Impaired control of the contact system in hereditary angioedema with normal C1-inhibitor.

BACKGROUND: Hereditary angioedema (HAE) comprises HAE with C1-inhibitor deficiency (C1-INH-HAE) and HAE with normal C1-INH activity (nl-C1-INH-HAE), due to mutations in factor XII (FXII-HAE), plasminogen (PLG-HAE), angiopoietin 1 (ANGPT1-HAE), kininogen 1 genes (KNG1-HAE), or angioedema of unknown origin (U-HAE). The Italian network for C1-INH-HAE (ITACA) created a registry including different forms of angioedema without wheals. OBJECTIVE: We analyzed clinical and laboratory features of a cohort of Italian subjects with nl-C1-INH-HAE followed by ITACA to identify specific biomarkers. METHODS: A total of 105 nl-C1-INH-HAE patients were studied. Plasma concentrations of cleaved high-molecular-weight kininogen (cHK), vascular endothelial growth factors (VEGFs), angiopoietins (Angs), and secreted phospholipase A2 enzymes (sPLA2 ) were evaluated. RESULTS: We identified 43 FXII-HAE patients, 58 U-HAE, and 4 ANGPT1-HAE. We assessed a prevalence of 1:1.4 × 106 for FXII-HAE and 1:1.0 × 106 for U-HAE. cHK levels in U-HAE patients were similar to controls in plasma collected using protease inhibitors cocktail (PIC), but they significantly increased in the absence of PIC. In FXII-HAE patients, cHK levels, in the absence of PIC, were significantly higher than in controls. We found a significant increase of VEGF-A, VEGF-C, and Ang1 levels in U-HAE patients compared to controls. In FXII-HAE, only VEGF-C levels were increased. Ang2 concentrations and sPLA2 activity were not modified. The levels of these mediators in ANGPT1-HAE patients were not altered. CONCLUSIONS: Our results suggest that pathogenesis of FXII-, ANGPT1-, and U-HAE moves through an unbalanced control of kallikrein activity, with bradykinin as most likely mediator. VEGFs and Ang1 participate in the pathophysiology of U-HAE increasing the basal vascular permeability.

Mutation of the angiopoietin-1 gene (ANGPT1) associates with a new type of hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a rare genetic disease usually caused by mutation in the C1 inhibitor or the coagulation Factor XII gene. However, in a series of patients with HAE, no causative variants have been described, and the pathophysiology of the disease remains unknown (hereditary angioedema with yet unknown genetic defect [U-HAE]). Identification of causative genes in patients with U-HAE is valuable for understanding the cause of the disease. OBJECTIVE: We conducted genetic studies in Italian patients with U-HAE to identify novel causative genes. METHODS: Among patients belonging to 10 independent families and unrelated index patients with U-HAE recruited from the Italian Network for C1-INH-HAE (ITACA), we selected a large multiplex family with U-HAE and performed whole-exome sequencing. The angiopoietin-1 gene (ANGPT1) was investigated in all patients with familial or sporadic U-HAE. The effect of ANGPT1 variants was investigated by using in silico prediction and plasma and transfected cells from both patients and control subjects. RESULTS: We identified a missense mutation (ANGPT1, c.807G>T, p.A119S) in a family with U-HAE. The ANGPT1 p.A119S variant was detected in all members of the index family with U-HAE but not in asymptomatic family members or an additional 20 patients with familial U-HAE, 22 patients with sporadic U-HAE, and 200 control subjects. Protein analysis of the plasma of patients revealed a reduction of multimeric forms and a reduced ability to bind the natural receptor tunica interna endothelial cell kinase 2 of the ANGPT1 p.A119S variant. The recombinant mutated ANGPT1 p.A119S formed a reduced amount of multimers and showed reduced binding capability to its receptor. CONCLUSION: ANGPT1 impairment is associated with angioedema, and ANGPT1 variants can be the basis of HAE.

Characterization of patients with angioedema without wheals: the importance of F12 gene screening.

