Spectrum of F8 gene mutations in haemophilia A patients from a region of Italy: identification of 23 new mutations.

SUMMARY: Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by a lack or decrease of coagulation factor VIII activity. The molecular diagnosis of HA is challenging and a variety of different mutations have been identified throughout the F8 gene. Our aim was to detect the causative mutation in 266 HA patients from Emilia-Romagna region (Italy) and in all suspected carriers. Molecular analysis of F8 in 201 HA patients (152 index cases) was performed with a combination of several indirect and direct molecular approaches, such as long distance polymerase chain reaction, multiplex ligation-dependent probe amplification, denaturing high performance liquid chromatography and direct sequencing. The analysis revealed 78 different mutations, 23 of which were novel, not having been reported in national or international databases. The detection rate was 100%, 86% and 89% in patients with severe, moderate and mild HA, respectively. The information provided by this registry will be helpful for monitoring the treatment of HA patients in Emilia-Romagna and also for reliable genetic counselling of affected families in the future.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

A web-based registry of inherited bleeding disorders in the region of Emilia-Romagna: results at three and a half years.

A Registry of inherited bleeding disorders was set up in the Region of Emilia-Romagna (RER) to collect information about these diseases and to improve the quality of care. From January 2003, the eight Haemophilia Centres (HC) in the RER began to use computerized clinical records; every 6 months, they send data to Parma Hospital to be processed and published in a website (http://www.registroemofiliarer.it). Great efforts are made to ensure high quality of data. Results of general interest are included in a free 'public area' and more sensitive data in a 'reserved area' (open only to HC and to health authorities). A total of 610 individuals are included: 249 haemophilia A (HA), 63 haemophilia B (HB), 173 von Willebrand's disease, 69 rare bleeding disorders, seven platelet disorders and 49 haemophilia carriers; 131 were genotyped, 188 were tested for inhibitors (16 affected). The most frequent bleeding was haemarthrosis. The joint score (evaluated in 104 haemophiliacs) was higher in severe HA. There were 22 HIV-positive and 182 hepatitis C virus-positive patients (21% have chronic hepatitis, two hepatocellular carcinoma). In 2005, two patients received primary prophylaxis, 47 secondary prophylaxis, four children were on immune-tolerance induction. From 2003 to 2005 the use of recombinant products was greatly increased and the majority of patients received them. The mean clotting factor consumption for prophylaxis was higher than on-demand treatment. The main features of registry are to collect high quality and comprehensive data of all patients followed by HC, to improve quality of care and it's availability on the web.

Dynamical slowdown of polymers in laminar and random flows.

The influence of an external flow on the relaxation dynamics of a single polymer is investigated theoretically and numerically. We show that a pronounced dynamical slowdown occurs in the vicinity of the coil-stretch transition, especially when the dependence on polymer conformation of the drag is accounted for. For the elongational flow, relaxation times are exceedingly larger than the Zimm relaxation time, resulting in the observation of conformation hysteresis. For random smooth flows, hysteresis is not present. Yet, relaxation dynamics is significantly slowed down because of the large variety of accessible polymer configurations. The implications of these results for the modeling of dilute polymer solutions in turbulent flows are addressed.

Experimental study for the feasibility of a crystalline undulator.

We present an idea for creation of a crystalline undulator and report its first realization. One face of a silicon crystal was given periodic microscratches (grooves) by means of a diamond blade. The x-ray tests of the crystal deformation due to a given periodic pattern of surface scratches have shown that a sinusoidal-like shape is observed on both the scratched surface and the opposite (unscratched) face of the crystal; that is, a periodic sinusoidal-like deformation goes through the bulk of the crystal. This opens up the possibility for experiments with high-energy particles channeled in a crystalline undulator, a novel compact source of radiation.

Lorenz-like systems and classical dynamical equations with memory forcing: an alternate point of view for singling out the origin of chaos.

