Search

In hemophilia A, F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favor inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTC) may contribute to immune tolerance by producing full-length proteins through the insertion of amino acid subset(s). To quantitatively evaluate the readthrough output in vitro, we developed a very sensitive luciferase-based system to detect very low full-length FVIII synthesis from a wide panel (n=45; ~60% patients with PTC) of F8 nonsense variants. PTC not associated with inhibitors displayed higher readthrough-driven expression levels than inhibitor-associated PTC, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six hemophilia A patients with PTC, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTC not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (4 out of 57). These original insights into the molecular genetics of hemophilia A, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favor PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.

The new European Union animal health law and its rule concerning brucellosis in cattle and buffaloes The authors examine the latest European Union (EU) rules concerning eradication and surveillance of brucellosis and animal infectious diseases of EU concern. The Italian rules concerning brucellosis in cattle and buffaloes Italy is included in the EU co-financed compulsory eradication and surveillance programmes for brucellosis in cattle and in buffaloes in the frame of the EU and the related Italian laws, which allowed reaching the Brucellosis Free status without vaccination (former "Officially Brucellosis Free - OBF" status) in the majority of the northern and middle Italian Regions and in some middle and southern Provinces included in Regions where the infection persists. Epidemiology of brucellosis in the EU and in Italy In the Italian Province of Caserta, the highest prevalence of brucellosis in buffaloes is reported; in 2017-2021, a total of 314 outbreaks occurred, in which 39,163 heads tested positive. Here, brucellosis is threatening not only human health and the widespread buffaloes breeding but also the important satellite activities concerning the Protected Designation of Origin (P.O.D.) cheese “Mozzarella di Bufala Campana". The authors also discuss the reemerged brucellosis in cattle in the Molise Region, which despite bordering the Province of Caserta, shows different hydrographic, orographic, and breeding characteristics. In Molise, the reemerged brucellosis had a very different epidemiological course, which allowed it to limit the adoption of One Health measures. The One Health approach to tackle brucellosis in buffaloes In order to tackle brucellosis in Campania Region and in its Province of Caserta, the One Health approach has been predisposed through strict control of animal health, human health, and the environment. The adopted model could be exportable to territories having similar characteristics.

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5′ splice site (5′ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.