Maria Luisa Serino

Low Folate Levels and Thermolabile Methylenetetrahydrofolate Reductase as Primary Determinant of Mild Hyperhomocystinemia in Normal and Thromboembolic Subjects

Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.

Factor XIII V34L polymorphism modulates the risk of chronic venous leg ulcer progression and extension

Low Factor XIII (FXIII) activity has been reported in the blood of patients with chronic venous leg ulcer (CVU). In vivo studies have described increased wound healing in CVU patients treated with FXIII concentrate, and in vitro studies have shown increased regenerative capacity in FXIII‐treated fibroblasts. In addition, a common G‐to‐T polymorphism in the FXIIIA‐subunit gene (V34L) significantly increases the activity and modifies the cross‐linking properties of the FXIII molecule and this variant has been investigated as a protective factor against thrombosis, a recognized risk factor for CVU establishment. Therefore, the role of FXIII levels, FXIII V34L, FVR506Q, and FIIG20210A, common gene polymorphisms in the pathogenesis of CVU was investigated. Ninety‐one patients with CVU and 195 healthy controls (91 of them sex‐ and age‐matched) were PCR‐genotyped for the FXIIIV34L, FVR506Q, and FIIG20210A substitutions and FXIIIA‐subunit levels were determined by immuno‐electrophoresis. The extent of the venous ulcer surface in patients was measured by computer software. The allele frequency and the genotype distribution of the FXIII polymorphism did not show significant differences between the whole group of cases and controls as well as prothrombin variants did. On the contrary, the FVR506Q variant (FV Leiden) allele was more frequent in patients, yielding a significant OR value of 5.93 (95 percent CI, 1.83–19.17; p= 0.003). Considering only CVU cases secondary to a post–thrombotic syndrome (n= 24), FV Leiden yielded a greater OR value of 16.08 (95 percent CI, 4.33‐59.6; p < 0.0001). When the CVU cases were stratified by the three possible FXIII genotypes, a significant trend toward a lower mean value of the ulcerated area was clearly evident as the number of the polymorphic alleles (L34) increased in the genotype of patients (VV = 11.9 cm2,± 23.6; VL = 6.1 cm2,± 6.9; LL = 4.1 cm2,± 2.8; p= 0.01). On the other hand, FXIIIA antigen levels were similar between CVU cases and matched controls, but 11 percent of cases had FXIII deficiency (FXIIIA ≤ 0.65 U/ml; p= 0.003) and they showed a greater mean extension of the lesion if compared with the remaining cases without FXIIIA deficiency (14.5 cm2, ± 20.2 vs. 9.0 cm2, ± 6.3; p= 0.08). We conclude that FXIII antigen levels and FXIII V34L polymorphism may play a crucial role in the complex cascade of CVU pathophysiology, being significantly related to the CVU progression and extension because of the direct effects they have on the FXIII molecular activity.

Factor XIII contrasts the effects of metalloproteinases in human dermal fibroblast cultured cells

Matrix metalloproteinases (MMPs) are overexpressed in venous leg ulcers, determining a breakdown of the main extracellular matrix (ECM) components owing mainly to collagenase activities, and so playing a crucial role in ulcer pathogenesis. The authors studied the effects of coagulation factor XIII (FXIII), which cross-links collagen and other ECM components, in human fibroblast cultured cells in the presence and in the absence of matrix metalloproteinases from Clostridium histolyticum collagenase. Clostridium collagenase at concentrations of 2.0, 1.0, and 0.5 mg/mL was added to normal human dermal fibroblasts cultured in the presence of 0.0, 1.0, and 5.0 U/mL of FXIII concentrate (Fibrogammin P, Aventis Behring). Cell counting and metabolically active fibroblast evaluation in the cultures were monitored for 72 hours, by means of trypan-blue dye and MTT test, respectively. The MTT test showed that at the highest collagenase concentration (2.0 mg/mL), the cell number decreased more than 95% in 72 hours of treatment and no significant differences were observed regardless of the FXIII concentrations utilized. At lower collagenase concentration (1.0 mg/mL), in absence or in presence of FXIII (1.0 U/mL), the cell number decreased by about 80% in 72 hours. In contrast, in the presence of higher FXIII levels (5.0 U/mL), cells suffered globally significantly less collagenase effects (p=0.011) and the gain was appreciable at each time tested. Finally, at 0.5 mg/mL of collagenase concentration, in the absence of FXIII, the cell number decreased by about 60% in 72 hours, whereas in presence of FXIII 1.0 U/mL and 5.0 U/mL, cells decreased significantly less, by about 35% and 20%, respectively (p<0.025 and p<0.01, respectively). These data were also confirmed by direct cell counting utilizing the trypan-blue test. Factor XIII contrasts effectively the detrimental action of Clostridium collagenases in human fibroblast cultured cells. These results support several in vivo reports about the effectiveness of its topical application in order to enhance the venous ulcer healing processes.

