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R. Leaci, M. Galetti, P. Petronini, S. Gomarasca, A. Carta; From Phenotype to Genotype in Moebius Syndrome: Institution of a Dna Bank for Collaborative Genetic Studies . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2945.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To create a DNA bank from patients affected by Moebius Syndrome (MBS) referred to our Department from the Italian Association of Moebius Syndrome (www.moebius–italia.it). Methods: To date, a total of 28 patients have been followed in our Department. The 10 most severe cases have been invited, after informed consent, to partecipate in the creation of a DNA bank for MBS. Peripheral blood mononuclear cells (PBM) have been obtained from probands and their relatives (mother and father) by centrifugation of heparinized venous blood over sodium diatriazoate/Ficoll gradients. Afterwards PBM were separated into T cell–enriched and B cell–enriched populations by incubating the mononuclear cell suspension with phytohemagglutinin and cyclosporine A respectively. The resultant population of T cells were incubated with Il–2, while B cells were immortalized using Epstein Barr Virus (EBV). T– and B– cells were then frozen in liquid nitrogen. Time from initiation of a B cell culture to cryoconservation was 8 weeks; this period was necessary for banking of large numbers of lymphoblastic cells. Results: Immortalization by EBV was effective in producing a long–term growth of human B–lymphocytes. The overall success rate in establishing permanent cell lines was 70%. Actually, a permanent line of immortalized cells is available for DNA testing in MBS. Conclusions: To our knowledge, this is the first DNA bank created in a large series of cases with severe phenotypical characterization. The present work is a call for future collaboration with laboratories interested in testing candidate genes which may play a key role in the pathogenesis of MBS.
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