 Hi, my name is Mirco Pinotti and I'm a professor of biochemistry at the University of Ferrara. Behind me you can see the department in which I'm working, which is the Department of Biochemistry and Molecular Biology. By this video I would like to walk you through the study we have conducted and that has been recently published in Human Mutation. The topic is a nonsense mutation and the point we would like to discuss and we have addressed is the question whether the nonsense mutations are truly and always new mutations and then later in a while I will show you the data and I try to demonstrate that in some cases nonsense mutations are associated to residual expression, that in some pathologies can have pathophysiological implications. So I decided to use some slides to make the concept more clear and to show you the essential data supporting the conclusion of our paper. So as you can see I just drawn a picture cartoon in which there is a stop mutation and for women who are not familiar with nonsense mutations, this mutation implies the insertion of a premature top codon into the coding mRNA. And in this way the nonsense mutation is associated to the production of truncated molecules that most of the time are not functional. And in some cases, especially for early nonsense mutations, this change leads to mRNA decay. In the scientific community nonsense mutations are commonly considered neuro-genetic conditions. But we have to consider that the translation termination, it means the stop of the protein synthesis at the nonsense triplets is not perfect. And in some cases also with a low efficiency the ribosome can read through over the nonsense triplet and give rise to a process which is called ribosome read through, which can lead to the synthesis of full length molecules even in the presence of nonsense mutation. Of course this process is not that efficient but even traces of full length protein and especially if they are functional can have pathophysiological implication depending on the disease we are considering. Our field is coagulation factor deficiency in which one of the factors involved in coagulation cascade is affected by the mutation. And we have addressed this issue on imophilia B which is the disease caused by mutations in the factor 9G. In this deficiency, nonsense mutations are relatively frequent, something around 10-15% and that are associated to the most severe cases. And in this disease even traces of full length protein can have implication for the clinical manifestation and particularly for the development of inhibitory antibodies. So we decided to investigate four different nonsense mutations that have been found in the database and are associated to severe imophilia B. As you can see there are four different mutations which are occurring at different point of the CNA. They are changing, they are at different position in the protein and they have different sequence. And based on previous data we know that the sequence and the sequence context of the nonsense mutation is an important determinant of the efficiency of rethrough. And as you can see the predicted rethrough over this mutation is different with the mutation 162 which is expected to undergo rethrough with the most efficient. So the first part of the study was addressed in plasma from patients. And I would like to remind you that factor 9 is the coagulation factor which is circulating plasma which makes easier for us to investigate the residual expression in the physiological side. As you can see here are the bands and by western blotting we were able to detect traces. You can see in this with the mutation 298 or with the 294 we were able to detect traces of full-length protein. But to coagulate this data which were obtained in patients we wanted also to in parallel to express all these recombinant variant and to check whether in geocreatic cells these nonsense mutations are also associated to residual levels of full-length protein. So we made mutagenesis into the phatonized DNA, we inserted the mutation and we expressed these recombinant proteins in the eukaryotic cells. As you can see in medium we detected level, appreciable levels of protein but by western blotting we discovered that the majority of these molecules were truncated as expected for nonsense mutation. However when we focused on the part of the gel in which is corresponding to the side of the full-length molecule we discovered that the mutation 294, 298 and 162 that I remind you is the mutation with the sequence context more susceptible to rethrow we were able to detect the full-length protein. At various one of these mutations the 103 even in patient and also in recombinant protein was not giving any, any appreciable trace of full-length protein. So in conclusion all this data for the first time for a secreted protein demonstrated that the ribotry fluid is occurring in vivo and may account for a residual level of full-length protein. And this finding if we consider hemophilia and correlation phatodeficiency can have pathophysiological implication. I would like just to thank all the people involved in this study and Francesco Bernardi for his illuminating suggestion. Pierparo Colusa, Alessandro Canella and Matteo Campioni who perform most of the work in expression study and western blotting analysis. And the group of Giuseppe Targariello and Castaman who provided us the sample from patients and the clinical picture. And last I would like to thank the agency that funded the study and particularly Telethon, the Foundation in Carife and Ministry of the University. And I thank you for your attention. I hope I was clear enough to convey the main message of our study. And I hope in the future to present some new data on how this process can, to demonstrate how can this process have really implication for the diagnosis and interpretation of the relationship between the molecular defects and the clinical phenotype.