Sporadic and familiar forms of non-histaminergic angioedema and normal C1 inhibitor encompass a group of disorders possibly caused by bradikinin. We aimed to study the subgroups of hereditary angioedema with FXII mutation (FXII-HAE), unknown genetic defect (U-HAE) and idiopathic non-histaminergic acquired angioedema (InH-AAE). We screened the F12 locus in our cohort and delineated the clinical, laboratory and genetic features. Four families carried the p.Thr309Lys mutation in F12 gene. Haplotyping confirmed the hypothesis of a common founder. Six families were affected by U-HAE and 13 patients by sporadic InH-AAE. C4 levels were significantly lower in FXII-HAE than in InH-AAE. In the FXII-HAE group, none had attacks exclusively in high estrogenic states; acute attacks were treated with icatibant. Prophylaxis with tranexamic acid reduced the attack frequency in most patients. Our study provides new data on the diagnosis, clinical features and treatment of non-histaminergic angioedema, underlying the role of the screening for F12 mutations.

Mutational spectrum of the c1 inhibitor gene in a cohort of Italian patients with hereditary angioedema: description of nine novel mutations.

Hereditary angioedema (HAE) is an autosomal dominant disease due to mutations in the C1 inhibitor gene (C1NH) that affects protein synthesis (HAE type I) or function (HAE type II). In 45 subjects affected by HAE diagnosed through clinical features and C1 inhibitor deficiency from the south of Italy (38 with type I and 7 with type II HAE), the whole C1NH coding region was screened for mutations by direct DNA sequencing. A severity score based on clinical manifestation, age at disease onset, and need for long-term prophylaxis was used to investigate possible genotype-phenotype correlations. A series of 22 different mutations was identified: nine missense (40.9%), five nonsense (22.7%), six frameshift (27.3), one small deletion (4.5%), and one splicing defect (4.5%). Nine C1NH mutations have not been previously described. No correlation was found between C1 inhibitor function level and severity score or age at first attack. Moreover, there was no correlation between different types of mutations and clinical phenotype. The number of different mutations identified highlights the heterogeneity of C1 inhibitor deficiency and supports the hypothesis that HAE clinical phenotype is not strictly related to the type of mutation but rather depends on unknown factors.

McKusick-Kaufman or Bardet-Biedl syndrome? A new borderline case in an Italian nonconsanguineous healthy family.

McKusick-Kaufman syndrome (MKS, OMIM #236700) is a rare syndrome inherited in an autosomal recessive pattern with a phenotypic triad comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP), and congenital cardiac disease (CHD). The syndrome is caused by mutations in the MKKS gene mapped onto chromosome 20p12 between D20S162 and D20S894 markers. Mutations in the same gene causes Bardet-Biedl-6 syndrome (BBS-6, OMIM #209900) inherited in an autosomal recessive pattern. BBS-6 comprises retinitis pigmentosa, polydactyly, obesity, mental retardation, renal and genital anomalies. HMC, CHD, and PAP defects can also occur in BBS-6, and there is a significant clinical overlap between MKS and BBS-6 in childhood. We describe a new borderline case of MKS and BBS syndrome and suggest insights for understanding correlation between MKKS gene mutations and clinical phenotype. Here, we report the results of molecular analysis of MKKS in a female proband born in an Italian nonconsanguineous healthy family that presents HMC and PAP. The mutational screening revealed the presence of two different heterozygous missense variants (p.242A>S in exon 3, p.339 I>V in exon 4) in the MKKS gene, and a nucleotide variation in 5'UTR region in exon 2 (-417 A>C).

Role of the M2 haplotype within the annexin A5 gene in the occurrence of pregnancy-related venous thromboembolism.

OBJECTIVE: Knowledge about risk factors for venous thromboembolism (VTE) is still limited. A recently found haplotype within the natural anticoagulant protein annexin A5 (ANXA5) exerts an important modulating effect on gene expression. STUDY DESIGN: Eighty-three nonanticoagulated patients with a documented pregnancy-related VTE and 195 controls were investigated. The presence of the ANXA5 haplotypes was determined. RESULTS: Twenty-seven patients (32.5%) carried the M2 haplotype. Among them, 17 (63.0%) had a history of VTE in puerperium and 10 (37.0%) during pregnancy. The prevalence of the M2 haplotype was different as compared with that recorded among controls (odds ratio, 2.7; 95% confidence interval, 1.5-4.9, P < .001). A logistic regression analysis, correcting for potential confounders (age at which the thrombotic event occurred, factor V Leiden, and factor IIA20210 variants) showed a significant increase (odds ratio, 3.4; 95% confidence interval, 1.7-6.7) of the occurrence of VTE in carriers of the M2 haplotype as compared with noncarriers. CONCLUSION: The M2 haplotype within the ANXA5 gene may represent a new thrombophilic risk factor for pregnancy-related VTE.

