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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.

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.

Context Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. Objectives We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype–phenotype correlation in individuals of two different families. Design and methods To verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. Results The amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. Conclusions The strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.

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.

Development of inhibitory antibodies is perhaps the most serious complication of FVIII replacement therapy, precluding efficient clinical management of patients with haemophilia A (HA). The development and function of immune system are also regulated by microRNAs (miRNAs). Mutations and changes in the level of expression of some miRNA genes have been associated with the onset and progression of immunological disorders. The aim of this study was to investigate new genetic polymorphisms in loci for miRNA and their targets to evaluate whether these SNPs may confer susceptibility to inhibitor development in patients with HA. Italian HA patients with and without inhibitors and healthy controls were recruited in this study. For SNP analysis, standard DNA sequencing method was used. We have studied four SNPs, i.e. rs36101366, rs34683807, rs1803603 and rs3024496 located in the 3'UTR of F8 and IL-10 genes. These SNPs have been checked for their frequencies in patients with and without inhibitors, but no statistically significant differences were found. Then, we have searched for other genetic variants in loci for haematopoietic-specific miRNAs, i.e. hsa-mir-150, hsa-mir-155, hsa-mir-146a, hsa-mir-142, hsa-mir-181a and in a specific miRNA, hsa-mir-1184, i.e. predicted to be located in the intron 22 of F8 gene. For all miRNAs selected, we did not identify any sequence variation in our study population. This is the first study to demonstrate that there was no association between selected SNPs in miRNAs and their targets and the susceptibility to inhibitor development in people affected by HA.

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.

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.

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 63 kDa 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 60 kDa 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 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. 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.

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Δ mutant strain was constructed. Coupled mitochondria isolated from flx1Δ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Δ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

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).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.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.

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.

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.

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.

One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFalpha, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

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.Eighty-three nonanticoagulated patients with a documented pregnancy-related VTE and 195 controls were investigated. The presence of the ANXA5 haplotypes was determined.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.The M2 haplotype within the ANXA5 gene may represent a new thrombophilic risk factor for pregnancy-related VTE.