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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.

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.

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).

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.

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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1106–1113, 2012

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.

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.