Lucia Evangelisti

Genetic determinants of fasting and post-methionine hyperhomocysteinemia in patients with retinal vein occlusion

INTRODUCTION Moderate hyperhomocysteinemia is considered a risk factor for both venous and arterial thrombosis. A prevalence of up to 30% of fasting hyperhomocysteinemia has been recently reported in patients with retinal vein occlusion (RVO) whereas conflicting data exist on the role of C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene as a risk factor for RVO. No report has been published on cystathionine beta-synthase (CBS) 844ins68 polymorphism (another genetic determinant of blood Hcy levels) in RVO patients. Moreover, scarce information is available on the usefulness of measuring homocysteine also after methionine loading to increase the diagnostic efficacy of hyperhomocysteinemia in RVO patients. MATERIALS AND METHODS In 55 consecutive patients with diagnosis of RVO and 65 matched controls, plasma fasting total homocysteine (Hcy) levels and CBS and MTHFR polymorphisms were evaluated. In patients with normal fasting Hcy levels, post-methionine Hcy levels were determined. RESULTS Moderate fasting hyperhomocysteinemia was detected in 18/55 patients (32.7%). In the remaining 37 patients, Hcy was measured again post-methionine loading (PML). Only 3/37 (8.1%) patients had PML hyperhomocysteinemia. Thus, the total prevalence of moderate hyperhomocysteinemia in this cohort of RVO patients was 21/55 (38.2%). The prevalence of homozygosity for C677T MTHFR genotype, but not that of heterozygosity for CBS844ins68, was significantly higher in RVO patients than in controls. CONCLUSIONS Differently from what has been reported for arterial and/or venous thrombosis, a single fasting Hcy measurement is able to detect most of RVO patients (85.7%) with moderate hyperhomocysteinemia. C677T MTHFR, but not CBS 844ins68, genotype may play a role as risk factor for RVO.

Fibrillin-1 (FBN1) gene frameshift mutations in Marfan patients: genotype-phenotype correlation

Marfan syndrome (MFS) is a multisystemic disease associated with mutations in the fibrillin‐1 gene. Most of the reported mutations are missense substitutions mainly affecting the epidermal growth factor (EGF)‐like protein domain structure and the calcium‐binding (cb) site. The aim of our study was to investigate the correlation between fibrillin‐1 frameshift mutations and the clinical phenotype in patients affected by MFS. In 48 out of 66 Marfan patients a pathogenetic mutation was found. We detected novel mutations causing premature termination codon in exons 19, 37, 40 and 41 of four Italian patients. The first mutation in exon 19 (cbEGF #8 domain) results in a clinical phenotype involving mainly the skeletal and cardiovascular systems. Interestingly, we noticed that, while mutations in exons 37 and 41 (eight cysteine domains #4 and #5) are milder, the mutation in exon 40 (cbEGF #24 domain) is more severe and causes major cardiovascular involvement with thoracic and abdominal aortic aneurysms. It is noteworthy that the degree of the severity in the phenotype of one of our patients and another from the literature carrying a mutation in exon 41 could be explained with alterations in mRNA expression.

FBN1 mutation screening of patients with Marfan syndrome and related disorders: detection of 46 novel FBN1 mutations

Fibrillin‐1 gene (FBN1) mutations cause Marfan syndrome (MFS), an inherited connective tissue disorder with autosomal dominant transmission. Major clinical manifestations affect cardiovascular and skeletal apparatuses and ocular and central nervous systems. We analyzed FBN1 gene in 99 patients referred to our Center for Marfan Syndrome and Related Disorders (University of Florence, Florence, Italy): 85 were affected by MFS and 14 by other fibrillinopathies type I. We identified mutations in 80 patients. Among the 77 different mutational events, 46 had not been previously reported. They are represented by 49 missense (61%), 1 silent (1%), 13 nonsense (16%), 6 donor splice site mutations (8%), 8 small deletions (10%), and 3 small duplications (4%). The majority of missense mutations were within the calcium‐binding epidermal growth factor‐like domains. We found preferential associations between The Cys‐missense mutations and ectopia lentis and premature termination codon mutations and skeletal manifestations. In contrast to what reported in literature, the cardiovascular system is severely affected also in patients carrying mutations in exons 1–10 and 59–65. In conclusion, we were able to detect FBN1 mutations in 88% of patients with MFS and in 36% of patients with other fibrillinopathies type I, confirming that FBN1 mutations are good predictors of classic MFS.