DHFR 19-bp insertion/deletion polymorphism and MTHFR C677T in adult acute lymphoblastic leukaemia: Is the risk reduction due to intracellular folate unbalancing?

Folate and its derivatives are pivotal for cell cycle and proliferation. They facilitate the crosstalk between DNA synthesis and methylation crucial processes in cancer establishment [1]. Dietary folate or supplements (e.g., folic acid) must be fully reduced by dihydrofolate reductase (DHFR) before entering cell metabolism [1]. DHFR is responsible for dihydrofolate (DHF) to tetrahydrofolate (THF) conversion, as well as for assisting the generation of additional partially reduced folates (i.e., methylene-THF and formyl-THF), which are then transformed into the fully active folate (i.e., methyl-THF) with the help of methylenetetrahydrofolate reductase (MTHFR). As the main folate isoforms involved in DNA synthesis and methylation are handled by these two key enzymes, alterations in DHFR and/or in MTHFR functions may have detrimental effects on DNA stability and cancer susceptibility [2-5].

A Modified Functional Global Test to Measure Protein C, Protein S Activities and the Activated Protein C-Resistance Phenotype

Identifying a defect affecting the protein C/protein S (PC/PS) anticoagulant system, using a single global test, has recently become possible thanks to a new methodological approach based on the activation of endogenous plasma PC by Protac, derived from Agkistrodon Contortix snake venom (ACV). The introduction of a commercial test (ProC Global), ACV-based, provides a useful tool for the screening of thrombotic patients since the most frequent causes of inherited thrombophilia are found in the PC/PS system. The test provides information only on the global activity of the anticoagulant pathway but not on PC and PS activity or on the factor V related conditions (e.g., FV Leiden). The present study shows that by carrying out the test alternating the presence of PC-, PS-, or FV-deficient plasma and using appropriate amounts of ACV, it is possible to increase the specificity of the test to correctly evaluate respectively the PC or PS activities or the activated protein C resistance condition (APC-R). These simple modifications applied to the original commercial test allow to detect exactly, using a single, basic methodology, the principal defects affecting the PC/PS anticoagulant pathway. Furthermore, carrying out the tests on an automated coagulometer, in combination or not with the classic ProC Global assay, it is possible to use a unique reagent profile to simultaneously investigate in the same or different samples, the PC, PS, and APC-R defect.

Coexistence of antithrombin deficiency, factor V Leiden and hyperhomocysteinemia in a thrombotic family

We report a thrombotic family with combined type I antithrombin deficiency and factor V Leiden (factor V-R506Q) in which the proposita, affected by recurrent venous and arterial thrombosis, was also characterized by mild hyperhomocysteinemia (28 micromol/l; normal <18.5 micromol/l). Her two thrombotic sisters, with normal antithrombin levels and factor V molecules, showed hyperhomocysteinemia (51 and 30 micromol/l, respectively). Four other members of the family had the combined antithrombin/factor V Leiden defect and two of them had thrombosis. The common A223V mutation in the methylenetetrahydrofolate reductase gene, responsible for the thermolabile variant of the enzyme, was found to be heterozygous in the proposita; the two sisters were homozygous and heterozygous, respectively. The heterozygous sister also had a high titre of antiphospholipid antibodies (85 units of immunoglobulin G antiphospholipid antibody/ml). Furthermore, low plasma folate levels were found in the three hyperhomocysteinemic subjects of the family. This family with several prothrombotic defects is a clear example of the polyfactorial nature of thrombophilia.