Genetic basis of thrombosis.

Venous thrombosis (VT) represents a common and serious disorder that occurs as the result of clotting of the blood in the venous system and venous obstruction. Environmental risk factors and genetic predisposition play an important role in the development of thrombosis. It is therefore seen as a classic example of a complex common disease. We have focused on the role of genetic risk factors, primarily related to the hemostatic system, in triggering thrombotic events. Since the identification of antithrombin deficiency in 1965, major efforts have been made during the past 15 years to identify other genetic entities that lead to increased thrombotic risk. Results of early genetic studies demonstrated that two types of genetic defects cause VT: loss of function mutations in the natural anticoagulants antithrombin, protein C and protein S and gain of function mutations in procoagulant factors V (FV Leiden) and II (prothrombin G20210A). The high incidence of these mutations in Caucasians induced a shift from family studies to case-control association studies. Several investigations have been performed on the role of other candidate genetic risk factors predisposing to VT, including such variants in FXIII, FIX and fibrinogen genes. Moreover, the contribution of genetic variation in genes encoding less-well studied proteins that are part of the anticoagulant pathways has been evaluated. Recently, different genome-wide association studies have been performed in which several single nucleotide polymorphisms were investigated and related to the risk of VT. However, further studies are needed to identify additional genetic causes of thrombosis and to assess functional molecular mechanisms.

A beta3 Asp217-->Val substitution in a patient with variant Glanzmann Thrombasthenia severely affects integrin alphaIIBbeta3 functions.

A dysfunctional Glanzmann Thrombasthenia (variant) is a rare bleeding disorder due to qualitative abnormalities of platelets alphaIIBbeta3 heterodimers. Dynamically conformational change of alphaIIBbeta3 is a complex mechanism that is not fully understood. For these reasons, genotyping and functional analysis of variant Glanzmann Thrombasthenia is important to elucidate the molecular basis of alphaIIBbeta3 receptor functions. In this report, we have analyzed the molecular effects of an A>T substitution leading to an amino acid change, D217>V, in the beta3 integrin gene identified in patients with variant Glanzmann Thrombasthenia. As the D217 residue is highly conserved among all seven beta integrin subunits and among beta3 integrins of different species, we tested the effect on the phenotype of the D217V mutation by cotransfecting the beta3 mutant (V217) or wild-type beta3 (D217) construct with the wild-type alphaIIb into eukaryotic Chinese hamster ovary cells. Levels of mutant alphaIIBbeta3 heterodimers on Chinese hamster ovary cell surface were lightly reduced as compared with the wild type. Functional investigation of alphaIIBbeta3 V217 on Chinese hamster ovary cell surface was carried out, as fibrinogen binding, adhesion and aggregation tests showed a substantial reduction in respect to the control sample. Our results confirm ex-vivo data and suggest that the D217 amino acid is required for alphaIIBbeta3 receptor interactions with fibrinogen.

A novel mutation in human ether-a-go-go-related gene, alanine to proline at position 490, found in a large family with autosomal dominant long QT syndrome.

Long-QT syndrome is a rare disease characterized by prolonged ventricular repolarization. The clinical presentation of long-QT syndrome is the occurrence of syncope, seizures, or cardiac arrest in young patients. Previous studies have demonstrated locus heterogeneity, with causative mutations reported in >or=8 different genes, including the human ether-a-go-go-related gene. This study was conducted in 26 members of a 4-generation family with long-QT syndrome. The proband was a 14-year-old female patient referred to the emergency department for the evaluation of recurrent syncope associated with a prolonged QT interval on electrocardiography at rest. There was a family history of sudden death in a 27-year-old woman. Sequencing of the entire coding regions of the human ether-a-go-go-related gene and the intron and exon boundaries of the proband identified a single base-pair substitution (guanine to cytosine at nucleotide 1468). This mutation resulted in a novel missense mutation, alanine to proline at position 490 (Ala490Pro), in the inner loop of the S2 and S3 domains. The proband was heterozygous for the Ala490Pro mutation. To address whether the mutational change detected in the patient would be a polymorphism, 100 control subjects from the same ethnical background were investigated. None showed the Ala490Pro substitution. Of 26 family members, 9 were mutation carriers, and none had normal electrocardiographic results. The penetrance of this pedigree was assumed to be 100%. In conclusion, the Ala490Pro mutation of the human ether-a-go-go-related gene is a rare, novel mutation that was inherited in this family, leading to Romano-Ward syndrome with complete penetrance.

