Hways involved in valve formation have been unraveled through the unexpected phenotype encountered in mice lacking the Nfatc1 gene [8,9]. NFATC1 (Nuclear Factor for Activated T-Cells) belongs to the Rel/NF-kB family of transcription factors that were first described as being key regulators of T-cells’ activation. Five members (NFATC1-5) are found in mammals; all playing different non-redundant roles during embryonic and postnatal development [10,11,12,13]. All five members share a conserved DNA-binding domain at the Cterminus of the protein that binds specifically to the consensus (A/ T)GGAAA sequence [14]. In addition, they harbor at the Nterminal region a series of conserved serine-proline residues (S/P) that when dephosphorylated Title Loaded From File unmasked a nuclear localization signal allowing the translocation of NFATC proteins from the cytoplasm to the nucleus [15,16,17,18]. All NFATC proteins except NFATC5 are dephosphorylated by the calcium dependent phosphatase calcineurin (PPP3CA/PPP3CB) at the N-terminus triggering the translocation process. Although NFATC proteinsNFATC1 and Tricuspid Atresiaare weak transactivators, their transcriptional potency is boosted through their interactions with different classes of transcription factors Title Loaded From File mainly the AP-1 family members, c-Fos and Jun, the MADS family, and the GATA zinc finger proteins [19,20,21,22,23]. NFATC1 was shown to be expressed in numerous cell types including lymphocytes, osteoclasts, neurons, and myotubes [17,24,25,26,27]. The first in vivo assessment of the role of the gene came however from the inactivation of the gene in mice. Two independent reports showed that Nfatc12/2 mice die at midgestation stage (e14.5) due to lack of EC growth and remodeling [8,9]. While Ranger AM et al showed a selective defect in the semilunar valves, the Nfatc12/2 embryos generated by de la Pompa Jl et al had severe defects in both atrioventricular and semilunar valves. Although this discrepancy might be linked to the genetic background and/or knock-out strategy, the fact that in both phenotypes the endocardial cushions are hypoplastic do point out to a major role for NFATC1 in endocardial cushion formation and proliferation. This role is even highlighted by the inactivation of PPP3CB, which encodes the calcineurin regulatory subunit, specifically in the endocardium and that results in a mirror-image phenotype identical to that of the Nfatc12/2 knock-out [28,29]. This intrinsic requirement for endocardial expression of NFATC1 in endocardial cushion formation is dispensable for endocardial-mesenchymal transformation since in both Ppp3cb2/2 and Nfatc12/2 embryos, mesenchymal cells are found in the cardiac jelly. The Calcineurin/NFAT pathway is however required in myocardial cells to control EMT through the repression of secreted VEGF. Given the fact that NFATC1 is at the center of valve formation in mammals, 15857111 we hypothesize that mutations in the gene encoding it would be associated with valve malformations in humans. We have previously shown that a tandem repeat in the intronic region of NFATC1 is associated with ventricular septal defects but with no valvular phenotype [30]. We therefore screened for such mutations in patients with different valve diseases registered at the congenital heart disease genetics program at the American University of Beirut Medical Center. Results showed 2 novel missense (P66L, I701L) single nucleotide polymorphisms (SNPs) in only one patient with tricuspid atresia. Functional analyses.Hways involved in valve formation have been unraveled through the unexpected phenotype encountered in mice lacking the Nfatc1 gene [8,9]. NFATC1 (Nuclear Factor for Activated T-Cells) belongs to the Rel/NF-kB family of transcription factors that were first described as being key regulators of T-cells’ activation. Five members (NFATC1-5) are found in mammals; all playing different non-redundant roles during embryonic and postnatal development [10,11,12,13]. All five members share a conserved DNA-binding domain at the Cterminus of the protein that binds specifically to the consensus (A/ T)GGAAA sequence [14]. In addition, they harbor at the Nterminal region a series of conserved serine-proline residues (S/P) that when dephosphorylated unmasked a nuclear localization signal allowing the translocation of NFATC proteins from the cytoplasm to the nucleus [15,16,17,18]. All NFATC proteins except NFATC5 are dephosphorylated by the calcium dependent phosphatase calcineurin (PPP3CA/PPP3CB) at the N-terminus triggering the translocation process. Although NFATC proteinsNFATC1 and Tricuspid Atresiaare weak transactivators, their transcriptional potency is boosted through their interactions with different classes of transcription factors mainly the AP-1 family members, c-Fos and Jun, the MADS family, and the GATA zinc finger proteins [19,20,21,22,23]. NFATC1 was shown to be expressed in numerous cell types including lymphocytes, osteoclasts, neurons, and myotubes [17,24,25,26,27]. The first in vivo assessment of the role of the gene came however from the inactivation of the gene in mice. Two independent reports showed that Nfatc12/2 mice die at midgestation stage (e14.5) due to lack of EC growth and remodeling [8,9]. While Ranger AM et al showed a selective defect in the semilunar valves, the Nfatc12/2 embryos generated by de la Pompa Jl et al had severe defects in both atrioventricular and semilunar valves. Although this discrepancy might be linked to the genetic background and/or knock-out strategy, the fact that in both phenotypes the endocardial cushions are hypoplastic do point out to a major role for NFATC1 in endocardial cushion formation and proliferation. This role is even highlighted by the inactivation of PPP3CB, which encodes the calcineurin regulatory subunit, specifically in the endocardium and that results in a mirror-image phenotype identical to that of the Nfatc12/2 knock-out [28,29]. This intrinsic requirement for endocardial expression of NFATC1 in endocardial cushion formation is dispensable for endocardial-mesenchymal transformation since in both Ppp3cb2/2 and Nfatc12/2 embryos, mesenchymal cells are found in the cardiac jelly. The Calcineurin/NFAT pathway is however required in myocardial cells to control EMT through the repression of secreted VEGF. Given the fact that NFATC1 is at the center of valve formation in mammals, 15857111 we hypothesize that mutations in the gene encoding it would be associated with valve malformations in humans. We have previously shown that a tandem repeat in the intronic region of NFATC1 is associated with ventricular septal defects but with no valvular phenotype [30]. We therefore screened for such mutations in patients with different valve diseases registered at the congenital heart disease genetics program at the American University of Beirut Medical Center. Results showed 2 novel missense (P66L, I701L) single nucleotide polymorphisms (SNPs) in only one patient with tricuspid atresia. Functional analyses.