Evaluation of a new point-of-care celite-activated clotting time analyzer in different clinical settings: The i-STAT celite-activated clotting time test

Background Activated clotting time (ACT) is used to monitor heparin therapy during cardiopulmonary bypass, interventional cardiology, and hemodialysis. Traditionally, ACT is performed by use of the Hemochron system. Recently, a new device, the i-STAT system, has been introduced to measure ACT. The aim of this study was to correlate the performances of these two systems and to compare ACT values with heparin levels. Methods One hundred sixty-five samples from 29 patients undergoing cardiopulmonary bypass or hemodialysis were assayed in duplicate with two Hemochron and two i-STAT devices. Heparin levels were determined by anti–factor Xa assay. Results The Hemochron ACT ranged from 88 to 1,028 s, and the i-STAT ACT ranged from 80 to 786 s. Heparin plasma levels ranged from 0.01 to 10.8 U/mL. Bland–Altman analysis showed a mean difference between the two methods of 24 ± 101 s. Strong relationships between anti–factor Xa activity and Hemochron ACTs (r2 = 0.69, P < 0.001) and i-STAT ACTs (r2 = 0.79, P < 0.001) were observed. During cardiac surgery, significant correlations were found: Hemochron, r2 = 0.61, P < 0.001 and i-STAT, r2 = 0.74, P < 0.001. During hemodialysis, relationships between anti–factor Xa activity and ACTs were found: Hemochron, r2 = 0.62, P < 0.001 and i-STAT, r2 = 0.55, P < 0.001. Conclusions During cardiopulmonary bypass procedure and hemodialysis, i-STAT provides measurements of clotting time quite similar to Hemochron ACT, which were significantly correlated with heparin levels.

Evidence for oxidative stress in plasma of patients with Marfan syndrome

0.91–1.31 1.11 0.77–1.60 Metabolic syndrome — NCEP 1.16 0.99–1.35 1.56* 1.06–2.29 1.14 0.95–1.37 1.20 0.83–1.74 Abdominal obesity — IDF 1.27** 1.08–1.50 1.25 0.85–1.84 0.96 0.79–1.17 1.11 0.75–1.64 Abdominal obesity — NCEP 1.18* 1.01–1.39 1.26 0.86–1.85 1.20 0.99–1.44 0.95 0.65–1.39 Hypertension 1.35*** 1.14–1.59 1.21 0.82–1.80 1.15 0.94–1.41 1.02 0.69–1.53 Low high-density lipoprotein 0.92 0.74–1.14 1.98* 1.10–3.57 0.89 0.69–1.15 1.00 0.60–1.66 Elevated triglycerides 0.75 ** 0.61–0.92 0.92 0.57–1.47 0.93 0.73–1.17 1.31 0.83–2.06 Insulin resistance TG/HDL 0.87 0.75–1.02 1.55* 1.05–2.29 0.90 0.74–1.09 0.88 0.60–1.28 Hyperglycemia 1.34*** 1.14–1.57 1.38 0.95–2.01 1.13 0.94–1.36 1.00 0.69–1.46 Women Metabolic syndrome — IDF 1.52*** 1.34–1.75 1.44* 1.07–1.94 1.15* 1.00–1 .33 0.74 0.53–1.05 Metabolic syndrome — NCEP 1.54*** 1.35–1.77 1.35* 1.00–1.82 1.26** 1.09–1.45 0.86 0.61–1.22 Abdominal obesity — IDF 1.47*** 1.25–1.73 0.82 0.57–1.17 1.23* 1.03–1.46 0.73 0.51–1.06 Abdominal obesity — NCEP 1.56*** 1.36–1.78 1.00 0.82–1.22 1.34*** 1.16–1.56 0.88 0.63–1.23 Hypertension 2.15*** 1.84–2.52 1.23 0.90–1.68 1.19* 1.02–1.39 0.90 0.64–1.27 Low high-density lipoprotein 1.12 0.96–1.31 1.59* 1.11–2.27 0.98 0.83–1.16 1.01 0.68–1.50 Elevated triglycerides 1.08 0.89–1.30 0.94 0.62–1.42 1.01 0.82–1.24 1.32 0.83–2.11 Insulin resistance TG/HDL 1.15* 1.00–1.34 1.60** 1.14–2.26 1.04 0.89–1.22 0.93 0.63–1.37 Hyperglycemia 1.45*** 1.27–1.65 1.48** 1.11–1.98 1.16* 1.01–1.33 1.01 0.72–1.41

PPARgamma Promoter Polymorphisms and Acute Coronary Syndrome

BACKGROUND PPARgamma (PPARg) is a nuclear transcription factor involved in the control of lipid and glucose homeostasis. Two PPARg common polymorphisms, Pro12Ala and 161C>T, have been found to be associated with cardiovascular disease. In this study, in addition to PPARg coding region, we looked for genetic variations in promoters and their association with acute coronary syndrome (ACS). METHODS We studied 202 Italian patients with ACS, and 295 healthy Italian subjects by dHPLC (denaturing high-performance liquid chromatography), heteroduplex analysis and direct sequencing or RFLP (restriction fragment length polymorphism) analysis for screening mutations. RESULTS We identified 7 new and 2 already published polymorphisms in PPARg promoters. The C>T93695 (promoter 4) mutation showed significantly different genotype distribution and allele frequency between controls and ACS patients (p<0.001); the T allele conferred a protection against ACS at both univariate (OR: 0.45, 95% CI 0.29-0.69: p<0.001) and multivariate analysis adjusted for sex, age and traditional cardiovascular risk factors (OR: 0.44, 95% CI 0.25-0.76: p<0.005). Moreover, the 161C>T polymorphism allele frequency (p=0.03) and genotype distribution (p=0.015) resulted to be different in ACS group if compared to healthy controls. CONCLUSIONS The protective role of 93695C>T polymorphism in PPARg promoter in ACS suggests that PPARg genetic variants may affect the susceptibility to atherosclerotic diseases.

