Genetic factors contributing to late adverse musculoskeletal effects in childhood acute lymphoblastic leukemia survivors.

BACKGROUND: A substantial number of survivors of childhood acute lymphoblastic leukemia (ALL) suffer from treatment-related late adverse effects. While multiple studies have identified the effects of chemotherapeutics and radiation therapy on musculoskeletal outcomes, few have investigated their associations with genetic factors. METHODS: Here we analyzed musculoskeletal complications in relation to common and rare genetic variants derived through whole-exome sequencing of the PETALE cohort. Top-ranking associations were further assessed through stratified and multivariate analyses. RESULTS: DUOX2 variant was associated with skeletal muscle function deficit, as defined by peak muscle power Z score ≤ -2 SD (P = 4.5 × 10-5 for genotyping model). Upon risk stratification analysis, common variants in the APOL3, COL12A1, and LY75 genes were associated with Z score ≤ -2 SD at the cross-sectional area (CSA) at 4% radial length and lumbar bone mineral density (BMD) in high-risk patients (P ≤ 0.01). The modulation of the effect by risk group was driven by the interaction of the genotype with cumulative glucocorticoid dose. Identified variants remained significant throughout multivariate analyses incorporating non-genetic factors of the studied cohort. CONCLUSION: This exploratory study identified novel genetic variants associated with long-term musculoskeletal impairments in childhood ALL survivors. Replication in an independent cohort is needed to confirm the association found in this study.

Association study of candidate DNA-repair gene variants and acute graft versus host disease in pediatric patients receiving allogeneic hematopoietic stem-cell transplantation.

Acute Graft versus Host Disease (aGvHD) grades 2-4 occurs in 15-60% of pediatric patients undergoing allogeneic haematopoietic stem-cell transplantation (allo-HSCT). The collateral damage to normal tissue by conditioning regimens administered prior to allo-HSCT serve as an initial trigger for aGvHD. DNA-repair mechanisms may play an important role in mitigating this initial damage, and so the variants in corresponding DNA-repair protein-coding genes via affecting their quantity and/or function. We explored 51 variants within 17 DNA-repair genes for their association with aGvHD grades 2-4 in 60 pediatric patients. The cumulative incidence of aGvHD 2-4 was 12% (n = 7) in the exploratory cohort. MGMT rs10764881 (G>A) and EXO rs9350 (c.2270C>T) variants were associated with aGvHD 2-4 [Odds ratios = 14.8 (0 events out of 40 in rs10764881 GG group) and 11.5 (95% CI: 2.3-191.8), respectively, multiple testing corrected p ≤ 0.001]. Upon evaluation in an extended cohort (n = 182) with an incidence of aGvHD 2-4 of 22% (n = 40), only MGMT rs10764881 (G>A) remained significant (adjusted HR = 2.05 [95% CI: 1.06-3.94]; p = 0.03) in the presence of other clinical risk factors. Higher MGMT expression was seen in GG carriers for rs10764881 and was associated with higher IC50 of Busulfan in lymphoblastoid cells. MGMT rs10764881 carrier status could predict aGvHD occurrence in pediatric patients undergoing allo-HSCT.

SKIN DOSE MAPPING IN INTERVENTIONAL CARDIOLOGY: A PRACTICAL SOLUTION.

Numerous cases of radiation-induced tissue reactions following interventional cardiology (IC) procedures have been reported, resulting in the need for an optimized and personalized dosimetry. At present, there are many fluoroscopy units without Digital Imaging and Communications in Medicine (DICOM) Radiation Dose Structured Report globally installed. Many of these have not been updated yet, and may never be, therefore, the main objectives of this paper are to develop an offline skin dose mapping application, which uses DICOM headers for the peak skin dose (PSD) assessment and to compare the PSD assessment results to XR-RV3 Gafchromic film for common IC procedures. The mean deviation between the measured and the calculated PSD was 8.7 ± 26.3%. Simulated skin dose map showed good matching with XR-RV3 Gafchromic film. The skin dose mapping application presented in this paper is an elegant solution and a suitable alternative to XR-RV3 Gafchromic film.

Is there a relationship between vitamin D nutritional status and metabolic syndrome in childhood acute lymphoblastic leukemia survivors? A PETALE study.

