Comparative Analysis Of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B- domain Favours Readthrough- mediated Protein Output

Background: Nonsense mutations, inserting premature termination codons (PTCs), might undergo, with low frequency (<0.01%), spontaneous suppression (readthrough) with production of full-length proteins upon amino acid insertion at the PTC. This process, dictated by nucleotide/protein sequence features, might have implications for hemophilia A (HA) patients. Aims: To investigate residual factor VIII (FVIII) expression through complementary studies in HA patients’ plasma and exploiting a sensitive in-vitro expression platform. Methods: Detection of plasma FVIII levels (ELISA, aPTT), and expression studies (HEK293 cells) with a highly-sensitive naturally-secreted luciferase (Gaussia, GL) fused to FVIII (FVIII-GL). Results: Plasma samples from HA patients affected by six nonsense mutations (p.R446X, p.R814X, p.K1289X, p.W1726X, p.R1985X, p.R2135X) revealed traces of FVIII. Strikingly, the two B-domain variants (p.R814X, p.K1289X) showed the highest FVIII levels, suggesting a position-dependent effect. Expression studies with the FVIII-GL variants showed that those of the B-domain produced the highest luciferase activity levels, thus supporting in vivo findings. Accordingly, the predicted readthrough-deriving amino acid changes (R446W, R814W, K1289Q/Y, W1726Y, R1985W, R2135W) showed a minor impact for those affecting the B-domain. To verify further our hypothesis, the panel of F8 mutations was rationally expanded to be representative of the majority of patients with nonsense mutations (60%), including the most frequent (50% of patients) in the B-domain. Through our sensitive platform we observed that all F8 nonsense variants led to detectable luciferase activity (0.4-6%). Strikingly, when categorized in two groups (B-domain, n=21; other domains, n=26), secreted luciferase activity of B-domain variants was significantly higher (p<0.0001) as compared with variants located in the other FVIII domains. Conclusions: Our findings for the first time indicate that nonsense mutations in the B-domain, known to tolerate missense changes as those potentially arising from readthrough, are favoured in terms of readthrough-mediated protein output, which might have pathophysiological implications for HA patients

A novel de novo HCN1 loss-of-function mutation in genetic generalized epilepsy causing increased neuronal excitability

Abstract The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies

Dark Matter Annihilation around Intermediate Mass Black Holes: an update

The formation and evolution of Black Holes inevitably affects the distribution of dark and baryonic matter in the neighborhood of the Black Hole. These effects may be particularly relevant around Supermassive and Intermediate Mass Black Holes (IMBHs), the formation of which can lead to large Dark Matter overdensities, called {\em spikes} and {\em mini-spikes} respectively. Despite being larger and more dense, spikes evolve at the very centers of galactic halos, in regions where numerous dynamical effects tend to destroy them. Mini-spikes may be more likely to survive, and they have been proposed as worthwhile targets for indirect Dark Matter searches. We review here the formation scenarios and the prospects for detection of mini-spikes, and we present new estimates for the abundances of mini-spikes to illustrate the sensitivity of such predictions to cosmological parameters and uncertainties regarding the astrophysics of Black Hole formation at high redshift. We also connect the IMBHs scenario to the recent measurements of cosmic-ray electron and positron spectra by the PAMELA, ATIC, H.E.S.S., and Fermi collaborations.Comment: 12 pages, 7 figures. Invited contribution to NJP Focus Issue on "Dark Matter and Particle Physics

COVID-19-associated Guillain-Barré syndrome in the early pandemic experience in Lombardia (Italy)