Phenylalanine hydroxylase deficiency in south Italy: Genotype-phenotype correlations, identification of a novel mutant PAH allele and prediction of BH4 responsiveness.

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 33 Italian PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed by direct sequencing of the patients' genomic DNA. Thirty-three different disease causing mutations were identified in our patient group, including 19 missense, 6 splicing, 3 nonsense, 5 deletions, with a detection rate of 100%. The most prevalent mutation was the IVS10-11G>A, accounting for 12.1% of PKU alleles studied. Other frequent mutations were: p.R261Q (9.1%), p.P281L (7.6%), and p.R408W (6.1%). We also identified one novel missense mutation, p.H290Q. A spectrum of 31 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 13 were predicted to be BH4-responsive represented by thirteen PKU families. In addition, genotype-phenotype correlations were performed. This study reveals the importance of a full genotyping of PKU patients and the prediction of BH4-responsiveness, not only because of the definitive diagnosis and prediction of the optimal diet, but also to point out those patients that could benefit from new therapeutic approach. They may potentially benefit from BH4 therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions.

A novel congenital dysprothrombinemia leading to defective prothrombin maturation.

INTRODUCTION: Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening. AIM: Here we characterize a patient with a novel missense mutation in F2, c.1090T/A (p.Val322Glu), that causes severe dysprothrombinemia. METHODS: Coagulation assays, prothrombin Western Blotting, FII activation by Ecarin, fibrinogen degradation products quantification and thrombin generation assay were carried out to assess prothrombin expression and function. PCR followed by direct sequencing was carried out to characterize the mutation. In silico analysis for missense variant and molecular modeling were applied to predict the mechanism that leads to dysprothrombinemia. RESULTS AND CONCLUSIONS: The homozygous patient had a markedly prolonged prothrombin time, strongly reduced FII activity (0.82%) but normal antigen levels. In the thrombin generation assay the lag time and the peak height were unmeasurable, suggesting that the Val322Glu mutation results in the inability of the mutant prothrombin to be fully activated to thrombin. In fact, prothrombin activation by ecarin was defective, with a massive accumulation of the meizothrombin intermediate. Molecular modeling and dynamic simulation studies showed that the Val322Glu mutation interferes with protein flexibility at Arg271 and Arg320. This impairs the switch of the protein from zymogen to proteinase, thus preventing the formation of thrombin. Accumulated meizothrombin, however, maintains some fibrinogen-degrading activity, as shown by the formation of FDPs, and this probably explains the patient's mild bleeding phenotype.

SYBR green real time-polymerase chain reaction as a rapid and alternative assay for the efficient identification of all existing Escherichia coli biotypes approved directly in wastewater samples.

Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm-blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses SYBR green real time-polymerase chain reaction (RT-PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT-PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT-PCR analysis can encourage the testing of a higher number of samples.

Fatal pulmonary thromboembolism. A retrospective autopsy study: searching for genetic thrombophilias (Factor V Leiden (G1691A) and FII (G20210A) gene variants) and dating the thrombus.