BACKGROUND: Treatment of childhood acute lymphoblastic leukemia (cALL) has reached unprecedented success leading to survival rates reaching 90%. This is regrettably linked to increased risk of developing long-term health-related sequels into early adulthood. OBJECTIVE: This study aims at assessing the relationship between the vitamin D status and metabolic biomarkers in PETALE, a well-characterized cohort of cALL survivors. RESULTS: We demonstrate that 15.9% of the study participants exhibited 3 or more metabolic syndrome (MetS) risk factors. We also show a direct relationship between s25OHD3 and plasma HDL-Cholesterol concentrations in female but not male participants. CONCLUSION: Our data, from a metabolically well-described cohort, support a modest role for vitamin D in lipid metabolism in childhood leukemia survivors. The major outcome of this study is the strong association between HDL-Cholesterol concentration and s25OHD3 only in female subjects, thereby conveying vitamin D a gender-specific cardio-protective effect. cALL survivors represent a population at higher risk for secondary diseases. For this reason thorough nutritional evaluation, including vitamin D should be part of the regular follow-up.

Vitamin D nutritional status and bone turnover markers in childhood acute lymphoblastic leukemia survivors: A PETALE study.

BACKGROUND: The remarkable progress in the treatment of childhood acute lymphoblastic leukemia (cALL) has led to a survival rate reaching 90%. This success story is unfortunately linked to increased risk of impaired skeletal mass accumulation during childhood and adolescence, predisposing the patients to osteoporosis and pathological fractures at adulthood. OBJECTIVE: This study aims at characterizing the vitamin D status and bone health biomarkers in a well-characterized cohort of cALL survivors. RESULTS: Food frequency questionnaires reveal that (i) the total vitamin D intake varies greatly (44-2132 IU/d), (ii) only 16.8% of the participants consume vitamin D supplements, and (iii) 74% of survivors' intakes are below the Recommended Daily Intakes (400 IU/d). For the 42 participants taking vitamin D supplements, the median (2.5-97.5%iles) intake is 600 IU/d (21.2-1972 IU/d). Sixteen participants are vitamin D deficient (<30 nM) and 66 insufficient (≥30 - <50 nM). Serum 24,25(OH)2D3 concentrations are directly related to those of 25OHD3, and those of 3-epi-25OHD3 below the Lower Limit of Quantification in most samples. The participants' serum concentrations of cross-linked C-telopeptide of type-I collagen and intact amino-terminal pro-peptide of type-I collagen decrease steadily with age, leveling at adulthood, and are at all times higher in males. CONCLUSION: The present study shows that the prevalence of vitamin D insufficiency or deficiency is not greater in cALL survivors compared to the general Canadian population despite low vitamin D food and supplement intakes. Furthermore, there seem to be no overt imbalance in the gender- and age-adjusted serum bone turnover marker concentrations.

Impact of genetic polymorphisms determining leukocyte/neutrophil count on chemotherapy toxicity.

Neutropenia and infection are major dose-limiting side effects of chemotherapy. The risk of initial infection and subsequent complications are directly related to the depth and duration of neutropenia. Recent genome-wide association studies identified variants in DARC and CXCL2 genes, and in ORMDL3-GSDMA-CSF3 locus on chromosome 17q21 that influence white blood cell and neutrophil counts in healthy individuals. To investigate whether polymorphisms in these loci in conjunction with chemotherapy may modulate risk of treatment complications, we analyzed 21 SNPs across these genes for an association with chemotherapy-related neutropenia and infection in 286 Caucasian children with acute lymphoblastic leukemia. After correction for multiple testing, DARC polymorphism rs3027012 in 5'-UTR was associated with higher risk of low absolute phagocyte count (APC<500 and <1000 cells per microliter, P=0.001 and P<0.0005, respectively) and hospitalization due to febrile neutropenia (P=0.002). Protective effect was instead seen for DARC rs12075 A to G substitution (P=0.004). The SNP rs3859192 in the GSDMA were associated with hospitalization due to infection (P=0.004); infection was also modulated in the additive manner by the CXCL2 rs16850408 (P=0.002). This study shows for the first time that the variations in DARC, GSDMA and CXCL2 genes may play a role in the onset of chemotherapy complications.

How to interpret high levels of distress when using the Distress Thermometer in the long-term follow-up clinic? A study with Acute Lymphoblastic Leukemia survivors.

