Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

<p>Abstract</p> <p>Background</p> <p>Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the <it>HFE </it>gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for <it>HFE </it>mutations screening based on TaqMan technology and to determine the frequencies of <it>HFE </it>mutations in the Slovenian population.</p> <p>Methods</p> <p>Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of <it>HFE </it>mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing.</p> <p>Results</p> <p>The genotyping assay of the H63D, S65C and C282Y mutations in the <it>HFE </it>gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous <it>HFE </it>genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5 – 14.2%), 1.8% (95% CI 1.4 – 2.5%) and 3.6% (95% CI 3.0 – 4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions.</p> <p>Conclusion</p> <p>The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.</p

Microfabrication of a biomimetic arcade-like electrospun scaffold for cartilage tissue engineering applications

Designing and fabricating hierarchical geometries for tissue engineering (TE) applications is the major challenge and also the biggest opportunity of regenerative medicine in recent years, being the in vitro recreation of the arcade-like cartilaginous tissue one of the most critical examples due to the current inefficient standard medical procedures and the lack of fabrication techniques capable of building scaffolds with the required architecture in a cost and time effective way. Taking this into account, we suggest a feasible and accurate methodology that uses a sequential adaptation of an electrospinning-electrospraying set up to construct a system comprising both fibres and sacrificial microparticles. Polycaprolactone (PCL) and polyethylene glycol were respectively used as bulk and sacrificial biomaterials, leading to a bi-layered PCL scaffold which presented not only a depth-dependent fibre orientation similar to natural cartilage, but also mechanical features and porosity compatible with cartilage TE approaches. In fact, cell viability studies confirmed the biocompatibility of the scaffold and its ability to guarantee suitable cell adhesion, proliferation and migration throughout the 3D anisotropic fibrous network. Additionally, likewise the natural anisotropic cartilage, the PCL scaffold was capable of inducing oriented cell-material interactions since the morphology, alignment and density of the chondrocytes changed relatively to the specific topographic cues of each electrospun layer.publishe

Endothelial Differentiation of Human Stem Cells Seeded onto Electrospun Polyhydroxybutyrate/Polyhydroxybutyrate-Co-Hydroxyvalerate Fiber Mesh

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues

Screening for hemochromatosis

Background: Hereditary hemochromatosis is the most common autosomal recessive disorder in populations of northern European descent. Issues: Many experts consider hemochromatosis to be an almost ideal disease for population screening because it essentially fulfills almost all the criteria for screening proposed by the WHO. However, others disagree and suggest that more data are required particularly with regard to the natural history and penetrance of the disease. There is also disagreement about the best diagnostic/screening test for the disease and the performance of these tests in the context of screening. Other concerns are the variability and lack of standardization in screening test measurements, the selection of screening threshold values and the identification of false positive cases. The advent of a genetic test for the condition has brought other worries with regard to informed consent and the ethical, legal and social implications of screening particularly in relation to medical and general discrimination. Other important issues include compliance, cost effectiveness and the evidence that screening has lessened the burden of disease in the community. Conclusions: At the present time, we believe that further data regarding both the exact disease burden and the outcomes of screening studies particularly in the general community are required before widespread population screening is introduced. (C) 2002 Elsevier Science B.V. All rights reserved

Effects of fiber orientation on the frictional properties and damage of regenerative articular cartilage surfaces

Articular cartilage provides a low-friction, wear-resistant surface for diarthrodial joints. Due to overloading and overuse, articular cartilage is known to undergo significant wear and degeneration potentially resulting in osteoarthritis (OA). Regenerative medicine strategies offer a promising solution for the treatment of articular cartilage defects and potentially localized early OA. Such strategies rely on the development of materials to restore some aspects of cartilage. In this study, microfibrous poly(ɛ-caprolactone) scaffolds of varying fiber orientations (random and aligned) were cultured with bovine chondrocytes for 4 weeks in vitro, and the mechanical and frictional properties were evaluated. Mechanical properties were quantified using unconfined compression and tensile testing techniques. Frictional properties were investigated at physiological compressive strains occurring in native articular cartilage. Scaffolds were sheared along the fiber direction, perpendicular to the fiber direction and in random orientation. The evolution of damage as a result of shear was evaluated via white light interferometry and scanning electron microscopy. As expected, the fiber orientation strongly affected the tensile properties as well as the compressive modulus of the scaffolds. Fiber orientation did not significantly affect the equilibrium frictional coefficient, but it was, however, a key factor in dictating the evolution of surface damage on the surface. Scaffolds shear tested perpendicular to the fiber orientation displayed the highest surface damage. Our results suggest that the fiber orientation of the scaffold implanted in the joint could strongly affect its resistance to damage due to shear. Scaffold fiber orientation should thus be carefully considered when using microfibrous scaffolds