The accuracy of antemortem diagnosis of pulmonary embolism is within the range of just 10-30%, so representing one of the most frequent missed diagnosis in sudden, unexpected death. We describe 43 fatal cases of pulmonary embolism as confirmed by post-mortem examination. The aim of our study was to verify the systematic search for the most common genetic thrombophilias (Factor V Leiden (G1691A) and FII (G20210A) gene variants) and dating the thrombus. As a whole, 41 patients (95.3%) had at least one risk factor. Pre-existing symptoms are described just before fatal embolism in 18 (41.9%) out 43 patients. In 18 out of 43 (41.9%) it was not possible to find the thrombotic site. In 24 out of the remaining 25 cases the involvement of the deep veins of one leg was shown; in 1 case the thrombus was localised in the inferior caval vein. 10 (41.7%) were iliac vein thromboses, 7 (29.1%) femoral, 2 (8.3%) popliteal, 3 (12.6%) posterior-tibial, 1 (4.1%) anterior-tibial and 1 (4.1%) peroneal vein thromboses. In our cohort of patients, 4 (10%) out of 40 cases carried the 20210A prothrombin gene variant in heterozygosis. One (2.5%) out of 40 carried the Factor V Leiden (G1691A) gene variant in heterozygosis. Patients carrying these gene variants in homozygosis or carrying both were not present in our case-series. We strongly underline the relevance of a complete methodological approach, integrating clinical data by means of autopsy findings and histological study. On the contrary, investigating common inherited thrombophilia is not warranted.

Obstetric complications and pregnancy-related venous thromboembolism: the effect of low-molecular-weight heparin on their prevention in carriers of factor V Leiden or prothrombin G20210A mutation.

Whether the administration of low-molecular-weight heparin (LMWH) during pregnancy is effective in preventing obstetric complications and pregnancy-related venous thromboembolism (VTE) in women who are carriers of factor V Leiden (FVL) and/or prothrombin variant G20210A (PTm) is controversial. This observational study investigated the possible efficacy of pharmacological treatment with LMWH ± aspirin (ASA) in pregnancy outcomes in 1,011 pregnancies of 416 women with thrombophilia (FVL and/or PTm). Most patients were chosen on the basis of previous obstetrical complications (36%), or because of familial or personal history of venous/arterial thromboembolism (28% and 18%, respectively); 74 patients (18%) were incidentally identified. The outcome was evaluated according to the type of treatment and of the period of pregnancy when the treatment was started. After adjustment for observation before and after diagnosis of thrombophilia, previous miscarriages and VTE, parity, age and centre, we observed that LMWH had a protective effect on miscarriages (odds ratio [OR] 0.52, 95% confidence interval [CI] 0.29-0.94) and VTE (OR 0.05, 95% CI 0.01-0.21). ASA appeared to have no effect on the prevention of obstetric complications and VTE. A nested analysis performed in 116 women with two or more obstetric complications confirmed that the highest number of live births was recorded in the group under LMWH prophylaxis (OR 0.19, 95% CI 0.05-0.75). These results suggest that LMWH prophylaxis reduces the risk of obstetric complications in carriers of FVL and/or PTm, particularly in those with previous obstetric events. Furthermore, LMWH prophylaxis reduces the risk of pregnancy-related VTE.

The risk of occurrence of venous thrombosis: focus on protein Z.

Protein Z (PZ) is a vitamin K-dependent factor identified in human plasma in 1984 characterized by an homology with other vitamin K-dependent factors. PZ acts as the cofactor of the PZ dependent inhibitor (ZPI), in the inhibition of activated factor X bound on phospholipid surface. In humans, PZ is characterized by an unusual wide distribution in plasma partly explained by a genetic control. Several PZ gene polymorphisms influencing plasma concentration have been described. In mice, the disruption of PZ gene is asymptomatic, but in association with homozygous FV Leiden produced a severe prothrombotic phenotype. This review analyzes the results obtained from different studies so far published in order to understand whether PZ deficiency could be considered as a risk factor for venous thrombosis. The roles of PZ plasma level and PZ gene polymorphisms remain debated with conflicting results. Many of these studies reported low PZ levels in association with an increased risk of venous thrombosis. On the other side, some studies did not observe an association between low levels of PZ and thrombotic events. A relationship between PZ deficiency and pregnancy complications was also described but not confirmed by all studies. These discrepancies can be explained by the heterogeneity of populations chosen as control, by the PZ interindividual variability and by the small size of the cohorts in mainly retrospective studies. Large prospective studies remain to be done to investigate its possible role in thrombosis.

Gene polymorphisms and sport attitude in Italian athletes.