Risk factors for cardiovascular disease in renal transplant recipients: new insights

Abstract Long‐term survival of renal transplant recipients appears to be influenced by the occurrence of thromboembolic complications and cardiovascular disease. In order to investigate the prevalence of new hemostasis‐related risk factors for venous and arterial thrombosis, we investigated 63 renal transplant recipients and 66 age‐ and sex‐matched control subjects. We assayed antiphospholipid antibodies [lupus anticoagulant (LA) and anticardiolipin antibodies (aCL)], lipoprotein (a) [Lp(a)], plasminogen activator inhibitor‐1 (PAI‐1), and total homocysteine (tHcy) levels. We found a significantly higher prevalence of positivity for LA (P < 0.001); no difference was detected in the prevalence of aCL between patients and controls. PAI‐1 levels were significantly higher in renal transplant recipients than in controls [12.3 IU/ml (2‐45.5) vs 7.9 IU/ml (4‐18.0); P < 0.0001] with an odd ratio (OR) of 11.8 (4.9‐28.5) in univariate analysis and of 5.8 (2.1‐15.4) in multivariate analysis. Lp(a) levels were higher in patients then in controls [159 mg/l (1‐992) vs 100.5 mg/l (10‐412); P < 0.005] with an OR of 5.9 (1.9‐18.4) in univariate analysis and of 3.5 (0.9‐13.4) in multivariate analysis. Fasting levels of tHcy were significantly higher in renal transplant recipients [7.0 μmol/l (4.0‐68) vs 8.1 μmol/l (2.0‐24.0); P < 0.00001] with an OR of 40.4 (14.7‐111) in univariate analysis and of 33.1 (11.1‐115.5) in multivariate analysis. After methionine loading test, we documented levels of tHcy above the 90th percentile of controls in 60/63 patients (95 %). Finally, we found a significant correlation between tHcy and PAI‐1 plasma levels (r = 0.76; P < 0.000001). Our results show a high prevalence of hemostasis‐related risk factors for arterial and venous thrombosis in renal transplant recipients, suggesting the need for the investigation of these patients for the presence of these risk factors in order to improve their long‐term survival and to tailor therapy.

Fibroblast autofluorescence in connective tissue disorders: a future tool for clinical and differential diagnosis?

Marfan syndrome (MFS) is an inherited disorder of connective tissue due to mutations in FBN1 (90%) and TGFBR1 and TGFBR2 (5 to 10%) genes. Clinical and differential diagnosis is difficult because of the inter- and intrafamiliar marked heterogeneity and the variable onset age of clinical manifestations. Among the disorders, in differential diagnosis, thoracic aortic aneurysm (TAA) and Ullrich scleroatonic muscular dystrophy (UCMD) are reported. We evaluate the possibility of utilizing autofluorescence (AF) analysis as a diagnostic tool in the clinical and/or differential diagnosis of MFS and related disorders and in the investigation of the molecular mechanisms involved. Both multispectral imaging autofluorescence microscopy (MIAM) and autofluorescence microspectroscopy (AMS) have been used to characterize AF emission of fibroblasts from patients affected by inherited connective tissue disorders. Our preliminary results show significant differences in AF emission between normal and pathological fibroblasts, suggesting possible improvement in diagnostics of connective tissue disorders by AF analysis.

A single heterozygous nucleotide substitution displays two different altered mechanisms in the FBN1 gene of five Italian marfan patients

The Fibrillin-1 gene (FBN1; chromosome 15q21.1) encodes a major glycoprotein component of the extracellular matrix. Mutations in FBN1, TGFBR1, TGFBR2 are known to cause Marfan syndrome (MIM 154700), a pleiotropic disorder. In the present study, we describe five novel missense FBN1 mutations in five Marfan patients that have the peculiarity to activate two contemporary mutational mechanisms: a missense mutation and exon skipping.

Vascular and connective tissue features in 5 italian patients with homocystinuria

Homocystinuria is a metabolic disorder associated with defects in genes encoding for methionine metabolism enzymes. Vascular and connective tissue manifestations such as deep venous thrombosis, ectopia lentis and skeletal alterations are the major clinical features. We investigated the clinical manifestations of 5 Italian homocystinuric patients, performed mutation screening analysis on cystationine beta-synthase (CBS) gene and searched for genotype/phenotype correlations. We detected mild cardiovascular and skin connective tissue stigmas in these patients, never reported in homocystinuric patients before. We found 1 novel and 7 known mutations. Our patients carried no other mutation associated with venous thrombosis. Our data stress the importance of extending the clinical investigation for connective tissue manifestations in homocystinuric patients to all the organs/systems involved in Marfan syndrome, also suggesting long term follow-ups for cardiovascular manifestations.