to PDT-V optimization. As PDT-V benefit is determined by CNV photothrombosis, we have retrospectively studied several coagulation-balance gene polymorphisms as predictors of PDT-V efficacy. METHODS: Ninety Caucasian patients with neovascular AMD were subdivided in responder and nonresponder, on the basis of CNV responsiveness to PDT-V application. Six gene polymorphisms, that is factor V G1691A, prothrombin G20210A, factor XIII-A G185T, methylenetetrahydrofolate reductase C677T, methionine synthase A2756G, and methionine synthase reductase A66G, were genotyped in the entire cohort. RESULTS: Logistic regression models showed that PDT-V responders were more prevalent within patients with prothrombin G20210A mutation [odds ratio (OR)=5.6, 95% confidence interval (CI) (1.2, 27.2), P=0.03], and within methylenetetrahydrofolate reductase 677T carriers [OR=6.9, 95% CI (2.7, 18.1), P<0.001]. Conversely, PDT-V nonresponders were overrepresented in carriers for factor XIII-A 185T [OR=0.13, 95% CI (0.05, 0.36), P<0.001]. CONCLUSION: These results provide evidences for the presence of pharmacogenetic relationship between peculiar coagulation-balance gene polymorphisms and different levels of PDT-V effectiveness in patients with AMD-related CNV.

Poor responsiveness to clopidogrel: drug-specific or class-effect mechanism? Evidence from a clopidogrel-to-ticlopidine crossover study.

OBJECTIVES: This study was designed to investigate whether poor responders to thienopyridines after clopidogrel remain so even after ticlopidine administration (class effect) or whether a drug-specific effect exists between currently available thienopyridines. BACKGROUND: Whether clopidogrel poor responders also display inadequate platelet inhibition after ticlopidine administration remains undefined. METHODS: Platelet aggregation (PA) was measured in 143 patients, while they were taking aspirin, with light transmission aggregometry using adenosine diphosphate as an agonist at baseline (T0) and at clopidogrel steady state (T1). After T1) clopidogrel was stopped and substituted with ticlopidine. Then PA was assessed at ticlopidine steady state (T2). Resistance was defined as an absolute difference between T0 and after-treatment (T1 or T2) PA < or =10%. RESULTS: Clopidogrel and ticlopidine responsiveness was normally distributed; PA at T1 did not differ compared with T2. Thirty (21%) and 28 (19%) patients were clopidogrel and ticlopidine nonresponders, respectively. Only 5 patients (3.5%) were nonresponders to both clopidogrel and ticlopidine (class effect), whereas 25 patients (83%) who were clopidogrel nonresponders at T1 were responsive to ticlopidine, reaching a higher level of platelet inhibition at T2 (PA 69 +/- 15 vs. 44 +/- 18, p < 0.01) (drug-specific response). On the other hand, 23 patients who were responsive to clopidogrel showed resistance to ticlopidine at T2 (PA 46 +/- 15 vs. 70 +/- 15, p < 0.01) (drug-specific response). CONCLUSIONS: Poor responsiveness to either clopidogrel or ticlopidine at steady state was common, whereas nonresponders to both drugs were relatively infrequent (3.5%, 95% confidence interval 1.5% to 7.9%), suggesting that poor response to thienopyridines may frequently be a drug-specific mechanism.

Factor XIIIA-V34L and factor XIIIB-H95R gene variants: effects on survival in myocardial infarction patients.