Different Anticoagulant Response to Activated Protein C (APC test) and to Agkistrodon Contortix Venom (ACV test) in a Family with FV-R506Q Substitution

To identify the defect(s) responsible for the thrombotic condition affecting a 55-year-old male and his family, we have utilized a new methodological approach (ProC Global®, Istituto Behring, Milan, Italy) to screen the global anticoagulant activity of the protein C pathway, a defect that accounts for the majority of inherited thrombophilias. The test is based on the activation of endogenous protein C in plasma by Protac®, derived from Agkistrodon contortix snake venom (ACV test). Nineteen members of the family were investigated, 11 showed low responsiveness to ACV (normalized ACV ratios < 0.66; normal > 1. 12); in these individuals specific assays of protein C (PC) and protein S (PS) levels and normalized activated protein C ratios (n-APC-r) were performed. A second test evaluating response to APC, using the classic commercial APC test (n-APC-r 1), detected only 10 subjects with abnormal responses : the propositus and two members of the family with n-APC-r 1 values < 0.54, indicating the homozygous state for the R506Q factor V gene mutation, and seven with values ranging 0.69-0.83, consistent with the heterozygous condition (normal > 0.85). Although only ten subjects presented with low n-APC-r 1 values, DNA analysis, in agreement with the ACV test, detected 11 individual with factor V-R506Q substitution (two homozygotes and nine heterozygotes). Thus the classical APC test failed to identify the APC resistance phenotype in two heterozygous subjects whose values were clearly normal (1.05) in the first case and homozygous (0.53) in the second. The ACV test, however, and the modified APC test with test plasma 1/5 diluted in factor V-deficient plasma (n-APC-r 2) completely matched the DNA analysis. A phenotype/genotype correlation was observed in dilutions higher than 1/3 test plasma factor V-deficient plasma. The presence of unknown mechanisms that influence plasma response to exogenous preformed APC (normal at high factor V-deficient plasma dilutions) but not endogenous ACV activated PC was suspected. The suspected low levels of proteins C and S found in several R506Q members of the family were excluded by reassaying the anticoagulant activities at higher plasma dilution ; this supports the known influence of factor V Leiden on functional PC and PS clotting activity. We conclude that the ACV test is appropriate to evaluate the APC resistance condition, but for a firm diagnosis DNA analysis together with the modified APC test are strongly advised even in the presence of unquestionable APC-r values. Key Words: APC resistance-Factor V Leiden-APC test-ACV test-Diagnosis-Inherited thrombophilia.

Resistance to activated protein C and low levels of protein S activity in nine thrombophilic families: a correct diagnosis.

In order to define the thrombophilic conditions related to activated protein C resistance (APC-R) and protein S (PS) deficiency and to detect the possible combination of these defects, we studied nine unrelated patients selected because of low anticoagulant response to APC and reduced PS activity with at least one first degree relative having the same coagulation feature. The mean APC ratio was 0.64 (normal values > 0.80) and range 0.35-0.80. Three of the patients were heterozygous and two were homozygous for the Leiden mutation (FVR506Q); in the remaining four there was no mutation. The mean PS activity was 41.6% and range 32-54 (normal 65-150%). Five of the patients had low PS activity despite normal total and free antigenic levels, and normal activity when measured at higher plasma dilution; these were carrying at least one gene for the Leiden mutation. In the remaining four patients the crossed-immunoelectrophoresis and the Western-blotting analysis showed a type I PS deficiency confirmed by the familial restriction fragment length polymorphism analysis. Thus, four families were diagnosed with the type I PS defect and five with congenital APC-R. No combined PS/FV Leiden or type II PS defect was found. The only defect found was in the anticoagulant PC pathway. We therefore designed a procedure to diagnose thrombophilic conditions related to this pathway. This study indicates that a specific methodological approach must be used to accurately characterize APC-R and PS deficiency and that care is necessary to avoid the possibility of misdiagnosis.

Thrombosis of the cerebral veins and sinuses in acute promyelocytic leukemia after all-trans retinoic acid treatment: a case report.

Thrombosis of the cerebral veins or sinuses is a rare cerebrovascular disorder, which seldom represents a complication of acute promyelocytic leukemia. To the best of our knowledge, it never occurred during treatment with all-trans retinoic acid. We report a case of a 35-year-old woman affected by acute promyelocytic leukemia, who developed massive thrombosis of the cerebral sinuses and veins when she was in complete morphological and molecular remission after all-trans retinoic acid and idarubicin treatment. Anticoagulant therapy contributed to progressive dissolution of the thrombosis as documented by magnetic resonance imaging with complete disappearance of neurological signs without sequelae.

Coexistence of Factor V G1691A and Factor II G20210A Gene Mutations in a Thrombotic Family Is Associated with Recurrence and Early Onset of Venous Thrombosis

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject – the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations – showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus’ uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus’ two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.