OBJECTIVES: Recent guidelines recommend to assess emotional distress in pediatric oncology during treatment and in after care. One tool used to do this is the distress thermometer (DT), a simple tool which has almost exclusively been studied in its screening abilities. Given its increased used as a measure of distress per se, it is necessary to document its concurrent validity. The goal of this study was to identify clinical domains (eg, depression, anxiety) and individual symptoms associated with pediatric cancer survivors' rating on the DT. PARTICIPANTS: To do so we used data collected from 84 young (≤18 years old), and 120 older (>18 years old) survivors who were treated for pediatric leukemia. METHODS: Participants responded to self-report questionnaires as part of a research visit. RESULTS: Results from stepwise regressions show that in the younger group, high scores on the thermometer were associated with higher negative affectivity only. In adults, high scores were associated with higher anxiety, higher negative affectivity, and lower positive affectivity. When exploring associations with individual items, we found that the main emotional tone reflected by the thermometer score was anxiety. CONCLUSIONS: Interpreting ratings on the thermometer should probably focus on anxiety in childhood cancer survivors. This widely used tool also does not measure the same domains in young versus older survivors, so that age groups should be considered separately in future work.

A 2-year dyadic longitudinal study of mothers' and fathers' marital adjustment when caring for a child with cancer.

OBJECTIVE: Studies examining interrelationships within parental couples confronted with pediatric cancer are scarce. This study explored dyadic longitudinal associations between both partners' family functioning and mood at diagnosis, and marital adjustment 2 years later. METHOD: Parents of children (n = 47 couples) with acute lymphoblastic leukemia (ALL) completed the Family Well-Being Assessment and Profile of Mood States-Bipolar Form at diagnosis, and the Locke-Wallace Marital Adjustment Test 2 years post diagnosis. Multilevel linear models using the actor-partner interdependence model (APIM) and controlling for baseline marital adjustment were conducted to evaluate within subject and dyadic longitudinal effects. RESULTS: For mothers, better marital adjustment 2 years post diagnosis was associated with perception of greater family support and less role conflict and role overload at diagnosis. For fathers, better marital adjustment 2 years post-diagnosis was associated with perception of less role conflict, greater role ambiguity, and being more tired at diagnosis, as well as their partner's perception of less role conflict at diagnosis. CONCLUSIONS: These findings highlight the importance of considering both partners' perspectives in understanding marital adjustment across treatment phases in parents of children with ALL. Early interventions for couples should be tailored to meet each partner's needs in order to foster resilience within the couple.

Adverse neuropsychological effects associated with cumulative doses of corticosteroids to treat childhood acute lymphoblastic leukemia: A literature review.

Corticosteroids (CS) are an essential component of childhood acute lymphoblastic leukemia treatments (cALL). Although there is evidence that daily doses of CS can have neuropsychological effects, few studies have investigated the role of cumulative doses of CS in short- and long-term neuropsychological effects in cALL. The aims of this review were to identify the measures used for documenting adverse neuropsychological effects (ANEs) of CS treatment and to study the association between cumulative doses of CS and the presence of ANEs. Twenty-two articles met the inclusion criteria. A variety of measures were used to evaluate outcomes in the domains of emotion, behaviour, neurocognition, and fatigue/sleep. The results suggest that we cannot conclude in favour of an association between the cumulative dosage of CS and ANEs. Yet, several factors including the heterogeneity of measures used to evaluate outcomes and reporting biases may limit the scope of the results. We offer several recommendations that could help improve the future published evidence on ANEs in relation to CS treatment in cALL.

Influence of glutathione S-transferase gene polymorphisms on busulfan pharmacokinetics and outcome of hematopoietic stem-cell transplantation in thalassemia pediatric patients.

Hematopoietic stem-cell transplantation (HSCT) is currently the only curative therapeutic option for the treatment of thalassemia. In spite of the high cure rate, HSCT can lead to life-threatening adverse events in some patients. Busulfan (Bu) is a key component of the conditioning regimen prior to HSCT. Inter-individual differences in Bu pharmacokinetics (PK) are hypothesized to influence Bu efficacy and toxicity. Since Bu is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship of GSTA1 and GSTM1 genotypes with first-dose PK and HSCT outcomes in 44 children with thalassemia intermedia and thalassemia major. All children received a myeloablative conditioning regimen with IV Bu. Association analysis revealed a relationship between GSTA169C>T (or haplotype *A/*B) and first Bu dose PK that was dependent on sex and Pesaro risk classification (PRC). Among female patients and patients with PRC I-II, homozygous individuals for the GSTA1T-69 allele defining haplotype *B, had higher Bu exposure and lower clearance (P⩽0.01). Association with HSCT outcomes showed that patients with the GSTM1 null genotypes had higher occurrence of regimen-related toxicity (P=0.01). These results suggest that GST genotypes could be useful to tailor the first Bu dose accordingly to improve HSCT outcome.