AIM: The aim of this study was to evaluate whether the distribution of polymorphisms in the ACE, ACTN3, NOS3, UCP2, and UCP3 genes, which has been reported to be correlated with different physiological parameters, played a role in sport performance. We focused on a cohort of 82 Italian athletes: first of all, athletes were divided according to type of sport: team (n=72) versus individual (n=10), and subsequently, according to the performance, into "power" sports (n=29; sprinters, short distance swimmers, and volleyball players) and "intermittent" sports (n=53; football, basketball, and hockey players). RESULTS: All the populations studied were in Hardy-Weinberg equilibrium for the following polymorphisms: ACE (I/D), ACTN3 (R577X), NOS3 (-786 T/C), UCP2 (A55V), and UCP3 (-55 C/T). We observed that the frequency of NOS3-786 T and UCP2 C alleles was higher among power athletes compared with controls (p=0.011 and p=0.012, respectively); these alleles were also overrepresented in individual athletes (p=0.02 and p=0.045, respectively), although a small sample was analyzed. The frequency of NOS3 298G allele was higher among power athletes compared with controls (p=0.015); these data remained suggestive after correction for multiple testing. CONCLUSION: We found a suggestive association between NOS3 (-786 T/C; G298A) and UCP2 (A55V) polymorphisms and power athletes, whereas no significant correlation was found with UCP3 (-55C/T), ACE (I/D), and ACTN3 (R577X) polymorphisms, in contrast to previous studies. Analysis of multiple performance-associated genetic polymorphisms needs further examination to explain the relationship between genetic background and potential success in sport performance.

Sex modulation of the occurrence of jak2 v617f mutation in patients with splanchnic venous thrombosis.

BACKGROUND: The JAK2 V617F mutation is an independent risk factor for MPN and SVT. Gender-related differences in MPN distribution have been reported and, recently, variability in the JAK2 V617F allele burden between sexes has been suggested. We wondered whether gender would modulate the role of the JAK2 V617F mutation as susceptibility risk factor for SVT. MATERIALS AND METHODS: In 180 patients presenting with SVT, medical history was collected. The presence of the JAK2 V617F mutation and 46/1 haplotype was determined by polymerase chain reaction followed by TaqMan SNP genotyping assays. RESULTS: Among patients with SVT, 43 (23.9%; 95%-CI: 18.2-30.7) carried the JAK2 V617F mutation. The JAK2 V617F mutation was found more frequently in women (29/95: 30.5%; 95%-CI: 22.1-40.4) than in men (14/85: 16.5%; 95%-CI: 10.0-25.9; OR: 2.2; 95%-CI: 1.1-4.5). The distribution of 46/1 haplotype frequencies did not differ significantly between men and women. In women carrying the rs12343867 CC genotype, the frequency observed for the occurrence of the V617F mutation was significantly higher than that observed in those not carrying (60.0% [95% CI: 31.2-83.3] vs. 26.8% [95% CI: 18.4-37.4]; OR: 4.1; 95%-CI: 1.1-14.9). In men, a similar prevalence was found among carriers of the rs12343867 CC genotype (16.7% [95% CI: 3.5-46.0]) and in non carriers (16.4% [95% CI: 9.3-27.2]). The V617F allele burden was unrelated to clinical characteristics and significantly higher in carriers of the rs12343867 CC genotype. CONCLUSIONS: Present findings suggest that, in patients presenting with SVT, the JAK2 V617F mutation is frequently found in women and, possibly by interacting with the 46/1 haplotype, may represent a gender-related susceptibility allele for SVT.

Detection of new deletions in a group of Italian patients with Hemophilia A by multiplex ligation-dependent probe amplification.

AIM: Hemophilia A is an X-linked bleeding disorder caused by mutations widespread in the human coagulation F8 gene. Apart from common intrachromosomal translocations, most of the mutations in the F8 gene are detectable using genomic sequencing analysis. However, deletions of one or more exons or deletion encompassing the entire gene can go undetected, especially in heterozygous females. RESULTS: The multiplex ligation-dependent probe amplification is an efficient tool, new and fast, for discovering these rearrangements. In this study different deletions, which were detected using multiplex ligation-dependent probe amplification assay on 25 patients affected by severe hemophilia A, were classified as "mutation negative" by sequencing analysis. CONCLUSIONS: These data suggest that this screening could be systematically included in genetic screening of patients with Hemophilia A.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.