An alternate view for the emergence of chaos in Lorenz-like systems is presented in this paper. For such purpose, the Lorenz problem is reformulated in a classical mechanical form and it turns out to be equivalent to the problem of a damped and forced one-dimensional motion of a particle in a two-well potential, with a forcing term depending on the "memory" of the particle past motion. The dynamics of the original Lorenz system in the proposed particle phase space can then be rewritten in terms of a one-dimensional first-exit-time problem. The emergence of chaos turns out to be due to the discontinuous solutions of the transcendental equation ruling the time for the particle to cross the intermediate potential wall. The whole problem is tackled analytically deriving a piecewise linearized Lorenz-like system that preserves all the essential properties of the original model.

High-efficiency beam extraction and collimation using channeling in very short bent crystals.

A silicon crystal was used to channel and extract 70 GeV protons from the U-70 accelerator with an efficiency of 85.3+/-2.8%, as measured for a beam of approximately 10(12) protons directed towards crystals of approximately 2 mm length in spills of approximately 2 s duration. The experimental data follow very well the prediction of Monte Carlo simulations. This demonstration is important in devising a more efficient use of the U-70 accelerator in Protvino and provides crucial support for implementing crystal-assisted slow extraction and collimation in other machines, such as the Tevatron, RHIC, the AGS, the SNS, COSY, and the LHC.

Immunochemical identification of abnormal constituents in the dermis of pseudoxanthoma elasticum patients.

Pseudoxanthoma elasticum (PXE) is a connective tissue inherited disease characterized by dermal alterations and mineralization of the elastin fibres. To investigate its pathogenesis, which is still unknown, antibodies against the principal connective tissue components were assayed on ultrathin sections of dermis from 7 PXE subjects and 5 age matched controls. Both control and PXE elastin fibres were positive for heparan, dermatan and chondroitin 0-sulphates, decorin and biglycan. In PXE, elastin fibres were also highly positive for chondroitin 6-sulphate, vitronectin, fibronectin and serum amyloid antigen. Vitronectin and fibronectin were mostly concentrated in the areas of dense mineralization within the elastin fibres. The abnormal microfilament aggregates, often seen in PXE dermis, were positive for all the above mentioned molecular species as well as for collagen types I and III and fibrillin; on the contrary, they were always negative for elastin. The results suggest that PXE is a complex disorder, in which the whole extracellular matrix is deeply disturbed. Therefore, without excluding an elastin gene defect, the data seem rather to suggest that PXE is a disorder of the mechanisms controlling the production of matrix constituents and that elastin mineralization is caused by molecules abnormally produced and entrapped within the fibre during elastin fibrogenesis.

Aging of the human synovium: an in vivo and ex vivo morphological study.

Age-associated changes of the human synovium have been investigated by microarthroscopy, optical and electron microscopy, immunohistochemistry, and cytochemistry. The knee joints of nineteen 15- to 56-year-old subjects, classified as normal by inspection, were carefully examined by microarthroscopy; small synovial tissue biopsy specimens from both the suprapatellar pouch and the medial tibiofemoral gutter were taken. Microarthroscopy showed that the villi were more numerous and the vascular network and cell distribution and profiles less regular in aged individuals. These data were confirmed by scanning electron microscopy, which also showed large areas of the synovial surface devoid of cells and collagen bundles in contact with the joint cavity in aged subjects. Light and transmission electron microscopy confirmed these data and allowed evaluation of the number, distribution, shape, and internal organization of cells as well as the distribution of vessels and the organization of the extracellular matrix in the full thickness of the synovium (down to 2 mm). Particular attention was paid to synovial lining cells, among which three main phenotypes could be recognized: synthetic type (present at all ages and hypertrophied in aged subjects), macrophagelike (increasing with age), and fibroblastlike. Collagen increased with age. Further studies are needed for comprehensive understanding of age-associated changes in the human synovium.

Immunocytochemical localization of proteoglycans within normal elastin fibers.