Polymorphisms of ABCC5 and NOS3 genes influence doxorubicin cardiotoxicity in survivors of childhood acute lymphoblastic leukemia.

Anthracyclines are efficient chemotherapy agents. However, their use is limited by anthracycline-induced cardiotoxicity (CT). We investigated the influence of polymorphisms in doxorubicin metabolic and functional pathways on late-onset CT as estimated by echocardiography in 251 childhood acute lymphoblastic leukemia (cALL) patients. Association analyses revealed a modulating effect of two variants: A-1629 T in ABCC5, an ATP-binding cassette transporter, and G894T in the NOS3 endothelial nitric oxide synthase gene. Individuals with the ABCC5 TT-1629 genotype had an average of 8-12% reduction of ejection (EF) and shortening fractions (SF; EF: P<0.0001, and SF: P=0.001, respectively). A protective effect of the NOS3 TT894 genotype on EF was seen in high-risk patients (P=0.02), especially in those who did not receive dexrazoxane (P=0.002). Analysis of an additional cohort of 44 cALL patients replicated the ABCC5 association but was underpowered for NOS3. In summary, we identified two biomarkers that may contribute to cALL anthracycline CT risk stratification.

Determinants Of Oral corticosteroid Responsiveness in Wheezing Asthmatic Youth (DOORWAY): protocol for a prospective multicentre cohort study of children with acute moderate-to-severe asthma exacerbations.

INTRODUCTION: Oral corticosteroids are the cornerstone of acute asthma management in the emergency department. Recent evidence has raised doubts about the efficacy of this treatment in preschool-aged children with viral-induced wheezing and in smoking adults. The aims of the study were to: (1) document the magnitude of response to oral corticosteroids in children presenting to the emergency department with moderate or severe asthma; (2) quantify potential determinants of response to corticosteroids and (3) explore the role of gene polymorphisms associated with the responsiveness to corticosteroids. METHODS AND ANALYSIS: The design is a prospective cohort study of 1008 children aged 1-17 years meeting a strict definition of asthma and presenting with a clinical score of ≥4 on the validated Pediatric Respiratory Assessment Measure. All children will receive standardised severity-specific treatment with prednisone/prednisolone and cointerventions (salbutamol with/without ipratropium bromide). Determinants, namely viral aetiology, environmental tobacco smoke and single nucleotide polymorphism, will be objectively documented. The primary efficacy endpoint is the failure of emergency department (ED) management within 72 h of the ED visit. Secondary endpoints include other measures of asthma severity and time to recovery within 7 days of the index visit. The study has 80% power for detecting a risk difference of 7.5% associated with each determinant from a baseline risk of 21%, at an α of 0.05. ETHICS AND DISSEMINATION: Ethical approval has been obtained from all participating institutions. An impaired response to systemic steroids in certain subgroups will challenge the current standard of practice and call for the immediate search for better approaches. A potential host-environment interaction will broaden our understanding of corticosteroid responsiveness in children. Documentation of similar effectiveness of corticosteroids across determinants will provide the needed reassurance regarding current treatment recommendations. RESULTS: Results will be disseminated at international conferences and manuscripts targeted at emergency physicians, paediatricians, geneticists and respirologists. TRIAL REGISTRATION NUMBER: This study is registered at Clinicaltrials.gov (NCT02013076).

The association of cytochrome P450 genetic polymorphisms with sulfolane formation and the efficacy of a busulfan-based conditioning regimen in pediatric patients undergoing hematopoietic stem cell transplantation.