Objective To estimate the incidence and describe clinical characteristics and outcome of GBS in COVID-19 patients (COVID19-GBS) in one of the most hit regions during the frst pandemic wave, Lombardia. Methods Adult patients admitted to 20 Neurological Units between 1/3–30/4/2020 with COVID19-GBS were included as part of a multi-center study organized by the Italian society of Hospital Neuroscience (SNO). Results Thirty-eight COVID19-GBS patients had a mean age of 60.7 years and male frequency of 86.8%. CSF albuminocytological dissociation was detected in 71.4%, and PCR for SARS-CoV-2 was negative in 19 tested patients. Based on neurophysiology, 81.8% of patients had a diagnosis of AIDP, 12.1% of AMSAN, and 6.1% of AMAN. The course was favorable in 76.3% of patients, stable in 10.5%, while 13.2% worsened, of which 3 died. The estimated occurrence rate in Lombardia ranges from 0.5 to 0.05 GBS cases per 1000 COVID-19 infections depending on whether you consider positive cases or estimated seropositive cases. When we compared GBS cases with the pre-pandemic period, we found a reduction of cases from 165 to 135 cases in the 2-month study period in Lombardia. Conclusions We detected an increased incidence of GBS in COVID-19 patients which can refect a higher risk of GBS in COVID-19 patients and a reduction of GBS events during the pandemic period possibly due to a lower spread of more common respiratory infectious diseases determined by an increased use of preventive measures

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Protein engineering and pharmacological approaches to develop novel treatment strategies for coagulation disorders

Haemophilia A (HA) and B (HB) as well as factor VII (FVII) or X (FX) deficiencies are well-characterized haemorragic genetic disorders, whose etiology, even at the molecular level, has been extensively described. Treatment options for patients affected by these diseases are currently available and mainly consist of replacement therapy with the missing factor. However, there are still important limitations and research in the field is boosted towards the implementation of current therapeutic options or the discovery of innovative procedures. In the present work we aimed at providing consistent examples of novel treatment strategies for bleeding disorders, via protein engineering or pharmacological approaches. The different systems here proposed intend to (i) provide selection criteria for an individually-targeted therapy of patients carrying specific mutations (personalized medicine) and to (ii) ameliorate pharmacokinetic features of therapeutic proteins in order to reduce the burden of treatment. In the first part of the thesis, we studied the molecular event of ribosome readthrough in the framework of nonsense mutations causing HB or FVII deficiency. We took advantage of an in vitro platform for the expression of factor IX nonsense variants. We were able to show that specific mRNA and protein constraints limit the number of mutations that could be productively suppressed by readthrough induction. In particular, the potential re-insertion of the original residue in place of the premature stop codon strongly favors a productive functional output. Moreover, this correction approach is predicted to be efficient for those mutations that fall in a protein domain removed during biosynthesis (i.e. pre-peptide). We also studied ribosome readthrough in order to elucidate the molecular mechanisms underlying phenotypes’ severity in FVII deficiency, by two paradigmatic homozygous nonsense mutations. The data obtained suggest that the appreciable rescue for one mutation was driven by reinsertion of the wild-type residue, whereas the minimal function seen for the other one was explained by missense changes permitting FVII secretion and function. Together, these data may lay the foundation for the rational selection of patients who could benefit from treatment with readthrough-inducing drugs. In the second part of the thesis, we exploited protein engineering to extend coagulation factors half-life. Among the several known strategies, we choose the fusion with human serum albumin (HSA), a protein with an extremely long half-life due to a neonatal Fc receptor (FcRn)-mediated recycling mechanism. In the context of by-passing therapy for haemophilias, we aimed at providing next-generation recombinant activated FVII-albumin (rFVIIa-HSA) fusion proteins by taking advantage of albumin engineered variants with improved pharmacokinetic features. The rFVIIa-HSA variants showed a by-passing activity in hemophilic plasma comparable to that of rFVIIa alone, both in vitro and in vivo. Furthermore, the fusion proteins comprising engineered albumin bind to FcRn with increased affinity compared to wild-type albumin. So, these molecules would have the potential to last remarkably longer in circulation; if translated into patients, this would eventually mean needing less frequent injections, which represents a great achievement in terms of patients’ quality of life. Finally, we proposed a fusion protein between FX and albumin, in order to fulfill the unmet need of a recombinant product for FX deficiency therapy. We were able to show for the first time that fusion with albumin is compatible with a FX-HSA chimaera endorsed of robust procoagulant activity; we have also preliminarily demonstrated that albumin actually confers to FX prolonged persistence in circulation in a mouse model. Nevertheless, the molecule here proposed could be the platform for albumin engineering aimed at further improving pharmacokinetic properties of the therapeutic protein(s)

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5' splice site (5'ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5'ss is surrounded by various cryptic 5'ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5'ss and create/strengthen cryptic 5'ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5'ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5'ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5'ss (similar to 80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5'ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A

Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants

Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins