Povezanost inbreedinga i melanoma kod lipicanskih konja

The relationship between inbreeding and melanoma status (graded from 0 to 4) was analysed by various regression models. Analysed data referred to 296 grey Lipizzan horses originating from five state-owned studs (Austria, Croatia, Hungary, Slovakia and Slovenia) and with average inbreeding coefficient (F=0.107) calculated from extremely informative pedigrees (98% and 76% of horses had completely full pedigree in generation 10 and 20, respectively). In all regression models, in addition, the effects of stud (fixed) and age (covariate) were included. When all data were treated as one population, the estimates from linear and ancestral inbreeding models were not significant. Total inbreeding effect estimates (at F=0.125 and Fa=0.57) were 0.26 and 0.30 for the ancestral inbreeding and linear regression models, respectively. Heterogeneity among state-owned studs in inbreeding effects was also tested for both models and weak statistical significance was obtained for the interaction model with ancestral inbreeding (P=0.049). However, observed effect in the model with interaction was not consistent, did not yield in better model fitting and the obtained significance is probably just a statistical artefact. In general, although some indications about the relationship between ancestral inbreeding and melanoma were present, inbreeding does not appear to be a factor that substantially influences the expression of melanoma in Lipizzan horses.Regresijskim modelima analizirana je povezanost stadija melanoma (na skali od 0 do 4) i inbreedinga. Analizirani podaci odnose se na 296 sivih lipicanskih konja iz pet državnih ergela (Austrija, Hrvatska, Mađarska, Slovačka i Slovenija). Prosječna razina inbreedinga (F=0.107) izračunata je na temelju izuzetno informativnog pedigreea (98% jedinki imalo je potpuno poznat pedigre u generaciji 10, a 76% jedinki imalo je potpuno poznat pedigree u generaciji 20). U svim modelima uvažen je i utjecaj ergele (fiksni utjecaj) te starosti konja (kovarijabla). Kada se analiza koeficijenta regresije (regresija stadija melanoma na koeficijent inbreedinga) odnosila na populaciju kao jednu cjelinu, procjena linearnog koeficijenta regresije za inbreeding kao i za inbreeding predaka nije bila signifikantna. Kod razine inbreedinga F=0.125 i Fa=0.57, ukupna procjena linearnog utjecaja inbreedinga bila je 0.30, a utjecaja inbreedinga predaka bila je 0.26. Analizirana je i heterogenost utjecaja inbreedinga između državnih ergela i za interakcijski model sa inbreedingom predaka dobivena je slaba signifikantna interakcija (P=0.049). Ipak, dobiveni utjecaj u model sa interakcijom nije bio konzistentan, nije bolje objašnjavao nerazjašnjenu varijabilnost modela te je dobivena signifikantnost vjerojatno posljedica slučajnosti. Iz navedenog se može zaključiti, premda postoji indicija o povezanosti inbreedinga predaka i melanoma, da kod lipicanskih konja inbreeding nije čimbenik koji utječe na pojavu melanoma

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver

Background Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. Results We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. Conclusions We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.Peer reviewe

Von Willebrand factor propeptide and pathophysiological mechanisms in European and Iranian patients with type 3 von Willebrand disease enrolled in the 3WINTERS-IPS study

Background Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag. Objective To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity. Methods European and Iranian type 3 patients were enrolled in the 3WINTERS-IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients' diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann-Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges-Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman's rank correlation. Results Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2-2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0-4.2; P = .054]). FVIII:C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778). Conclusions An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype.Peer reviewe

Bleeding symptoms in patients diagnosed as type 3 von Willebrand disease : Results from 3WINTERS-IPS, an international and collaborative cross-sectional study

Background Type 3 von Willebrand's disease (VWD) patients present markedly reduced levels of von Willebrand factor and factor VIII. Because of its rarity, the bleeding phenotype of type 3 VWD is poorly described, as compared to type 1 VWD. Aims To evaluate the frequency and the severity of bleeding symptoms across age and sex groups in type 3 patients and to compare these with those observed in type 1 VWD patients to investigate any possible clustering of bleeding symptoms within type 3 patients. Methods We compared the bleeding phenotype and computed the bleeding score (BS) using the MCMDM-1VWD bleeding questionnaire in patients enrolled in the 3WINTERS-IPS and MCMDM-1VWD studies. Results In 223 unrelated type 3 VWD patients, both the BS and the number of clinically relevant bleeding symptoms were increased in type 3 as compared to type 1 VWD patients (15 versus 6 and 5 versus 3). Intracranial bleeding, oral cavity, hemarthroses, and deep hematomas were at least five-fold over-represented in type 3 VWD. A more severe bleeding phenotype was evident in patients having von Willebrand factor antigen levels <20 IU/dL at diagnosis in the two merged cohorts. In type 3 patients, there was an apparent clustering of hemarthrosis with gastrointestinal bleeding and epistaxis, whereas bleeding after surgery or tooth extraction clusters with oral bleeding and menorrhagia. Conclusions In the largest cohort of type 3 VWD patients, we were able to describe a distinct clinical phenotype that is associated with the presence of a more severe hemostatic defect.Peer reviewe