Cytochrome P450 enzymes (CYPs) and flavin-containing monooxygenases (FMOs) likely have a role in the oxidation of intermediate metabolites of busulfan (Bu). In vitro studies to investigate the involvement of these enzymes are cumbersome because of the volatile nature of the intermediate metabolite tetrahydrothiophene (THT) and the lack of sensitive quantitation methods. This study explored the association between the CYP2C9, CYP2C19, CYP2B6 and FMO3 genotypes and sulfolane (Su, a water soluble metabolite of Bu) plasma levels in children undergoing hematopoietic stem cell transplantation (HSCT). The relationship between these genotypes and the effectiveness of myeloablative conditioning was also analyzed. Sixty-six children receiving an intravenous Bu-based myeloablative conditioning regimen were genotyped for common functional variant alleles in CYP2C9 (*2 and *3), CYP2C19 (*2 and *17), FMO3 (rs2266780, rs2266782 and rs1736557) and CYP2B6 (*5 and *9). The plasma levels of Bu and its metabolite Su were measured after the ninth Bu dose in a subset of 44 patients for whom plasma samples were available. The ratio of Bu to Su was considered the metabolic ratio (MR) and was compared across the genotype groups. Higher MRs were observed in CYP2C9*2 and *3 allele carriers (mean±s.d.: 7.8±3.6 in carriers vs 4.4±2.2 in non-carriers; P=0.003). An increased incidence of graft failure was observed among patients with an MR>5 compared with those with MR values <5 (20% vs 0%; P=0.02). In contrast, a significantly higher incidence of relapse and graft failure (evaluated as event-free survival) was observed in patients with malignant disease who carried CYP2B6 alleles with reduced function on both chromosomes compared with carriers of at least one normal allele (100% vs 40%; P=0.0001). These results suggest that CYP2C9 has a role in the oxidation reactions of THT and indicate that it may be possible to predict the efficacy of Bu-based myeloablative conditioning before HSCT on the basis of CYP genotypes and Bu MRs.

The role of the MTHFR 677C>T polymorphism in methotrexate-induced liver toxicity: a meta-analysis in patients with cancer.

Methotrexate (MTX), one of the important pillars in the treatment of different forms of cancer, is associated with the development of hepatotoxicity. The 677C>T variant (rs1801133) in the methylenetetrahydrofolate reductase (MTHFR) gene might affect the development of hepatotoxicity. Results in literature are, however, contradictive. The aim of this study was to evaluate the role of the MTHFR 677C>T polymorphism in MTX-induced hepatotoxicity by analyzing a Dutch cohort of pediatric patients treated with high doses of MTX and subsequently performing a meta-analysis. Ninety-eight patients receiving 542 courses of high-dose MTX were genotyped for the MTHFR 677C>T variant. Hepatotoxicity was evaluated retrospectively according to common terminology criteria for adverse events-National Cancer Institute criteria. The influence of MTHFR 677C>T on hepatotoxicity was examined using a generalized estimating equation (GEE) analysis. A fixed-effect meta-analysis based on this and previous studies investigating the association between the MTHFR 677C>T polymorphism and uniformly coded hepatotoxicity was performed. The GEE analysis showed an increased risk of developing hepatotoxicity for T versus C allele (odds ratio (OR) 1.8; 95% confidence interval (CI) 1.0-3.2, P=0.04). This finding was not supported by the meta-analysis including seven studies and 1044 patients; the OR for the 677T versus C allele was 1.1 (95% CI 0.84-1.5, P=0.25). Heterogeneity between studies was observed, possibly related to differences in MTX dose and leucovorin rescue. In conclusion, in patients with cancer, the MTHFR 677T allele has only a minor role in the development of MTX-induced hepatotoxicity. Observed heterogeneity between studies warrants further study into (tailored) leucovorin rescue.

Glutathione S-transferase gene variations influence BU pharmacokinetics and outcome of hematopoietic SCT in pediatric patients.