It has been demonstrated recently that coagulation factor XIII (FXIII) plays an extraordinary role in myocardial healing after infarction, improving survival in a mouse model. Common FXIII gene variants (i.e. FXIIIA-V34L and FXIIIB-H95R) significantly influence the molecular activity. To evaluate whether there is a relationship between the two FXIII gene variants and survival in patients after myocardial infarction (MI), V34L and H95R were PCR-genotyped in a cohort of 560 MI cases and follow-up was monitored. Cases with ST-segment elevation MI (STEMI) were 416 (74.3%) and 374 of these were treated with primary percutaneous coronary intervention (PCI) (89.9%). The remaining 144 patients showed non-ST-segment elevation MI (NSTEMI) at enrollment. The combined endpoint was the occurrence of death, re-infarction, and heart failure. Kaplan-Meier analysis at one year yielded an overall rate for adverse events of 24.5% with a lower incidence in the L34-carriers (28.8% vs 17.1%; log-rank, P = 0.00025), similar to that of the 416 STEMI (23.8%) being (28.0% and 16.9%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Primary PCI-group had a slight lower incidence (22.9%) of adverse events (26.8% and 17.1%; VV34- and L34-carriers respectively; log-rank, P = 0.009). During hospitalization, 506 patients received PCI (374 primary PCI and 132 elective PCI). Significance was conserved also in the overall PCI-group (28.6% and 17.8%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Similar findings were observed at 30 days follow-up. Cases carrying both FXIII variants had improved survival rate (log-rank, P = 0.019). On the other hand, minor bleeding complications were found increased in L34-carriers (P = 0.0001) whereas major bleeding complications were not. Finally, more direct evidence on the role of FXIII molecule on survival might come from the fact that despite significant FXIII antigen reductions observed in cases after MI, regardless the FXIII genotype considered, L34-carriers kept almost normal FXIII activity (VV34- vs L34-carriers; P < 0.001). We conclude that FXIII L34-allele improves survival after MI in all the groups analyzed, possibly through its higher activity associated with assumable positive effects on myocardial healing and recovered functions. Genetically determined higher FXIII activity might influence post-MI outcome. This paves the way for using FXIII molecules to improve myocardial healing, recovery of functions, and survival after infarction.

Methylenetetrahydrofolate reductase C677T and A1298C gene variants in adult non-Hodgkin's lymphoma patients: association with toxicity and survival.

BACKGROUND AND OBJECTIVES: Common methylenetetrahydrofolate reductase gene variants (MTHFR C677T and A1298C) have been described to have opposite effects on cancer patients. They may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g. methotrexate) are utilized. We analyzed 110 patients with high-grade non-Hodgkin's lymphoma (NHL), 68 of whom were eligible for a chemotherapy combination containing methotrexate (MACOP-B) and 42 for chemotherapy without methotrexate (CHOP). DESIGN AND METHODS: Patients were genotyped by polymerase chain reaction and stratified by MTHFR variants. These data were related to the toxicity (WHO grade GO-4) that the patients suffered and their survival. Overall 64 cases (58.2%) developed some form of toxicity and 23 (20.9%) had grade 3/4 toxicity. RESULTS: When considering toxicity of any grade (grade 1-4), the 677TT genotype was significantly over-represented among cases with mucositis (OR=4.85; 95% CI, 1.47-15.97; p=0.009) and those with hepatic toxicity (OR=3.43; 95% CI, 0.99-11.86; p=0.052). Sub-analyses in the group treated with MACOP-B showed a slight increase in the risk of developing mucositis (OR=5.22; 95% CI, 1.20-27.27; p=0.03), and a strong increase in the risk of hepatic toxicity (OR=7.08; 95% CI, 1.38-36.2; p=0.019) and thrombocytopenia (OR=7.69, 95% CI 1.0-58.94; p=0.05). Interestingly, compared to the risk of developing toxicity of any grade, the risk of developing severe (grade 3/4) mucositis was almost doubled in the whole group of cases with 677TT (OR=8.13; 95% CI 1.61-41.04; p=0.011) and dramatically increased in the MACOP-B-treated cases with this gene variant (OR=24.6; 95% CI 2.49-87.41; p=0.001). There were significant results for 1298CC cases exclusively for mucositis (any grade, OR=5.33; 95% CI, 1.25-22.70; p=0.023 and OR=9.15; 95% CI, 1.14-73.41; p=0.037; for the whole group and the MACOP-B-treated group, respectively). Similarly, the risk of 1298CC patients developing severe mucositis increased (OR=9.24; 95% CI, 1.47-58.0; p=0.017 and OR=11.53; 0.93-143.18; p=0.057; in the whole group and in the MACOP-B-treated group, respectively). Event-free survival analysis revealed a lower probability of event-free survival at 5 years for 677T-carriers (log-ranks, p=0.05 and p=0.07 in the whole group and in the MACOP-B-treated group, respectively). More significant results were obtained when 1298CC cases were excluded from the reference group (log-ranks, p=0.03 and p=0.04, respectively). No significant associations were found in the CHOP-treated group. INTERPRETATION AND CONCLUSIONS: Our data suggest that MTHFR gene variants play a critical role in NHL outcome, possibly by interfering with the action of methotrexate with significant effects on toxicity and survival. Genotyping of folate pathway gene variants might be useful to enable reduction of chemotherapy toxicity and/or to improve survival by indicating when dose adjustments or alternative treatments are necessary.

Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY Study.

OBJECTIVES: The purpose of this study was to evaluate the value of platelet reactivity (PR) in predicting the response to treatment and outcome in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention assisted by glycoprotein (GP) IIb/IIIa inhibition. BACKGROUND: There is limited prognostic information on the role of spontaneous or drug-modulated PR in STEMI patients. METHODS: The PR was measured with Platelet Function Analyzer (PFA)-100 and light transmission aggregometry (LTA) using adenosine diphosphate as agonist in 70 consecutive STEMI patients at entry (PR-T0), 10 min after GP IIb/IIIa bolus (PR-T1), and discharge (PR-T2) and in 30 stable angina (SA) patients (PR-SA). Complete platelet inhibition (CPI) was based on closure time >300 s by PFA-100 and percentage inhibition of platelet aggregation >95% by LTA. Clinical, electrocardiographic, and angiographic responses to treatment during 1-year follow-up were collected. RESULTS: According to both techniques, PR-T0 was higher than: 1) PR-T2 and PR-SA; 2) in those without CPI at T1; and 3) in patients with final Thrombolysis In Myocardial Infarction (TIMI) flow grade <3. The PR-T0 assessed with PFA-100 correlated with: 1) corrected TIMI frame count (r = -0.6, p < 0.001); 2) ST-segment resolution (r = 45, p < 0.001); and 3) creatine kinase-MB (r = -0.47, p < 0.001). At 1 year, patients with high PR-T0 showed an adjusted 5- to 11-fold increase in the risk of death, reinfarction, and target vessel revascularization (hazard ratio [HR] 11, 95% confidence interval [CI] 1.5 to 78 [p = 0.02] in PFA-100; HR 5.2, 95% CI 1.1 to 23 [p = 0.03] in LTA). CONCLUSIONS: The PR at entry affects response to GP IIb/IIIa inhibition, mechanical treatment, and long-term outcome in STEMI patients undergoing primary intervention.

Influence of gene polymorphisms in ulcer healing process after superficial venous surgery.

OBJECTIVE: Role of superficial venous surgery in reducing the time it takes for ulcers to heal is still controversial, although all studies confirm a significant reduction in ulcer recurrences. Recently, the HFE-C282Y and FXIII-V34L gene variants demonstrated a role in the risk of venous ulceration in primary chronic venous disorder (CVD) and in modulating lesion size in chronic venous ulcer (CVU), respectively. This study was conducted to investigate the role of HFE-C282Y and FXIII (V34L and P564L) gene variants in ulcer healing time after superficial venous surgery, by assessing the outcome of a cohort of homogeneous CVU patients. METHODS: The study selected 91 patients affected by primary CVU (CEAP C6, Ep, Asp, Pr), with the exclusion of any other comorbidity factor involved in delayed healing process, who underwent surgery. We assessed the ulcer area and the healing time. Patients were genotyped by polymerase chain reaction for FXIII (V34L and P564L) and for HFE-C282Y substitutions. RESULTS: Globally, CVU cases had a postoperative mean healing time of 8.5 +/- 5.7 weeks. For the subset of cases above and below the median value (M = 8.0 weeks), FXIII-V34L genotype distribution significantly differed (P < .0001). In addition, Kaplan-Meier analysis yielded specific healing time profiles for the different FXIII-V34L classes of genotype (P = .00001), with an increased risk of delayed healing for the FXIII-VV genotype (hazard ratio, 4.14; 95% confidence interval, 2.1 to 8.2; P = .00005). Although FXIII-P54L genotype distributions did not differ, homozygous 564LL cases (P = .005) and double carriers for both FXIII variants (P < .0001), had a significantly reduced healing time vs wild types. No differences in healing time were observed between carriers and noncarriers of the HFE-C282Y variant, whereas when these cases were stratified by FXIII-V34L genotypes, the L34 carriers had a significantly shorter healing time, irrespective of the HFE genotype. CONCLUSION: The FXIII-34L variant was significantly associated with shorter healing time after superficial venous surgery, suggesting a role in the healing and tissue regeneration phases. Conversely, HFE-C282Y, despite its role in ulcer establishment, did not affect the postoperative healing time. In perspective, the identification of patients with a poor prognosis may give clinicians the opportunity to modify management and to target tailored therapies in the view of a new and alternative concept of treatment based on pharmacogenomics.

Prognostic role of factor XIII gene variants in nonhealing venous leg ulcers.

OBJECTIVE: Many factors impair healing of chronic venous ulcer (CVU), and many theories have been proposed to explain their pathogenesis. Coagulation factor XIII (FXIII) influences tissue regeneration and angiogenesis with effects on wound healing. Because FXIII properties depend upon its genetic variants, we investigated whether intragene polymorphisms may have modulating effects on the CVU area. METHODS: The study included 121 patients with nonhealing CVUs (CEAP clinical class C6) that included 67% with primary chronic venous disease (CVD), 26% with post-thrombotic ulcers, and 7% with mixed ulcer origin. Polymerase chain reaction was used to genotype them for Val34Leu, Pro564Leu, and Tyr204Phe variants in the FXIII-A subunit gene and for His95Arg variant in the FXIII-B subunit gene. The same variants were analyzed in 102 controls, healthy subjects who were case-matched by age and gender. RESULTS: Genotype distribution for all polymorphisms investigated was not significantly different between cases and controls. Conversely, our CVU cases had a mean ulcer area inversely related with the presence of both Leu34 and Leu564 alleles (ValVal, 12.3 +/- 22.4 cm2 vs LeuLeu, 3.9 +/- 2.6 cm2, P = .002; ProPro, 10.2 +/- 21.2 cm2 vs LeuLeu, 2.9 +/- 1.4 cm2, P = .002). In combined analysis, those cases who were wild-type for both variants (ValVal34/ProPro564) had a further increase in mean ulcer size compared with cases carrying both variants (Leu34/Leu564) (13.3 +/- 27.1 cm2 vs 5.2 +/- 5.6 cm2; P = .034). CONCLUSIONS: No correlation exists between FXIII genotypes and the prevalence of chronic venous ulcers, thus demonstrating that FXIII polymorphisms have no role in ulcer development. In contrast, FXIII-gene variants, in particular the non-wild-type alleles Leu34 and Leu564, were associated with a smaller venous ulcer surface and might have favorable effects on reparative processes.

The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogenesis.

Chronic venous stasis determines red blood cell extravasation and either dermal hemosiderin deposits or iron-laden phagocytes. Several authors have suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs) or else down-regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with chronic venous disease (CVD), cause lesions in only some individuals, whereas in others they do not. We hypothesized that such individual differences could be genetically determined and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD more than six times (OR = 6.69; 1.45-30.8; p = 0.01). Patients carrying the H63D variant have an earlier age of ulcer onset, by almost 10 years (p > 0.004). The increased risk of skin lesion and the early age of onset of the disease in HFE carriers confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than those of the wild type. We hypothesize that the physiologic iron protective mechanisms are affected by the HFE mutations and should be investigated in all diseases characterized by the combination of iron overload and inflammation.