Appreciable amounts of glycosaminoglycans have been found by immunocytochemistry within mature elastin fibers of human dermis. On thin sections, elastin fibers showed antigenic sites for monoclonal antibodies recognizing the unsaturated units remaining after digestion of hyaluronic acid with Streptomyces hyaluronidase and after digestion of dermatan and chondroitin sulfates with chondroitinase ABC. Moreover, sectioned elastin fibers were positive towards antibodies raised against synthetic peptides corresponding to amino acid sequences near the N-terminus of the protein core of small matrix proteoglycans PGI and PGII, respectively (Fisher et al., J. Biol. Chem. 262, 9702-9708 (1987)). This is the first demonstration that highly hydrophylic molecules are strictly associated with normally cross-linked elastin. The presence of highly hydrated molecules within the elastin polymer could greatly influence its physiological properties and behavior in pathology.

Dermal elastin and collagen in systemic sclerosis. Effect of D-penicillamine treatment.

Skin biopsies from 4 systemic sclerosis (SSc) patients, 6 SSc patients treated with D-penicillamine (from 8 to 60 months) and 4 normal subjects were analyzed by light and electron microscopy. By light microscopy, collagen bundles of SSc dermis were thicker and more compact than in age-matched controls; D-penicillamine treatment did not significantly modify their organization. On the contrary, a stereological analysis showed that the elastin volume density was higher in patients than in controls, and increased again after D-penicillamine treatment: moreover, the number of elastin fibers per unit area was significantly higher in the dermis of patients compared to controls, and became even higher after D-penicillamine treatment. The phenomena were evident in all strata of the dermis; however, the most significant increase of elastin in SSc patients compared to controls was in the superficial dermis, whereas after D-penicillamine treatment, all the strata of the dermis showed a significant increase in the percentage of elastin and in the number of elastin fibers per unit area compared to untreated patients and to controls. There were no relationships between elastin increase and time from the onset of SSc or time and dose of D-penicillamine treatment. At the ultrastructural level, collagen fibrils had rather heterogeneous diameters and formed more compact fibers, especially in the reticular and in the deep dermis of SSc patients compared to controls. After D-penicillamine, collagen fibril diameters in three of 5 patients examined were statistically wider and more heterogeneous than in controls and in untreated patients, whereas in the other 2 subjects both the mean diameter and the distribution of the class diameter were similar to both controls and untreated patients. This could suggest a specific individual reaction to the drug. Elastin fibers were smaller, more numerous and polymorphous in all patients compared to controls. After D-penicillamine, elastin fibers became even more numerous and smaller than in untreated patients. There was no correlation between the number and size of the elastin fibers and the time or dose of D-penicillamine. The internal organization of the elastin fibers was normal. These data indicate that the amount and distribution of collagen and elastin are altered in the dermis of SSc patients, and that D-penicillamine interferes with the deposition of both fibrous proteins in the dermal extracellular space.

Localization of human placenta lysyl oxidase on human placenta, skin and aorta by immunoelectronmicroscopy.

Polyclonal antibodies to human placenta lysyl oxidase (Kuivaniemi et al., 1984) were used to localize the enzyme at ultrastructural level in human placenta, skin and aorta, by using the indirect immunogold method. The antibodies were tested on thin sections of tissues fixed and embedded in various experimental conditions. With all methods employed, the immunoreaction was always positive on collagen fibers in all tissues examined, independently of the age of the subjects. In placenta, the reaction was also slightly positive on matrix microfilaments and cells. In dermis, fibroblasts and elastin were scarcely positive in a normal 5 day-old child, in a child with skin hyperelasticity, and in two babies with osteogenesis imperfecta type II; whereas they were negative in two 16 and 40 year-old normal subjects. In aorta, the immunoreaction was always positive on collagen, scarcely positive on cells and on elastin of a 24 week-old fetus, of a normal child, and of two babies who died of complications associated with O.I. type II; on the contrary, the reaction was negative on cells and elastin fibers of a 16 week-old fetus, and of a normal 19 year-old girl. When present on elastin, gold particles were localized mostly inside the fibers. Contrary to what was observed by Kagan and coworkers on bovine aorta by using antibodies against aortic lysyl oxidase (Kagan et al., 1986), no specific localization of gold particles could be observed on or adjacent to the elastin/associated microfibrils. The results indicate that antibodies raised against placenta lysyl oxidase recognize collagen-associated as well as elastin-associated epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)