BU is a key compound of conditioning regimens in children undergoing hematopoietic SCT (HSCT). Inter-individual differences in BU pharmacokinetics (PKs) might affect BU efficacy and toxicity. As BU is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship between GSTA1, GSTM1 and GSTP1 genotypes with first-dose BU PKs, and the relationship with HSCT outcomes in 69 children receiving myeloablative conditioning regimen. GSTM1 null genotype correlated with higher BU exposure and lower clearance in patients older than 4 years (P ≤ 0.04). In accordance with the suggested functional role, GSTA1*A2 haplotype was associated with lower drug levels and higher drug clearance (P ≤ 0.03). Gene-dosage effect was also observed (P ≤ 0.007). GSTA1 haplotypes were associated with HSCT outcomes. Patients with two copies of haplotype *A2 had better event free survival (P=0.03). In contrast, homozygous individuals for haplotypes *B and *B1 had higher occurrence of veno-occlusive disease (P=0.009). GSTM1 null individuals older than 4 years had more frequently graft versus host disease (P=0.03). In conclusion, we showed that GST gene variants influence BU PK and outcomes of HSCT in children. A model for the dosage adjustment with the inclusion of genetic and non-genetic factors should be evaluated in a future prospective validation cohort.

Polymorphism in multidrug resistance-associated protein gene 3 is associated with outcomes in childhood acute lymphoblastic leukemia.

Multidrug resistance-related proteins (MRPs) 2, 3 and 5 are involved in the efflux of drugs used in acute lymphoblastic leukemia (ALL) treatment. Polymorphisms of these genes were investigated for an association with treatment responses in 273 childhood ALL patients. The MRP3 A-189 allele of the regulatory AT polymorphism was associated with reduced event-free survival (P=0.01). The results remained significant after adjustment for multiple comparisons and in the multivariate analysis. Among patients with an event, the A-189 carriers had significantly higher methotrexate plasma levels (P=0.03). MRP3 A-189 also conferred four times higher risk of a relapse in central nervous system (P=0.01). Patients with this allele tended to have lower frequency of thrombocytopenia grade 2 (P=0.06). Gene reporter assay showed that the haplotype tagged by the A-189 had higher promoter activity (P≤0.01). In conclusion, MRP3 A-189 T polymorphism was associated with treatment responses in ALL, likely due to the change in MRP3 efflux.

Influence of GST gene polymorphisms on busulfan pharmacokinetics in children.

Busulfan (BU) is a key compound in conditioning myeloablative regimens for children undergoing hematopoietic stem cell transplantation (HSCT). There are wide interindividual differences in BU pharmacokinetics, which increase the risk of veno-occlusive disease, graft rejection and disease relapse. As BU is mainly metabolized by glutathione S-transferase (GST), it is hypothesized that functional polymorphisms in GST genes may explain in part the variability in BU pharmacokinetics. We analyzed polymorphisms in GSTA1 (C-69T, A-513G, G-631T, C-1142G), GSTM1 (deletion) and GSTP1 (A1578G, C2293T) genes in 28 children undergoing HSCT. All patients had individualized dosing based on pharmacokinetics after the first dose of intravenous BU. GSTM1-null individuals had higher drug exposure (P(Cmax)=0.008; P(AUC)=0.003; P(Css)=0.02) and lower clearance (P(CL)=0.001). Multivariate regression models showed that, other than the drug dose and age, the GSTM1 genotype was the best predictor of first-dose pharmacokinetic variability. GSTM1-null patients also received lower cumulative BU doses (P=0.02). No association was found between BU exposure and major GSTA1 or GSTP1 gene variants. In children, GSTM1 polymorphism seems to modify BU pharmacokinetics after intravenous drug administration.

Combining several polymorphisms of thymidylate synthase gene for pharmacogenetic analysis.

Thymidylate synthase (TS) is an essential enzyme in proliferating cells and an important target for several chemotherapeutics. Several TS gene polymorphisms correlate with variable TS expression: a double (2R) and triple (3R) 28-bp repeat element, a G to C substitution of the 3R allele and a 6 bp variation in 3'UTR. We have previously shown that childhood acute lymphoblastic leukemia (ALL) patients who are homozygous for the 3R allele had reduced event-free survival (EFS) probabilities. Here, we analyzed all three polymorphisms in an extended group of ALL patients (n=259). The effect of the 3R homozygosity on ALL outcome was confirmed (P=0.006), whereas 6 bp polymorphism did not influence EFS when analyzed separately. No significant difference among 3R3R genotype subgroups, as defined by a G to C substitution, was observed. The haplotype analysis revealed the higher frequency of the 3RC/6 bp+ haplotype (P=0.04) and the protective role of the 2R/6b p- (P=0.04). Consequently, homozygosity for the 6 bp- allele appeared to reduce an event-predisposing effect of 3R variant. Although of importance for translation into the clinical practice, these findings need confirmation in larger studies.